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A potential role for ICP, a Leishmanial inhibitor of cysteine peptidases, in the interaction between host and parasite
Abstract
The biological role of a natural inhibitor of cysteine peptidases (designated ICP) of Leishmania has been investigated by genetic manipulation of the parasite. Null mutants grew normally in vitro, were as infective to macrophages in vitro as wild-type parasites, but had reduced infectivity to mice. Mutants re-expressing ICP from a single gene gave partial restoration of virulence in vivo, whereas mutants overexpressing ICP secreted the inhibitor and showed markedly reduced virulence in mice. Promastigotes of the null mutants had similar cysteine peptidase activities as the wild-type parasites, suggesting that ICP is not required for the expression or processing of the enzymes. The only proteins found to bind to ICP in promastigote cell lysates were fully processed forms of CPA and CPB, showing that ICP does not bind in abundance either to zymogens of the cysteine peptidases or other leishmanial proteins. However, only a small proportion of ICP colocalized with CPA and CPB in the promastigote (in the endoplasmic reticulum and Golgi) and the majority of ICP resided in vesicles that are apparently distinct from endosomes and the multivesicular tubule (MVT)-lysosome. These data suggest that ICP has a role other than modulation of the activity of the parasite's own cysteine peptidases and their normal trafficking to the MVT-lysosome via the flagellar pocket. The finding that ICP partially colocalized with an endocytosed cysteine peptidase leads us to postulate that ICP has a role in protection of the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages.
... In the early 90`s, a single biochemical study pointed to the presence of heat-resistant activity in extracts of Leishmania, that was capable of inhibiting papain [5] but it was not until 2001 that a natural inhibitor of papain-like peptidases, chagasin, was characterized in T. cruzi [6]. Presently, the chagasin-family comprises several members, identified in protozoa and bacteria, and a growing number of studies in pathogenic protozoa identify these molecules as potent regulators of cysteine peptidases of both the parasite and the host, contributing to the outcome of infection [7][8][9][10][11][12][13]. ...
... A different picture emerged when the role of ICP was investigated in Leishmania. Unlike chagasin in T. cruzi, in L. mexicana only a small proportion of ICP was found colocalizing with CPs in the endoplasmic reticulum and Golgi and the majority of ICP resides in vesicles that are apparently distinct from endosomes and the multivesicular tubule (MVT)-lysosome [9]. Furthermore, L. mexicana mutant lines lacking ICP grew normally in vitro, were as infective to macrophages as wild-type parasites, but had reduced infectivity to mice. ...
... Furthermore, L. mexicana mutant lines lacking ICP grew normally in vitro, were as infective to macrophages as wild-type parasites, but had reduced infectivity to mice. These data led to the proposal that ICP has a role other than modulation of the activity of the parasite's own CPs and their normal trafficking to the MVT-lysosome via the flagellar pocket [9]. These findings indicate that natu-ral targets and the biological roles of ICPs might vary among trypanosomatids. ...
Trypanosomatids rely on peptidases as potent virulence factors and were recently found to contain a unique set of natural peptidase inhibitors not found in higher eukaryotes or in yeast, but present in a limited number of bacteria. Chagasin, identified in Trypanosoma cruzi, is a tight-binding, high affinity inhibitor of papain-like cysteine peptidases that has an Ig-like fold and inactivates target enzymes through a limited number of contacts mediated by a few conserved residues on three exposed loops. Chagasin homologues in other protozoa and bacteria are mostly single genes named ICPs, and together with chagasin compose family I42 at MEROPS, the peptidase and peptidase inhibitors database [http://merops.sanger.ac.uk/]. The biological function of chagasin/ICPs seems to vary depending on the organism, but generally, the current studies point to a role in controlling the activity of endogenous parasite CPs, influencing parasite differentiation, virulence and different aspects of the host response to infection. More recently, natural inhibitors of serine peptidases that share similarity to bacterial ecotins were identified in trypanosomatids and named ISPs. Ecotins are specific to trypsin-fold serine peptidases, enzymes which are not present in trypanosomatids. ISPs are limited to Trypanosomes and Leishmania and to date, only ISP2 proven to have an inhibitory function. In Leishmania, ISP2 seems to control the activity of host SPs at the initial stages of infection in order to ensure subsequent parasite survival in macrophages. The main aspects of chagasin/ICP biochemistry, structure and biological function and the recent findings on ISPs will be covered in this review.
... The first identified ICP was chagasin from Trypanosoma cruzi [17]. Subsequently, ICPs have been found in Trypanosoma brucei [18], Leishmania [19], Entamoeba histolytica [20], and all Plasmodium species analyzed thus far including human, rodent and avian Plasmodium species [21,22]. Related proteins have been described in Pseudomonas aeruginosa but are absent from multicellular eukaryotes [23]. ...
... Recently, the structure of the ICPs from Leishmania mexicana, T. cruzi, and Plasmodium berghei were described as immunoglobulin-like [24,25]. ICPs inhibit parasite proteases, in the case of T. cruzi [26] and T. brucei [27], and both parasite proteases and host cell proteases in the case of Leishmania [19]. ...
... The ICP of L. mexicana is secreted by the parasite and even though a lack of ICP had no impact on infectivity in vitro, ICP overexpressors, as well as knockout parasites, showed a reduced virulence and infectivity in vivo [19]. These observations suggested that LmICP regulates proteases of the host rather than those of the parasite. ...
Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.
... In L. mexicana the generation of lines lacking ICP ( icp) was used as tool to investigate its biological role [186]. The null mutant has reduced infectivity to mice, which was partially reversed by re-expressing a copy of the gene. ...
... The null mutant has reduced infectivity to mice, which was partially reversed by re-expressing a copy of the gene. On the other hand, overexpression of ICP also provoked reduced virulence to mice, with concomitant secretion of the inhibitor, leading to the proposal that secreted ICP could be inactivating cysteine peptidases of the mammalian host, therefore, interfering with the establishment of the infection [186]. Furthermore, localization studies showed that the majority of ICP does not co-localize with CPB and CPA, suggesting that the inhibitor is free to interact with host peptidases. ...
... Furthermore, localization studies showed that the majority of ICP does not co-localize with CPB and CPA, suggesting that the inhibitor is free to interact with host peptidases. Altogether, the data suggests that Leishmania ICP may not be directly involved in the modulation of the parasite's cysteine peptidases and could have a role in protection of the parasite from the hydrolytic milieu found in host macrophages and/or the sandfly gut [186]. This hypothesis was later assessed by following the development of infection of the invertebrate vector Lutzomyia longipalpis with the transgenic parasite lines. ...
Parasitic diseases caused by pathogenic protozoa remain a challenge for public health. Despite efforts to control transmission, to improve early diagnosis and to optimize patient care, millions of infected people, mainly in poor areas of the globe, develop debilitating pathologies that are often fatal. For most of those disorders, the current treatments are greatly unsatisfactory and the continuous search for alternative chemotherapies remains at the center of research. Over the last decades, cysteine peptidases of protozoa feature as highly promising drug targets and their validation in laboratory models of disease or experimental infections instigated growing efforts in medicinal chemistry, aiming at the development of compounds with therapeutical potential. More recently, it was uncovered that protozoa also express new families of endogenous proteinaceous peptidase inhibitors that act as potential virulence factors. In this review, we will cover the main findings that contributed to the validation of cysteine peptidases from Trypanosoma cruzi, Trypanosoma brucei and Leishmania as drug targets and the current knowledge of their biological roles in those organisms. We give an overview of the development of small molecule cysteine peptidase inhibitors with anti-parasite activity and describe the current background on natural peptidase inhibitors in trypanosomatids.
... In protozoan parasites, chagasin, a specific parasite-derived inhibitor of clan CA (family C1 cysteine peptidases), was identified in Trypanosoma cruzi (31). Chagasin and chagasin-like inhibitors (ICP [inhibitor of cysteine peptidases]) lack significant identity with proteins of the cystatin I25 family and are classified as chagasins (clan XI, family I42), which have been suggested to regulate endogenous and/or host cell proteases (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). However, little is known regarding the presence of cystatins of the I25 family in protozoan parasites or of their involvements in differentiation. ...
... Several cystatins have been found to inhibit cysteine proteases and to have immunomodulatory effects on nematodes (20)(21)(22)(23)(24)(25)(26)(27), and the regulatory effects of intracellular cysteine proteases have been found to protect against proteolytic damage in trematodes (28,29). In protozoan parasites, endogenous protease inhibitors have been found to regulate protease activity by blocking deleterious effects of host proteases from Plasmodium falciparum (34), Trypanosoma cruzi (31), Leishmania mexicana (32), and Entamoeba (36). In Plasmodium, falstatin, a cysteine protease inhibitor of P. falciparum, is required to facilitate red cell invasion (34), and PbICP (Plasmodium berghei inhibitor of cysteine proteases) has been reported to play important roles in sporozoite invasion and host cell survival (35). ...
... In Plasmodium, falstatin, a cysteine protease inhibitor of P. falciparum, is required to facilitate red cell invasion (34), and PbICP (Plasmodium berghei inhibitor of cysteine proteases) has been reported to play important roles in sporozoite invasion and host cell survival (35). In T. cruzi, the overexpression of chagasin, an inhibitor of cysteine proteases (including cruzain), decreased infectivity in cell culture (31), and in L. mexicana, virulence in mice was markedly abolished by disrupting the activities of cysteine protease inhibitors (32). However, these cysteine protease inhibitors were classified as members of the chagasins (family I42), and thus, no member of the cystatins (family I25) has yet been identified in protozoan parasites. ...
The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under
unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases
have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation
of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been
established in Acanthamoeba. In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with
lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin
B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity
during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction
in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases
and that it plays an essential role during the encystation of Acanthamoeba.
... A prominent example is chagasin, which is expressed by Trypanosoma cruzi and was the first identified member of a new superfamily of reversible, tight-binding cysteine protease inhibitors [35]. Structurally similar inhibitors were found in Trypanosoma brucei, Pseudomonas aeruginosa, Leishmania mexicana, Leishmania major, Entamoeba histolytica, P. falciparum and Toxoplasma gondii [36,37,38,39,40,41,42,43] . Chagasin-like inhibitors (also termed ICPs for inhibitor of cysteine proteases) are suggested to regulate both endogenous parasitederived cysteine proteases (T. ...
... brucei, T. cruzi, P. falciparum, E. histolytica) and/or host cell proteases (L. mexicana, P. aeruginosa, P. falciparum) [35,37,38,39,40,41,42,44,45,46]. The P. falciparum ICP (PfICP), termed falstatin, has been described previously for the blood stage of the human malaria parasite [40]. ...
... Endogenous L. mexicana ICP had no impact on the infectivity of the parasites in vitro. However, when LmICP null mutants and overexpressors were analysed in vivo in the mouse model system, both showed a reduced virulence and infectivity [38]. It is suggested therefore, that LmICP controls host-derived rather than parasite-derived proteases. ...
Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.
... The activity of cysteine proteinases of higher eukaryotes is controlled by a number of endogenous inhibitors, including cystatins and α2-Macroglobulin. No genes homologous to cystatins have been detected in protozoa, but several protozoa, including T. cruzi [13], T. brucei [14], Leishmania [15], E. histolytica [16], and P. falciparum [17] synthesize endogenous inhibitors with a novel conserved structure, called Inhibitor of Cysteine Proteinases or ICP. Related proteins have also been identified in bacteria but are absent in higher eukaryotes [18,19]. ...
... Related proteins have also been identified in bacteria but are absent in higher eukaryotes [18,19]. The structure of the L. mexicana ICP [15] and chagasin [20,21] were recently described and have a unique immunoglobulin-like fold. ICPs may inhibit parasite cysteine proteinases as in T. cruzi [13] and T. brucei [14] or host proteinases as in Leishmania [15]. ...
... The structure of the L. mexicana ICP [15] and chagasin [20,21] were recently described and have a unique immunoglobulin-like fold. ICPs may inhibit parasite cysteine proteinases as in T. cruzi [13] and T. brucei [14] or host proteinases as in Leishmania [15]. We now report the identification of genes encoding two cysteine protease inhibitors, toxostatin-1 and 2, which inhibit T. gondii cathepsin L and B in the nanomolar range. ...
Toxoplasma gondii is an obligate intracellular parasite of all vertebrates, including man. Successful invasion and replication requires the synchronized release of parasite proteins, many of which require proteolytic processing. Unlike most parasites, T. gondii has a limited number of Clan CA, family C1 cysteine proteinases with one cathepsin B (TgCPB), one cathepsin L (TgCPL) and three cathepsin Cs (TgCPC1, 2, 3). Previously, we characterized toxopain, the only cathepsin B enzyme, which localizes to the rhoptry organelle. Two cathepsin Cs are trafficked through dense granules to the parasitophorous vacuole where they degrade peptides. We now report the cloning, expression, and modeling of the sole cathepsin L gene and the identification of two new endogenous inhibitors. TgCPL differs from human cathepsin L with a pH optimum of 6.5 and its substrate preference for leucine (vs. phenylalanine) in the P2 position. This distinct preference is explained by homology modeling, which reveals a non-canonical aspartic acid (Asp 216) at the base of the predicted active site S2 pocket, which limits substrate access. To further our understanding of the regulation of cathepsins in T. gondii, we identified two genes encoding endogenous cysteine proteinase inhibitors (ICPs or toxostatins), which are active against both TgCPB and TgCPL in the nanomolar range. Over expression of toxostatin-1 significantly decreased overall cysteine proteinase activity in parasite lysates, but had no detectable effect on invasion or intracellular multiplication. These findings provide important insights into the proteolytic cascades of T. gondii and their endogenous control.
... C. albicans demonstrates pathogenicity by modifying host defense mechanisms that secondarily initiate transformations in the fungal behavior. Also, as the fungal cell is almost similar to human cells, targeted drug development is a very difficult task [27,28]. ...
... Therefore, cysteine proteases are the key virulence factor of protozoan parasites [27]. To counter, structure-based drugs have been designed against proteases like cathepsin L-like (CpL) and B-like (CpB) cysteine proteases in Leishmania sp [28]. All parasitic protozoa contain numerous proteases including plasmepsins or the aspartic proteases from Plasmodium which provide required nutrients for the growth of the parasite. ...
Background:
Proteases are important enzymes that can degrade proteins and are found in animals, plants, bacteria, fungi and viruses. The action of proteases can be controlled by Protease Inhibitors (PIs), chemical or proteinaceous in nature that can block the active site of protease. Since the step catalyzed by proteases may play important role in life cycle of microbes, hindering the action of proteases by PIs may act as therapeutic intervention for microbial infection.
Material and Methods:
A thorough study was performed and wide range of literature was surveyed to confirm our results of PIs showing antibacterial activity.
Results:
PIs have shown to be effective drugs against bacterial pathogens, pathogenic viruses- Human Immunodeficiency Virus (HIV), Herpes virus, Hepatitis Virus. PIs have recently been investigated for controlling protozoan parasites. Clinical value of proteases and their inhibitors has been studied in Helicobacter pylori which is the etiologic agent of gastritis.
Conclusion:
This review is intended to highlight the role of PIs in the Battle against Microbial Pathogens.
... ICP is present in all Leishmania species with sequenced genomes but it was studied only in L. mexicana [39]. The majority of ICP resides in cytoplasmic vesicles of promastigotes that are unrelated to the endosome or to the multi-vesicular tubule (MVT), and likely does not influence the traffic of endogenous proteases to the lysosome. ...
... Since re-expressing lines behaved similarly to Dicp in mice infections, it was difficult to evaluate the nature of the phenotypic changes related to lack of ICP. On the other hand, reduced infectivity was more drastic in ICP over-expressing parasites [39]. The correlation between increased ICP expression and loss of virulence in BALB/c mice was confirmed later on, and found to result from a switch of the host immunity to a protective Th1 response, with high levels of interferon g [40]. ...
Chagasin-type inhibitors comprise natural inhibitors of papain-like cysteine proteases that are distributed among Protist, Bacteria and Archea. Chagasin was identified in the pathogenic protozoa Trypanosoma cruzi as an approximately 11 kDa protein that is a tight-binding and highly thermostable inhibitor of papain, cysteine cathepsins and endogenous parasite cysteine proteases. It displays an Imunoglobulin-like fold with three exposed loops to one side of the molecule, where amino acid residues present in conserved motifs at the tips of each loop contact target proteases. Differently from cystatins, the loop 2 of chagasin enters the active-site cleft, making direct contact with the catalytic residues, while loops 4 and 6 embrace the enzyme from the sides. Orthologues of chagasin are named Inhibitors of Cysteine Peptidases (ICP), and share conserved overall tri-dimensional structure and mode of binding to proteases. ICPs are tentatively distributed in three families: in family I42 are grouped chagasin-type inhibitors that share conserved residues at the exposed loops; family I71 contains Plasmodium ICPs, which are large proteins having a chagasin-like domain at the C-terminus, with lower similarity to chagasin in the conserved motif at loop 2; family I81 contains Toxoplasma ICP. Recombinant ICPs tested so far can inactivate protozoa cathepsin-like proteases and their mammalian counterparts. Studies on their biological roles were carried out in a few species, mainly using transgenic protozoa, and the conclusions vary. However, in all cases, alterations in the levels of expression of chagasin/ICPs led to substantial changes in one or more steps of parasite biology, with higher incidence in influencing their interaction with the hosts. We will cover most of the findings on chagasin/ICP structural and functional properties and overview the current knowledge on their roles in protozoa.
... Interestingly, some prokaryotes, such as Pseudomonas aeruginosa, apparently lack endogenous CPs despite the presence of an ICP (19), and in Leishmania, CPs and ICPs do not colocalize (30). These findings indicate that ICPs may also play additional roles, for instance, in host-pathogen interaction. ...
... These findings indicate that ICPs may also play additional roles, for instance, in host-pathogen interaction. Null mutants of Leishmania mexicana show normal infectivity of macrophages in vitro but reduced infectivity to mice (30), and overexpression of chagasin in T. cruzi led to reduced infectivity in vitro and impaired the capacity to differentiate into trypomastigotes (31). ...
Unlabelled:
Malaria is transmitted when motile sporozoites are injected into the dermis by an infected female Anopheles mosquito. Inside the mosquito vector, sporozoites egress from midgut-associated oocysts and eventually penetrate the acinar cells of salivary glands. Parasite-encoded factors with exclusive vital roles in the insect vector can be studied by classical reverse genetics. Here, we characterized the in vivo roles of Plasmodium berghei falstatin/ICP (inhibitor of cysteine proteases). This protein was previously suggested to act as a protease inhibitor during erythrocyte invasion. We show by targeted gene disruption that loss of ICP function does not affect growth inside the mammalian host but causes a complete defect in sporozoite transmission. Sporogony occurred normally in icp(-) parasites, but hemocoel sporozoites showed a defect in continuous gliding motility and infectivity for salivary glands, which are prerequisites for sporozoite transmission to the mammalian host. Absence of ICP correlates with enhanced cleavage of circumsporozoite protein, in agreement with a role as a protease regulator. We conclude that ICP is essential for only the final stages of sporozoite maturation inside the mosquito vector. This study is the first genetic evidence that an ICP is necessary for the productive motility of a eukaryotic parasitic cell.
Importance:
Cysteine proteases and their inhibitors are considered ideal drug targets for the treatment of a wide range of diseases, including cancer and parasitic infections. In protozoan parasites, including Leishmania, Trypanosoma, and Plasmodium, cysteine proteases play important roles in life cycle progression. A mouse malaria model provides an unprecedented opportunity to study the roles of a parasite-encoded inhibitor of cysteine proteases (ICP) over the entire parasite life cycle. By precise gene deletion, we found no evidence that ICP influences disease progression or parasite virulence. Instead, we discovered that this factor is necessary for parasite movement and malaria transmission from mosquitoes to mammals. This finding in a fast-moving unicellular protozoan has important implications for malaria intervention strategies and the roles of ICPs in the regulation of eukaryotic cell migration.
... The first to be identified in L. major was an inhibitor of cysteine peptidases (ICP), which is a member of the chagasin family of inhibitors first identified in Trypanosoma cruzi (Monteiro et al., 2001) and subsequently found in a variety of bacterial and protozoan pathogens (Rigden et al., 2002;Sanderson et al., 2003). In T. cruzi and Trypanosoma brucei chagasin/ICP is a modulator of parasite differentiation (Santos et al., 2005;2007), while L. major ICP is thought to play a role in the hostparasite interaction (Besteiro et al., 2004). L. major ICP and T. cruzi chagasin have an unusual immunoglobulinlike fold with a cystatin-like mechanism of inhibition, which distinguishes them from all other known peptidase inhibitors (Salmon et al., 2006;Smith et al., 2006). ...
... The PCR fragments were subcloned into pGEM-T and then released by restriction digest with HindIII/SalI for the 5′ FR, and XmaI/BglII for the 3′ FR. The fragments were sequentially cloned into similarly digested hygromycin-resistant plasmid pGL792 (Besteiro et al., 2004) to give pGL959. To produce a phleomycin-resistant knockout construct the hygromycin cassette from pGL959 was replaced with the SpeI/BamHI phleomycin-resistance cassette to give pGL961. ...
Ecotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major. Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vector-borne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with K(i)s between 7.7 and 83 nM. L. major ISP2-ISP3 double null mutants (Deltaisp2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Deltaisp2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Deltaisp2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Deltaisp2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.
... Cysteine peptidases (CPs) play important roles in L. mexicana virulence, where the knockout of its natural inhibitor (CPI) increased infectivity and the generation of marked, non-healing lesions in mice [139]. However, overexpression of ICP generated attenuated L. mexicana species, with increased Th1 response in mice [135]. ...
Despite decades of research devoted to finding a vaccine against leishmaniasis, we are still lacking a safe and effective vaccine for humans. Given this scenario, the search for a new prophylaxis alternative for controlling leishmaniasis should be a global priority. Inspired by leishmanization—a first generation vaccine strategy where live L. major parasites are inoculated in the skin to protect against reinfection—live-attenuated Leishmania vaccine candidates are promising alternatives due to their robust elicited protective immune response. In addition, they do not cause disease and could provide long-term protection upon challenge with a virulent strain. The discovery of a precise and easy way to perform CRISPR/Cas-based gene editing allowed the selection of safer null mutant live-attenuated Leishmania parasites obtained by gene disruption. Here, we revisited molecular targets associated with the selection of live-attenuated vaccinal strains, discussing their function, their limiting factors and the ideal candidate for the next generation of genetically engineered live-attenuated Leishmania vaccines to control leishmaniasis.
... Gene expression of CPA is maximal level in amastigote stage, while lower expression is in promastigote [73]. Also, CPB is expressed at high levels in the amastigote, is expressed at a very few level in procyclic-promastigotes [75]. CPB1 and CPB2 are expressed in higher levels of metacyclic promastigote, while CPC in procyclicpromastigote [70]. ...
Leishmaniasis is still considered to be a global health problem, which spreads in most countries in the world. Leishmania is an intracellular obligate protistan parasite that causes different clinical symptoms in infected humans and other animals. There are clinically different types of the disease including: visceral, cutaneous or muco-cutaneous leishmaniasis. Approximately, two million new infections occurring annually; 0.7 to 1.2 million cases are recorded with cutaneous leishmaniasis and 200,000-400,000 cases return for visceral leishmaniasis. However, Cutaneous leishmaniasis considers one of uncontrolled wobbling endemic diseases, especially in Iraq, which occurs at the skin to cause a dermal lesion. Usually, the lesion is spontaneously healed to leave a colorless depressed scar and permanent immunity.
... British investigators observed that Leishmania promastigotes null mutants in the gene encoding chagasin were similar to WT parasites. Host-parasite interaction via modulating host CPs activity was suggested as a main role for Leishmania chagasin [111] . As previously described for chagasin structure, the investigators claimed that it was the only endogenous CPI with a cadherin-like immunoglobulin domain to be discovered in a non-metazoan. ...
... In protozoa, few CYSs were reported in T. cruzi (chagasin), L. mexicana, E. histolytica (EhICP), P. falciparum (falstatin), T. brucei (TbICP), T. gondii (toxostatins), C. parvum (cryptostatin), Acanthamoeba spp. (AcStefin) and T. vaginalis (trichocystatins 1-3) [38][39][40][41][42][43][44][45][46][47] . All are considered virulence factors due to their major role in CPs-mediated pathogenesis in their respective diseases (will be reviewed later). ...
... In addition, a study conducted in UK showed that CPI has a role other than modulation of the activity of the parasite's own CPs and their normal trafficking to the multi-vesicular tubule via the flagellar pocket. These results suggested that CPI has a role in protection of the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages (92) . On the other hand, CPIs (aziridine-2,3-dicarboxylates: 13b and 13e) impaired promastigote growth and decreased the infection rate of peritoneal macrophages. ...
... NMR relaxation measurements shows that the DE loop is highly flexible and structural-functional studies highlight that amino acids present in the DE and FG loops are essential for protease binding and inhibition. Lm-ICP colocalizes with endogenous proteases suggesting that Lm-ICP is involved in modulating the proteolytic activity of the host proteases [46] and its over-expression results in reduced virulence on a mice model [47]. ...
The number of protein folds in nature is limited, thus is not surprising that proteins with the same fold are able to exert different functions. The cysteine protease inhibitors that adopt an immunoglobulin-like fold (Ig-ICPs) are inhibitors encoded in bacteria and protozoan parasites. Structural studies indicate that these inhibitors resemble the structure of archetypical proteins with an Ig fold, like antibodies, cadherins or cell receptors. The structure of Ig-ICPs from four different protozoan parasites clearly shows the presence of three loops that form part of a protein-ligand interaction surface that resembles the antigen binding sites of antibodies. Thus, Ig-ICPs bind to different cysteine proteases using a tripartite mechanism in which their BC, DE and FG loops are responsible for the main interactions with the target cysteine protease. Ig-ICPs from different protozoan parasites regulate the enzymatic activity of host or parasite's proteases and thus regulate virulence and pathogenesis.
... However, the Kunitz family possesses Kunitz domains, which are relatively small active domains of proteins that bind strongly to the protease, blocking its active site and instantly forming an irreversible compound (http://smart.embl-heidelberg.de/). Similar to other parasites, Leishmania species also encode protease inhibitors in their genome, for example, Leishmania major cysteine protease inhibitor (ICP), which is predicted to participate in the hostparasite interaction [12]. The L. major genome has been shown to consist of three genes encoding ecotin-like ISPs [13,14]. ...
Aims:
This study aims to identify, purify, and characterize an endogenous serine protease inhibitor from an Indian strain of Leishmania donovani, which causes the fatal visceral leishmaniasis.
Main methods:
(i) Reverse zymography was used to identify the serine protease inhibitor by inhibiting the gelatinolytic activity of serine protease. (ii) Purification was performed by combining heat treatment, ultracentrifugation, and affinity and gel permeation chromatography. (iii) Spectrophotometric assays were conducted to quantify and compare the inhibitory activity of the Leishmania donovani serine protease inhibitor (LdISP). (iv) Further, the protein was identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS).
Key findings:
An endogenous inhibitor with an apparent molecular weight of 21.8 kDa, which is acidic in nature, having a pI of 5.9 was identified. The Ki value of the inhibitor for trypsin was determined to be in the nanomolar range. The protein has the following features: (i) ecotin-like nature, (ii) cross-organism functionality, that is, an inhibitory effect on the serine proteases of higher organisms other than its own, and (iii) homology with other such proteins from a different species of Leishmania on conducting protein mass fingerprinting after MALDI ToF MS.
Significance:
The inhibitor shows varying and entirely contrasting efficacies towards serine proteases of its own as well as of higher organisms. This indicates that it accelerates disease progression and drives parasite survival as it inhibits the activities of the host serine proteases.
... This is not surprising, given that 523 most T. vaginalis genes lack introns (Carlton et al., 2007). 524 It is remarkable that T. vaginalis has three cystatin-type 525 inhibitors, which is characteristic of higher organisms such as 526 nematodes (Newlands et al., 2001;Murray et al., 2005;Wasilewski 527 et al., 1996), instead of ICP-like inhibitors, as in other protozoan 528 parasites (Monteiro et al., 2001;Besteiro et al., 2004; ...
... In addition, the natural cysteine protease inhibitor, cystatin has been found to be very promising antileishmanial agent, as it induces protective response in experimental visceral leishmaniasis [101][102][103] . Moreover, leishmanial inhibitor of cysteine peptidases (ICP) is thought to protect the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages 104,105 . The natural biflavonoids compounds show anti-leishmanial activity and antiproteolytic activity against recombinant cysteine protease B 106 . ...
Leishmaniasis is a deadly protozoan parasitic disease affecting millions of people worldwide. The treatment strategy of Leishmania infection depends exclusively on chemotherapy till date. But the treatment of the disease is greatly hampered due to high cost, toxicity of the available drugs and more importantly emergence of drug resistance. Hence the potential new drugs are highly needed to combat this disease. The first and foremost step of the drug discovery process is to search and select the putative target in a specific biological pathway in the parasite that should be either unambiguously absent in the host or considerably different from the host homolog. Importantly, Leishmania genome sequences enrich our knowledge about Leishmania and simultaneously reinforce us to identify the ideal drug targets that distinctly exist in the parasite as well as to develop the effective drugs for leishmaniasis. Though the leishmanial research has significantly progressed during the past two decades, the identification of suitable drug targets or development of effective drugs to combat leishmaniasis is far from satisfactory. Enzymatic systems of Leishmania metabolic and biochemical pathways are essential for their survival and infection. Concurrently, it is noteworthy that Leishmania proteases, especially the cysteine proteases, metalloproteases and serine proteases have been extensively investigated and found to be indispensable for the survival of the parasites and disease pathogenesis. Herein, we have discussed the importance of few enzymes, particularly the Leishmania proteases and their inhibitors as promising candidates for potential development of anti-leishmanial drugs.
... The differentially expressed protein was identified by biological mass spectrometry at the Sir Henry Wellcome Functional Genomics Facility (University of Glasgow). Peptide tandem mass spectra were obtained by liquid chromatography-electrospray tandem mass spectrometry (LC-ES-MS/MS) as described previously (Drummelsmith et al., 2003;Besteiro et al., 2004) using LCQ DecaXP (Thermo Scientific) and ABI QStar (Invitrogen) quadropole ion trap instruments equipped with nanoelectrospray interfaces. The resulting peptide MS/MS spectra were utilised for interrogation of the NCBInr protein database using MASCOT 2.2.0.7 (www.matrixscience.com) ...
This study examined the potential biochemical and mechanical structures that may contribute to egg adhesion in European whitefish Coregonus lavaretus. Experiments showed that eggs from a population of C. lavaretus from Loch Eck remained non-adhesive in a solution chemically similar to ovarian fluid but became adhesive seconds after contact with water. Examination of the ultrastructure of the chorion showed that the morphology changed significantly after contact with water, with nodule-like protuberances attached to connective filaments on the surface present in water-hardened but not non-water hardened eggs. Biochemical analysis showed the presence of Chain A, RNase ZF-3e proteins in the chorion of water-hardened but not non-water hardened eggs. Histochemical staining of the chorion of C. lavaretus eggs showed that the externa, but not the interna, stained positively for the presence of glycoproteins. From these results, it was concluded that C. lavaretus from Loch Eck possess both anatomical and biochemical adhesive mechanisms that have been undocumented in this species so far.
... Other effector proteins with inhibitory activities against host hydrolytic enzymes, such as proteases, glucanases , and chitinases have been identified in Phytophthora infestans, P. sojae and Cladosporium fulvum [147]. Further examples of proteinase inhibitors that are believed to function as pathogenic determinants are found in animal parasites, such Trypanosoma cruzi [149], Leishmania mexicana [150], Entamoeba histolytica [151], leeches and blood-sucking insects. The inhibitors are mostly employed to inactivate the proteinases of the blood-clotting cascade, thereby preventing blood-clotting and permitting the invader to feed freely. ...
Enzyme inhibition is a fundamental process to preserve the orderly sequence of events required for life, from seed germination to apoptosis. This review will verse on recent findings showing the various ways in which enzyme inhibition has been incorporated into the arsenal of many organisms, either as an effective defensive weapon or as a factor needed for the establishment of infection, parasitism and/or symbiotic associations, and how this diverse functionality can be exploited for therapeutic uses. It will describe several non-proteinaceous enzyme inhibitors isolated from natural sources or synthesized on the basis of structural or functional similarity to biosynthetic enzymes that have been employed for effective treatment against infections and/or aggresive diseases, such as cancer and AIDS, given their ability to disrupt critical functions of the target organisms or malignant cells. A relation of novel plant proteinase inhibitors (PPIs) with multiple and/or novel functions or engineered to have a highly selectivity on their target enzymes in order to avoid deleterious effects in a multi-trophic level that negatively affect insect pollinators or predators, is included too. This development could eliminate or reduce one of the most pressing concerns about the use of PIs as transgenes in many important crops. Finally, an account of the highly specific inhibition of enzymatic activity by biotechnological (e.g. gene silencing) or biochemical means, which have proven to be powerful tools to reveal novel functions for a variety of enzymes will be made in the context of defense, development, senescence and programmed cell death in plants.
... The PCR fragments were subcloned into pGEM-T and then released by restriction digest with HindIII/SalI for the 5′ FR and XmaI/BglII for the 3′ FR. The fragments were sequentially cloned into a similarly digested hygromycin-resistant plasmid pGL792 (Besteiro et al., 2004 ). To produce blasticidin-and nourseothricinresistant plasmids, the hygromycin cassette was replaced with the SpeI/BamHI resistance cassettes from pGL 434 and pGL227 to give plasmids pGL1027 and pGL1028. ...
Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.
... In light of these data it was chosen to examine the expression profile of the L. mexicana SPT. Utilising the cross reactive anti-LmjLCB2 antibody [14,39] the expression of LmxLCB2 was probed using Western blotting ( Figure 5). The levels of the constitutively expressed N-myristoyltransferase (NMT) were used as a loading control [24]. ...
Leishmania species are the causative agents of the leishmaniases, a spectrum of neglected tropical diseases. Amastigote stage parasites exist within macrophages and scavenge host factors for survival, for example, Leishmania species utilise host sphingolipid for synthesis of complex sphingolipid. In this study L. mexicana endocytosis was shown to be significantly upregulated in amastigotes, indicating that sphingolipid scavenging may be enhanced. However, inhibition of host sphingolipid biosynthesis had no significant effect on amastigote proliferation within a macrophage cell line. In addition, infection itself did not directly influence host biosynthesis. Notably, in contrast to L. major, L. mexicana amastigotes are indicated to possess a complete biosynthetic pathway suggesting that scavenged sphingolipids may be nonessential for proliferation. This suggested that Old and New World species differ in their interactions with the macrophage host. This will need to be considered when targeting the Leishmania sphingolipid biosynthetic pathway with novel therapeutics.
... For example, re-introduction of the gene encoding the CPA cysteine peptidase in an L. infantum Δcpa mutant increased the in vitro infectivity of the cells but it did not improve the virulence of the mutant [5]. Similarly, the reduced infectivity to mice of a null mutant for the ICP gene, encoding another cysteine peptidase, was not restored by re-expressing the gene from either an episomally or a chromosomally integrated copy [2]. Given the temperature-dependent expression of L. infantum HSP70-II [8] , the reduced virulence of the Δhsp70- II mutant may be related, in part, to an impaired capacity for growth at the temperatures of the mammalian host. ...
The 70-kDa heat shock protein (HSP70) is highly conserved among both prokaryotes and eukaryotes and plays essential roles in diverse cellular functions not only under stress but also under normal conditions. In the protozoan Leishmania infantum, the causative agent of visceral leishmaniasis, HSP70 is encoded by two HSP70 genes. Here, we describe the phenotypic alterations of HSP70-II-deficient (Deltahsp70-II) promastigotes. The absence of HSP70-II caused a major alteration in growth as the promastigotes reached stationary phase. In addition, aberrant forms were frequently observed in Deltahsp70-II mutant cultures. An accumulation of cells in the G2/M phase in cultures of the Deltahsp70-II mutant was determined by flow cytometry. Furthermore, Deltahsp70-II promastigotes showed a limited capacity of multiplication within macrophages, even though attachment to and uptake by macrophages did not differ significantly from the wild-type. Moreover, Deltahsp70-II was highly attenuated in BALB/c mouse experimental infections. In mutants re-expressing HSP70-II, the growth rate was restored, the normal morphology was recovered, and interactions with macrophages increased. However, promastigotes re-expressing HSP70-II did not recover their virulence. Overall, these data highlight the essential role played by HSP70-II expression in Leishmania virulence, pointing to this gene as a promising target for therapeutic interventions.
... The expected rescue of wild-type phenotype by complementation of the null parasite was observed for some, but not all, of the parameters tested. Partial or failed rescuing of wild-type phenotypes by genetic complementation in Leishmania has been observed previously, partly as a consequence of poor regulation of transgene expression in complemented cells (Besteiro et al., 2004;Denise et al., 2006;Folgueira et al., 2008;McKean et al., 2001). The expression of META2 protein in meta2 À /+META2, as judged by immunofluorescence, had the same distribution pattern as the stationary-phase parental line (data not shown), but protein steady-state levels were increased approximately 6-fold. ...
The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L. amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock.
... Applied strategies of structure-based drug design against a family of cathepsin L-like (CpL) and cathepsin B-like (CpB) cysteine proteases in Leishmania sp. [3,4] should be addressed for the chemotherapy of Leishmaniasis [5]. ...
With the increasing evidence of protease involvement in several diseases, novel strategies for drug development involve the use of protease inhibitors (PIs). The local balance between protease inhibitors and proteases is an important determinant of the occurrence and progression of a particular disease. Hence, enzymes and their cognate inhibitors are finding their applications as diagnostic and prognostic markers. PIs are widely implicated for their use in host defense against infection, tissue repair and matrix production, blood coagulation, cancer, and they are, therefore, the current focus as therapeutic alternatives for major diseases such as AIDS and Alzheimer's diseases. This review is a brief summary of the varied role of protein protease inhibitors in controlling the activity of aberrant enzymes in several diseases afflicting mankind today.
... In mammals, cystatins function as a protection against lysosomal peptidases released occasionally at normal cell death or phagocyte degranulation, or intentionally by proliferating cancer cells or by invading organisms [2]. In addition, endogenous cysteine protease inhibitors have been described in parasites such as Trypanosoma cruzi [16], Leishmania mexicana [6], Entamoeba histolytica [19] and malaria parasites, Plasmodium falciparum [18]. These studies showed that Hb degradation occured in the food vacuole. ...
Although the actions of cysteine proteases are controlled in part by endogenous tight-binding cysteine protease inhibitors from the cystatin superfamily, regulatory mechanisms used by ticks to control protease activities are unknown. We report here the interaction of 2 endogenous midgut cysteine protease inhibitors, Hlcyst-1 and Hlcyst-2, with an endogenous midgut cysteine protease, HlCPL-A in Haemaphysalis longicornis. In vitro inhibition assays demonstrated that the hydrolytic activity of HlCPL-A was inhibited by Hlcyst-1 and Hlcyst-2 in dose dependent manner. Immunofluorescent studies revealed that Hlcyst-1 and Hlcyst-2 are co-localized with HlCPL-A in the epithelial cells of the midgut. The hemoglobin degradation activity of HlCPL-A was dose-dependently inhibited by Hlcyst-1 and Hlcyst-2. These results strongly indicate that, Hlcyst-1 and Hlcyst-2 are possible inhibitor of HlCPL-A and play a key role in regulatory mechanisms of hemoglobin degradation process in ticks.
... Details of primers used are given in Section 2.5.3. The 5' flank was ligated into the PCR-Script cloning vector and the 3' flank was ligated into the TOPO cloning vector, then both were inserted into the knock out construct that had previously been used to generate the CPB array and ICP null mutants (Mottram et al., 1996b;Besteiro et al., 2004), to give pGL1693. The HYG gene in pGL1693 was then replaced with the streptothricin acetyltransferase (SAT) gene to give pGL1792. ...
Peptidases of Leishmania are acknowledged virulence factors. It is hypothesised that peptidases are crucial for the survival of Leishmania in its hosts and that many could be potential targets for new antileishmanial drugs. As such, the investigation of peptidase activity in live Leishmania promastigotes was proposed as a valuable approach by which to increase knowledge on particular peptidases. In order to complete this investigation, it was decided to use short peptidyl fluorogenic substrates, which only fluoresce once the bond linking the peptide to the fluorescent moiety is cleaved. These allow detection of peptidase activity by quantifying the release of the fluorescent moiety. Detection of peptidase activity in live Leishmania using the fluorogenic substrate Bz-R-AMC proved fruitful, enabling study of the activity of the serine peptidase oligopeptidase B (OPB) in live L. major promastigotes. OPB is a member of the Family S9 peptidases, the prolyl-oligopeptidases, which are taxonomically restricted to plants, bacteria and trypanosomatid flagellates. In African and American trypanosomes, OPB has been shown to have important roles: OPB is a virulence factor in Trypanosoma cruzi, mediating entry into host cells, and OPB is released into the serum by African trypanosomes, where it cleaves host blood factors. In this study, the inhibition profile of L. major OPB has been determined and OPB has been localised to the cytosol, the site of hydrolysis of Bz-R-AMC. Immunoprecipitation of OPB confirmed that OPB was the sole peptidase responsible for the hydrolysis of Bz-R-AMC and anti-OPB antibodies were found to inhibit the hydrolysis of Bz-R-AMC. Inhibitors of OPB could also kill Leishmania promastigotes, suggesting OPB could be a valuable drug target. However, genetic manipulation of OPB was successful, with mutants over-expressing OPB and opb null mutants produced. OPB is thus not essential for the growth of promastigote L. major, though the opb null mutants did have a defect in metacyclogenesis, in survival in macrophages and a reduced ability to induce lesions on the footpads of mice. A role in amastigote differentiation or survival in macrophages was also suggested. OPB is thus likely to be a virulence factor, though not essential, and thus not suitable as a primary drug target. A number of avenues require further investigation, including the need for re-expression of OPB in the opb null mutants to confirm that lack of OPB is indeed responsible for the phenotypic deficiencies of the null mutants. Other important areas requiring attention are investigation of the role of OPB in amastigote differentiation or survival, investigation of the reported release of OPB by promastigotes, and identification of the physiological substrate of OPB.
... In the re-expresser line, the ICP was re-expressed in icp3 from an integrative vector, providing a level of expression similar to that in the wild type. In the icp [pXG ICP] line, the ICP was expressed from an extrachromosomal vector, and this resulted in a Þve-fold higher expression in procyclic promastigotes than in the wild-type parasites through selection with neomycin (G418; Gibco, Karlsruhe, Germany) at 50 g/ml (for more details see Besteiro et al. 2004). Parasites were maintained in medium 199 (Sigma, St. Louis, MO) supplemented with 20% (vol:vol) fetal calf serum (Gibco) and gentamycin (50 g/ml). ...
It has been proposed that the natural cysteine peptidase inhibitor ICP of Leishmania mexicana protects the protozoan parasite from insect host proteolytic enzymes, thereby promoting survival. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. No significant differences were found between the wild-type parasites, two independently derived ICP-deficient mutants, or mutants overexpressing ICP; all lines developed similarly in the sand fly midgut and produced heavy late-stage infections. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of the midgut extracts in vitro. We conclude that ICP has no major role in promoting survival of L. mexicana in the vectorial part of its life cycle in L. longipalpis.
... If so, it could serve to protect the cytosol against cysteine protease molecules released by damaged intracellular vesicles. By contrast , leishmanial ICP, which does contain a signal sequence and an extended prodomain, has a vesicular distribution and is suggested to play a role in the interaction between host and parasite [24]. Whatever its function, EhICP1 seems to be present at substoichiometric amounts compared to the endogenous cysteine proteases [6]. ...
Based on the Entamoeba histolytica genome project (www.sanger.ac.uk/Project/E_histolytical/) we have identified a cysteine protease inhibitor, EhICP1 (amoebiasin 1), with significant homology to chagasin. Recombinant EhICP1 inhibited the protease activity of papain and that of a trophozoite lysate with Ki's in the picomolar range. By immunocytology, we localized the endogenous approximately 13 kDa EhICP1 in a finely dotted subcellular distribution discrete from the vesicles containing the amoebic cysteine protease, EhCP1 (amoebapain). In an overlay assay, we observed binding of recombinant EhICP1 to EhCP1. As a heptapeptide (GNPTTGF) corresponding to the second conserved chagasin motif inhibited the protease activity of both papain (K) 1.5 microM) and trophozoite extract (Ki in sub-mM range), it may be a candidate for the rational development of anti-amoebiasis drugs.
... In addition to Kazal-like motifs, we also discovered a cDNA that encodes a secreted protein with similarity to the cystatin class of cysteine protease inhibitors [79]. Cysteine protease inhibitors, such as chagasin, have been reported in animal parasites, mainly trypanosomids, and are thought to target proteases of the insect vector or the mammalian host808182 . Perhaps, inhibition of host proteases is a widespread counterdefense strategy in animal and plant pathogenic eukaryotes. ...
The oomycete Saprolegnia parasitica is one of the most economically important fish pathogens. There is a dramatic recrudescence of Saprolegnia infections in aquaculture since the use of the toxic organic dye malachite green was banned in 2002. Little is known about the molecular mechanisms underlying pathogenicity in S. parasitica and other animal pathogenic oomycetes. In this study we used a genomics approach to gain a first insight into the transcriptome of S. parasitica.
We generated 1510 expressed sequence tags (ESTs) from a mycelial cDNA library of S. parasitica. A total of 1279 consensus sequences corresponding to 525944 base pairs were assembled. About half of the unigenes showed similarities to known protein sequences or motifs. The S. parasitica sequences tended to be relatively divergent from Phytophthora sequences. Based on the sequence alignments of 18 conserved proteins, the average amino acid identity between S. parasitica and three Phytophthora species was 77% compared to 93% within Phytophthora. Several S. parasitica cDNAs, such as those with similarity to fungal type I cellulose binding domain proteins, PAN/Apple module proteins, glycosyl hydrolases, proteases, as well as serine and cysteine protease inhibitors, were predicted to encode secreted proteins that could function in virulence. Some of these cDNAs were more similar to fungal proteins than to other eukaryotic proteins confirming that oomycetes and fungi share some virulence components despite their evolutionary distance
We provide a first glimpse into the gene content of S. parasitica, a reemerging oomycete fish pathogen. These resources will greatly accelerate research on this important pathogen. The data is available online through the Oomycete Genomics Database.
... 18 Along similar lines, recent data suggest that chagasin homologues in Leishmania mexicana modulate the outcome of host-parasite interactions. 19 Threading and comparative modelling provided predictions of the structure of chagasin-like proteins. 20 Based on these theoretical studies, it was hypothesized that chagasin-like ICPs adopt an immunoglobulin-like (Ig-like) fold. ...
A Trypanosoma cruzi cysteine protease inhibitor, termed chagasin, is the first characterized member of a new family of tight-binding cysteine protease inhibitors identified in several lower eukaryotes and prokaryotes but not present in mammals. In the protozoan parasite T.cruzi, chagasin plays a role in parasite differentiation and in mammalian host cell invasion, due to its ability to modulate the endogenous activity of cruzipain, a lysosomal-like cysteine protease. In the present work, we determined the solution structure of chagasin and studied its backbone dynamics by NMR techniques. Structured as a single immunoglobulin-like domain in solution, chagasin exerts its inhibitory activity on cruzipain through conserved residues placed in three loops in the same side of the structure. One of these three loops, L4, predicted to be of variable length among chagasin homologues, is flexible in solution as determined by measurements of (15)N relaxation. The biological implications of structural homology between chagasin and other members of the immunoglobulin super-family are discussed.
The regulation of the activity of proteases by endogenous inhibitors is a common trend in almost all forms of life. Here, we review the endogenous inhibitors of cysteine proteases of three major pathogenic parasitic protozoa. The review focuses on members of the genus Plasmodium, Entamoeba, and Leishmania. Research in this domain has revealed the presence of only chagasin-like inhibitors of cysteine proteases that house a β-barrel immunoglobulin-fold and inhibit the target proteases using a 3-loop inhibitory mechanism in these pathogens. Inhibitors of cysteine proteases are highly evolvable enzymes that target a broad spectrum of pathogenic cysteine proteases with a proclivity for those involved in host-parasite interactions. A common trend reflects a limited sequence homology between cysteine proteases and their inhibitors. The inhibitors are also known to participate in other housekeeping functions of the parasites. Generalizations about their roles are thus best avoided. In this review, the reader will find comprehensive information on the cellular localization of inhibitors of cysteine proteases, their structure, function, and the associated mechanisms of action. The reader will also find a thorough analysis of the role of these inhibitors in parasite pathology and the common trends interlinking them with parasite biology and evolution.
Introduction:
In Thailand, Leishmania martiniquensis is the predominant species causing cutaneous and visceral leishmaniasis. Its incidence has been increasing among immunocompetent and immunocompromised hosts. We developed a prototype DNA vaccine using a partial consensus sequence of the cysteine protease B (cpb) gene derived from L. martiniquensis from Thai patients.
Methodology:
The laboratory inbred strain of albino BALB/c mice were immunized intramuscularly three times at 2-week intervals (weeks 0, 2, and 4) with cpb plasmid DNA (pcDNA_cpb) with or without the adjuvant, monoolein (pcDNA_cpb-MO). Mice were challenged at week 6 with L. martiniquensis promastigotes. Sera were analysed for IgG1, IgG2a, interferon gamma and interleukin 10 (IFN-γ and IL-10, respectively) levels at weeks 0, 4, and 9. Additionally, livers and spleens were also analysed for parasite burden using immunohistochemistry and real-time polymerase chain (qPCR) assays.
Results:
Three weeks after promastigote challenge, vaccinated mice showed significantly increased levels of IgG2a and IFN-γ while IL-10 level was significantly reduced when compared with those in the control group (p < 0.01). Parasite burden in the livers and spleens of vaccinated mice significantly decreased. In addition, a significant increase in mature granuloma formation in the livers when compared with those of the control group (p < 0.05) was found, indicating increased T-helper cells (Th1)-induced inflammation and destruction of amastigotes. Monoolein produced a booster effect to enhance the mouse Th1 protective immunity.
Conclusions:
The prototype DNA vaccine could induce a Th1 immune response that conferred potential protection to the L. martiniquensis promastigote challenge in BALB/c mice.
Prokaryotes were the earliest life forms on Earth but, although they originated an estimated 2 billion years before eukaryotic cells, both prokaryotes and eukaryotes share the same basic molecular mechanisms, indicating that both stem from a primordial ancestor. Bacteria, including cyanobacteria, produce a vast variety of secondary metabolites capable of controlling multiple eukaryotic cell functions. Some metabolites direct morphogenetic processes (in both cyanobacteria and higher algal taxons), while others are toxic to eukaryotes, helping prokaryotes colonize a wider variety of ecological niches. In addition, bacteria often use secondary metabolites to control other bacterial groups. On the other hand, eukaryotic cells can also synthesize secondary metabolites with either bacteriostatic or bactericidal capabilities, to counteract either cyanobacterial or eubacterial organisms. Cyanobacteria are an ancient lineage of photosynthetic microorganisms, but their study was neglected for many years. Recent publications demonstrate that cyanobacterial genomes encode a large variety of natural products, with broad mechanisms of action, many of which are probably yet unknown. Current advances in genome sequencing, making it faster and cheaper, should see a great increase in the number of completed cyanobacterial genomes, which promises many interesting discoveries in the near future.
Oxidative stress is one of the physiological conditions where the reactive oxygen species (ROS) production overcomes the capacity of antioxidant defences and causes damage to key biomolecules. The major global parasites bare some key factors involved in the pathogenesis of parasitic diseases regarding oxidative stress. In this context, parasitic proteases play central roles in host-parasite interactions. Both parasitic proteases and host proteases are deeply implicated in the clinical manifestation of parasite survival and in the host immune modulation. Additionally, the involvement of endogenous parasitic protease inhibitors in the understanding of progression of infection under oxidative stress of host cannot be ignored as well. Therefore, the present review provides some insights into some important parasitic proteases with the contribution of the versatile roles of parasitic endogenous protease inhibitors in regulation of host proteases in oxidative stress in the context of host-parasite interaction.
In the oral cavity, reactive oxygen and nitrogen species are continuously generated by bacterial metabolism and host-mediated cellular factors. These reactive species facilitate a critical function that regulates the outcomes of both oral and systemic diseases, such as dental caries, periodontitis, and the maintenance of blood pressure. As a result, the protective effects of ROS and RNS are being explored for future therapeutic applications.
Malaria is a devastating infectious disease affecting mostly tropical and sub-tropical regions. Owning to the emergence of resistance to the existing chemotherapy, the development of anti-malarial drugs as novel chemotherapeutics remains unavoidable. Malaria parasite, Plasmodium, experiences oxidative stress throughout its life cycle upon infection, and underlying redox metabolism is quite complex. Alterations in the redox homeostasis occur during host-pathogen interactions. Parasite is highly vulnerable to such alterations in redox homeostasis. To circumvent this, the parasites engage in an efficient redox system having protective roles towards the turbulence faced by the parasite. Targeting the redox system of malaria parasite is tempting in developing novel antimalarial drugs. On the other hand, oxidative stress, generated during anti-malarial drug metabolism, acts as a source of inhibition against progression of this outrageous parasite. This review aims to provide updated knowledge on redox networks of parasite and structural insights of redox system enzymes, underpinning the balance between antioxidant and pro-oxidant candidates throughout the host-parasite interactions. Furthermore, it also highlights the importance of reactive oxygen species generation during anti-malarial drug metabolism. This review summarises on the vulnerabilities of the malaria parasite due to oxidative stress and the potential cues towards development of the novel antimalarial drugs.
Cystatins are important regulators of papain‐like cysteine proteases. In the protozoan parasite Giardia intestinalis, papain‐like cysteine proteases play an essential role in the parasite's biology and pathogenicity. Here, we characterized a cysteine protease inhibitor of G. intestinalis that belongs to type‐I‐cystatins. The parasite cystatin is shown to be a strong inhibitor of papain (Ki ≈ 0.3 nM) and three parasite cysteine proteases (CP14019, CP16160 and CP16779, Ki ≈ 0.9 – 5.8 nM), but a weaker inhibitor of human cathepsin B (Ki ≈ 79.9 nM). The protein localizes mainly in the cytoplasm. Together, these data suggest that cystatin of G. intestinalis plays a role in the regulation of cysteine protease activities in the parasite and, possibly, in the interaction with the host. This article is protected by copyright. All rights reserved.
Cysteine proteases (CPs) are the essential virulent factor of Entamoeba histolytica. Although the physiological and pathological roles of CPs have been demonstrated, the molecular basis of intracellular trafficking of CPs has only begun to be unveiled. Recent work has revealed the mechanisms of intra- and extracellular transport of CPs and other soluble lysosomal proteins in E. histolytica. Such proteins involved in the mechanisms include Rab small GTPases, their effectors, the intrinsic inhibitor of CPs, and a unique family of receptors responsible for lysosomal transport. In this chapter, we give an overview of the current understanding of molecules and mechanisms involved in the transport of CPs and other soluble lysosomal proteins in E. histolytica.
The Trypanosomatidae family, assorted on the Kinetoplastida order, consists of distinct genera of eukaryotic monoflagellated protozoa. Among the trypanosomatids with a digenetic life cycle, some species stand out: Trypanosoma cruzi, Trypanosoma brucei and several Leishmania species. These parasites are the causative agents of Chagas’ disease, African sleeping sickness and leishmaniasis, respectively. Likewise, some species belonging to the Phytomonas genus can induce serious diseases in plants, which indicates the economical importance of these trypanosomatids, a problem especially affecting developing countries. Trypanosomatid cysteine proteases have been implicated in several processes including proliferation, differentiation, nutrition, host cell infection, and evasion of the host immune responses. For instance, Leishmania spp. possess three major cysteine proteases of the papain family (designated clan CA, family C1), namely, the cathepsin L-like CPA and CPB and the cathepsin B-like CPC, which are directly linked to the parasite survival inside macrophage cells and modulation of host immune response. In addition, cruzipain is a major cysteine protease, expressed in all developmental forms of T. cruzi, being highly immunogenic in patients with chronic Chagas’ disease. Since cysteine proteases are present in trypanosomatids and their catalytic properties can vary considerably from those of the host enzymes they are considered important targets for new chemotherapeutical intervention. Typical cysteine protease inhibitors like E-64, leupeptin and K777 are able to inhibit both the cell-associated and released cysteine proteases produced by trypanosomatid cells in different extensions. Moreover, the superfamily of cystatins (stefins, cystatins and kininogens), which are endogenous proteins and tight-binding reversible competitive inhibitors of clan CA, family C1 papain-like cysteine inhibitors, interfere with different physiological aspects of trypanosomatid cells. In this sense, the present chapter will summarize the knowledge about the inhibitory effects of cystatins directly on cysteine proteases produced by pathogenic trypanosomatids or indirectly by potentiating the host immune cells.
Trypanosomatids rely on peptidases as potent virulence factors and were recently found to contain a unique set of natural peptidase inhibitors not found in higher eukaryotes or in yeast, but present in a limited number of bacteria. Cha-gasin, identified in Trypanosoma cruzi, is a tight-binding, high affinity inhibitor of papain-like cysteine peptidases that has an Ig-like fold and inactivates target enzymes through a limited number of contacts mediated by a few conserved residues on three exposed loops. Chagasin homologues in other protozoa and bacteria are mostly single genes named ICPs, and to-gether with chagasin compose family I42 at MEROPS, the peptidase and peptidase inhibitors database [http://merops.sanger.ac.uk/]. The biological function of chagasin/ICPs seems to vary depending on the organism, but generally, the current studies point to a role in controlling the activity of endogenous parasite CPs, influencing parasite differentiation, virulence and different aspects of the host response to infection. More recently, natural inhibitors of serine peptidases that share similarity to bacterial ecotins were identified in trypanosomatids and named ISPs. Ecotins are spe-cific to trypsin-fold serine peptidases, enzymes which are not present in trypanosomatids. ISPs are limited to Trypano-somes and Leishmania and to date, only ISP2 proven to have an inhibitory function. In Leishmania, ISP2 seems to control the activity of host SPs at the initial stages of infection in order to ensure subsequent parasite survival in macrophages. The main aspects of chagasin/ICP biochemistry, structure and biological function and the recent findings on ISPs will be covered in this review.
Cysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with K
i's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.
The morphological events involved in the Leishmania major promastigote cell cycle have been investigated in order to provide a detailed description of the chronological processes
by which the parasite replicates its set of single-copy organelles and generates a daughter cell. Immunofluorescence labeling
of β-tubulin was used to follow the dynamics of the subcellular cytoskeleton and to monitor the division of the nucleus via
visualization of the mitotic spindle, while RAB11 was found to be a useful marker to track flagellar pocket division and to
follow mitochondrial DNA (kinetoplast) segregation. Classification and quantification of these morphological events were used
to determine the durations of phases of the cell cycle. Our results demonstrate that in L. major promastigotes, the extrusion of the daughter flagellum precedes the onset of mitosis, which in turn ends after kinetoplast
segregation, and that significant remodelling of cell shape accompanies mitosis and cytokinesis. These findings contribute
to a more complete foundation for future studies of cell cycle control in Leishmania.
Trophozoites of E. histolytica are equipped with two chagasin-like cysteine protease inhibitors, EhICP1 and EhICP2, also known as amoebiasin 1 and 2. Expression studies using E. invadens as model organism showed that corresponding mRNAs were detectable in both life stages of the parasite, cyst and trophozoite state. Unlike EhICP1 known to act in the cytosol, EhICP2 co-localized with cysteine protease EhCP-A1 in lysosome-like vesicles, as demonstrated by immunofluorescence microscopy. Silencing or overexpressing of the two inhibitors did not show any effect on morphology and viability of the trophozoites. Overexpression of the EhICPs, however, although dramatically dampening the proteolytic activity of cell extracts from the corresponding cell lines, did not influence expression rate or localization of the major amoebic cysteine proteases as well as phagocytosis and digestion of erythrocytes. Activity gels of cell extracts from strains overexpressing ehicp1 showed a drastically reduced activity of EhCP-A1 suggesting a high affinity of EhICP1 towards this protease. From these data, we propose that EhCP-A1 accidentally released into the cytosol is the main target of EhICP1, whereas EhICP2, beside its role in house-keeping processes, may control the proteolytic processing of other hydrolases or fulfils other tasks different from protease inhibition.
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-12-2006
Leishmania mexicana cysteine peptidases (CPs) have been identified as important parasite virulence factors. More recently, a natural inhibitor of CPs (ICP) from L. mexicana has been characterized, and ICP mutants have been created. Infection of BALB/c mice with ICP null mutants or ICP reexpressing mutants resulted in nonhealing, progressively growing lesions albeit slightly attenuated compared with the growth of lesions produced by wild-type parasites. In contrast, BALB/c mice infected with mutants overexpressing ICP were able to significantly control lesion growth or heal. While BALB/c mice infected with wild-type parasites, ICP null mutants, or ICP reexpressing mutants produced significant antibody responses, including immunoglobulin E (IgE), no Th1 response, as indicated by antigen-induced splenocyte gamma interferon (IFN-gamma) production, could be demonstrated. In contrast, BALB/c mice infected with mutants overexpressing ICP produced significantly less antibody, particularly IgE, as well as significantly reduced splenocyte interleukin-4 and enhanced IFN-gamma production. BALB/c mice were able to resolve infection following infection with one ICP overexpressing clone, which was subsequently used for vaccination studies with BALB/c mice. However, no protection was afforded these mice when they were challenged with wild-type parasites. Nevertheless, two other mouse strains susceptible to L. mexicana, C3H and C57BL/6, vaccinated with overexpressing ICP mutants were able to control challenge infection associated with an enhanced Th1 response. This study confirms that L. mexicana CPs are virulence factors and that ICPs have therapeutic potential.
Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.
A calcium-activated protease caldonopain in the cytosolic fraction of Leishmania donovani has been found to digest different endogenous proteins when subjected to SDS-PAGE. Gelatin-embedded gel electrophoresis confirms presence of calcium-dependent protease activity. Ca(2+) affects proteolytic activity after 10 h. When host-parasite interaction was conducted in vitro, caldonopain was found to be active after 10 h of incubation with calcium. A 67-kDa protein is specifically digested during this time and two new proteins of 45 and 36 kDa appeared in SDS-PAGE electrophoregram. This belated action of calcium towards protease activity may be pre-requisite to facilitate invasion of host tissues and thereby mediate protein metabolism during survival of this pathogen both independently and intracellularly. It is likely that calcium metabolism in promastigotes and amastigotes does not propagate in the same manner. Involvement of calcium to initiate caldonopain activity may be critically associated with signal transduction pathways which may be responsible for the pathobiological action of this parasite. We propose that caldonopain could be a potential target to develop new chemotherapeutic approach against leishmaniasis.
Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected
diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens but are absent
from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes
of CP inhibitor. Recent data suggest that Leishmania mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicana by NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria.
The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile.
Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the
mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for
CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides
structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism.
A cdc2-related protein kinase gene, crk3, has been isolated from the parasitic protozoan Leishmania mexicana. Data presented here suggests that crk3 is a good candidate to be the leishmanial cdc2 homologue but that the parasite protein has some characteristics which distinguish it from mammalian cdc2. crk3 is predicted to encode a 35.6-kDa protein with 54% sequence identity with the human cyclin-dependent kinase cdc2 and 78% identity with the Trypanosoma brucei CRK3. The trypanosomatid CRK3 proteins have an unusual, poorly conserved 19-amino acid N-terminal extension not present in human cdc2. crk3 is single copy, and there is 5-fold higher mRNA in the replicative promastigote life-cycle stage than in the non-dividing metacyclic form or mammalian amastigote form. A leishmanial suc-binding cdc2-related kinase (SBCRK) histone H1 kinase, has previously been described which binds the yeast protein, p13(suc1), and that has stage-regulated activity (Mottram J. C., Kinnaird, J., Shiels, B. R., Tait, A., and Barry, J. D. (1993) J. Biol. Chem. 268, 21044-21051). CRK3 from cell extracts of the three life-cycle stages was found to bind p13(suc1) and the leishmanial homologue p12(cks1). CRK3 fused with six histidines at the C terminus was expressed in L. mexicana and shown to have SBCRK histone H1 kinase activity. Depletion of histidine-tagged CRK3 from L. mexicana cell extracts, by Ni-nitrilotriacetic acid agarose selection, reduced histone H1 kinase activity binding to p13(suc1). These data imply that crk3 encodes the kinase subunit of SBCRK. SBCRK and histidine-tagged CRK3 activities were inhibited by the purine analogue olomoucine with an IC50 of 28 and 42 microM, respectively, 5-6-fold higher than human p34(cdc2)/cyclinB.
A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigote-like forms in Schneider's Drosophila medium supplemented with 20% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32-33 degrees C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5.4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.
The cpb genes ofLeishmania mexicana encode stage-regulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel
electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first
two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3–cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective
to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation
in the COOH-terminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection ofcpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to
be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the
activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into thecpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability
to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the
isoenzymes implicates five residues located in the mature domain (Asn18, Asp60, Asn61, Ser64, and Tyr84) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct
roles in the parasite’s interaction with its host. This complexity reflects the adaptation of cathepsin L-like cysteine proteinases
to diverse functions in parasitic protozoa.
Leishmania mexicana mutants lacking cysteine proteinase genes cpa (delta cpa), cpb (delta cpb), or both cpa and cpb (delta cpa/cpb) have been generated by targeted gene disruption. Delta cpa mutants produce a disease phenotype in BALB/c mice close to that of wild-type L. mexicana, but delta cpb mutants are much less infective, producing very slowly growing small lesions, and delta cpa/cpb double mutants do not induce lesion growth. Immunologic analysis of Ab isotype during infection and splenocyte IFN-gamma, IL-2, and IL-4 production following stimulation with Leishmania Ag or Con A indicates that there was a significant shift from a predominantly Th2-associated immune response in mice infected with wild-type L. mexicana to a Th1-associated response in mice inoculated with delta cpb or delta cpa/cpb. Significantly, delta cpa altered the balance of the immunologic response to a lesser extent than did the other mutants. Similar disease outcomes and switches in the Th1/Th2 balance were also observed when other L. mexicana-susceptible mouse strains were infected with the mutants. BALB/c and C57BL/6 mice vaccinated with delta cpa/cpb and CBA/Ca mice vaccinated with delta cpb or delta cpa/cpb were subsequently more resistant, to varying degrees, than were untreated mice to infection with wild-type parasites, as measured by development of lesions and parasite burden. These data implicate leishmanial cysteine proteinases not only as parasite virulence factors but also in modulation of the immune response and provide strong encouragement that cysteine proteinase-deficient L. mexicana mutants are candidate attenuated live vaccines.
We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast
Saccharomyces cerevisiae
growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243–251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.
Trypanosome protozoa, an early lineage of eukaryotic cells, have proteases homologous to mammalian lysosomal cathepsins, but the precursor proteins lack mannose 6-phosphate. Utilizing green fluorescent protein as a reporter, we demonstrate that the carbohydrate-free prodomain of a trypanosome cathepsin L is necessary and sufficient for directing green fluorescent protein to the lysosome/endosome compartment. A proper prodomain/catalytic domain processing site sequence is also required to free the mature protease for delivery to the lysosome/endosome compartment. A nine-amino acid prodomain loop motif, implicated in prodomain-receptor interactions in mammalian cells, is conserved in the protozoa. Site-directed mutagenesis now confirms the importance of this loop to protease trafficking and suggests that a protein motif targeting signal for lysosomal proteases arose early in eukaryotic cell evolution.
Leishmania species are obligate intracellular parasites of cells of the macrophage-dendritic cell lineage. Indeed, the ability to survive and multiply within macrophages is a feature of a surprising number of infectious agents of major importance to public health, including Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes, Salmonella typhimurium, Toxoplasma gondii and Trypanosoma cruzi. The relationship between such organisms and their host cells is particularly intriguing because, not only are macrophages capable of potent microbicidal activity, but in their antigen-presenting capacity they can orchestrate the developing immune response. Thus, to initiate a successful infection parasites must gain entry into macrophages, and also withstand or circumvent their killing and degradative functions. However, to sustain a chronic infection, parasites must also subvert macrophage-accessory-cell activities and ablate the development of protective immunity. The leishmanias produce a wide spectrum of disease in mice, and as such they have provided excellent models for studying problems associated with intracellular parasitism. In recent years, largely using these organisms, we have made enormous progress in elucidating the mechanisms by which successful intracellular infection occurs. Furthermore, characterization of immunological pathways that are responsible for resistance or susceptibility to Leishmania has given rise to the Th1/Th2 paradigm of cellular/humoral dominance of the immune response.
Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.
Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates. Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa. To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype. Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose. The mutants are unable to synthesize phosphoglycan repeats [-6-Gal beta 1-4Man alpha 1-PO(4)-] and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose. The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development. L. mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function.
The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4(+) T cell differentiation. A 6-fold increase in NO production was observed with 0.5 microM chicken cystatin, a natural cysteine protease inhibitor, in IFN-gamma-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-gamma. In contrast to the case with promastigotes, cystatin and IFN-gamma inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-gamma or anti-TNF-alpha Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-gamma treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4(+) differentiation from Th2 to Th1, indicative of long-term resistance.
Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.
The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.
Organelles in the endocytic pathway are composed of a mosaic of structural and functional regions. These regions consist, at least in part, of specialized protein-lipid domains within the plane of the membrane, or of protein complexes associated with specific membrane lipids. Whereas some of these molecular assemblies can be found in more than one compartment, a given combination seems to be unique to each compartment, indicating that membrane organization might be modular.
Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas' heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.
We demonstrate the presence of a glycosylphosphatidylinositol (GPI) anchor-specific endosomal pathway in the protozoan pathogen Trypanosoma brucei. In higher eukaryotes evidence indicates that GPI-anchored proteins are transported in both the endocytic and exocytic systems by mechanisms involving sequestration into specific membrane microdomains and consequently sorting into distinct compartments. This is potentially extremely important in trypanosomatids as the GPI anchor is the predominant mechanism for membrane attachment of surface macromolecules, including the variant surface glycoprotein (VSG). A highly complex developmentally regulated endocytic network, vital for nutrient uptake and evasion of the immune response, exists in T. brucei. In common with mammalian cells an early endosomal compartment is defined by Rab5 small GTPases, which control transport processes through the endosomal system. We investigate the function of two trypanosome Rab5 homologues. TbRAB5A and TbRAB5B, which colocalize in the procyclic stage, are distinct in the bloodstream form of the parasite. TbRAB5A endosomes contain VSG and transferrin, endocytosed by the T. brucei GPI-anchored transferrin receptor, whereas TbRAB5B endosomes contain the transmembrane protein ISG(100) but neither VSG nor transferrin. These findings indicate the presence of trypanosome endosomal pathways trafficking proteins through specific routes depending on the mode of membrane attachment. Ectopic expression of mutant TbRAB5A or -5B indicates that TbRAB5A plays a role in LDL endocytosis, whereas TbRAB5B does not, but both have a role in fluid phase endocytosis. Hence TbRAB5A and TbRAB5B have distinct functions in the endosomal system of T. brucei. A developmentally regulated GPI-specific endosomal pathway in the bloodstream form suggests that specialized transport of GPI-anchored proteins is required for survival in the mammalian host.
The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs.
Chicken egg white cystatin was first described in the late 1960s. Since then, our knowledge about a superfamily of similar proteins present in mammals, birds, fish, insects, plants and some protozoa has expanded, and their properties as potent peptidase inhibitors have been firmly established. Today, 12 functional chicken cystatin relatives are known in humans, but a few evolutionarily related gene products still remain to be characterized. The type 1 cystatins (A and B) are mainly intracellular, the type 2 cystatins (C, D, E/M, F, G, S, SN and SA) are extracellular, and the type 3 cystatins (L- and H-kininogens) are intravascular proteins. All true cystatins inhibit cysteine peptidases of the papain (C1) family, and some also inhibit legumain (C13) family enzymes. These peptidases play key roles in physiological processes, such as intracellular protein degradation (cathepsins B, H and L), are pivotal in the remodelling of bone (cathepsin K), and may be important in the control of antigen presentation (cathepsin S, mammalian legumain). Moreover, the activities of such peptidases are increased in pathophysiological conditions, such as cancer metastasis and inflammation. Additionally, such peptidases are essential for several pathogenic parasites and bacteria. Thus cystatins not only have capacity to regulate normal body processes and perhaps cause disease when down-regulated, but may also participate in the defence against microbial infections. In this chapter, we have aimed to summarize our present knowledge about the human cystatins.
To understand the trafficking of endocytosed hemoglobin (Hb) in Leishmania, we investigated the characteristics of in vitro fusion between endosomes containing biotinylated Hb (BHb) and avidin-horseradish peroxidase (AHRP). We showed that early endosome fusion in Leishmania is temperature and cytosol dependent and is inhibited by ATP depletion, ATPgammaS, GTPgammaS and N-ethylmaleimide treatment. The Rab5 homolog from Leishmania donovani, LdRab5, was cloned and expressed. Our results showed that homotypic fusion between the early endosomes in Leishmania is Rab5 dependent. Early endosomes containing BHb fused efficiently with late endosomes in a process regulated by Rab7, whereas no fusion between early and late endosomes was detected using fluid phase markers. Pre-treatment of early endosomes containing BHb with monoclonal antibody specific for the C-terminus of the Hb receptor (HbR) or the addition of the C-terminal cytoplasmic fragment of the HbR specifically inhibited the fusion with late endosomes, suggesting that signal(s) mediated through the HbR cytoplasmic tail promotes the fusion of early endosomes containing Hb with late endosomes.
Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.
Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be com pared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.
SYPRO Orange and SYPRO Ruby staining methods, modified for use with large-format-two dimensional (2-D) gels, are compared to the manufacturer's recommended protocols to determine sensitivity and reproducibility of the new methods. This study examines the critical aspects of fixation, washing, and staining to develop an optimized fluorescent staining method. It was determined that careful control of sodium dodecyl sulfate (SDS) levels and pH in the gel was critical for successful staining with SYPRO Orange. Overnight fixation in 40% ethanol/2% acetic acid/0.0005% SDS preserved protein content, eliminated ampholyte-generated staining artifacts, and had no detrimental effects on staining. Three one-hour washes in 2% acetic acid/0.0005% SDS, followed by staining with SYPRO Orange diluted 1:5000 with washing solution for 3 or more hours, produced high sensitivity, tow background images using a STORM 860 laser scanner. Gels viewed two years after staining showed no significant changes with respect to the initial protein patterns, and allowed successful mass spectrometric postgel characterization of protein spots. Protocol changes applied to SYPRO Ruby staining improved the contrast of STORM 860-generated images, but had little impact on staining sensitivity. A comparison of the cost benefits of staining with SYPRO Orange vs. SYPRO Ruby is also discussed.
We demonstrate the presence of a glycosylphosphatidylinositol (GPI) anchor-specific endosomal pathway in the protozoan pathogen Trypanosoma brucei. In higher eukaryotes evidence indicates that GPI-anchored proteins are transported in both the endocytic and exocytic systems by mechanisms involving sequestration into specific membrane microdomains and consequently sorting into distinct compartments. This is potentially extremely important in trypanosomatids as the GPI anchor is the predominant mechanism for membrane attachment of surface macromolecules, including the variant surface glycoprotein (VSG). A highly complex developmentally regulated endocytic network, vital for nutrient uptake and evasion of the immune response, exists in T. brucei. In common with mammalian cells an early endosomal compartment is defined by Rab5 small GTPases, which control transport processes through the endosomal system. We investigate the function of two trypanosome Rab5 homologues. ThRAB5A and ThRAB5B, which colocalize in the procyclic stage, are distinct in the bloodstream form of the parasite. TbRAB5A endosomes contain VSG and transferrin, endocytosed by the T. brucei GPI-anchored transferrin receptor, whereas ThRAB5B endosomes contain the transmembrane protein ISG(100) but neither VSG nor transferrin. These findings indicate the presence of trypanosome endosomal pathways trafficking proteins through specific routes depending on the mode of membrane attachment. Ectopic expression of mutant ThRAB5A or -5B indicates that TbP-kB5A plays a role in LDL endocytosis, whereas TbRAB5B does not, but both have a role in fluid phase endocytosis. Hence TbRAB5A and TbRAB5B have distinct functions in the endosomal system of T. brucei. A developmentally regulated GPI-specific endosomal pathway in the bloodstream form suggests that specialized transport of GPI-anchored proteins is required for survival in the mammalian host.
Leishmaniasis is caused by a wide range of parasites that are transmitted by an even wider range of sand fly vectors. The phlebotomine vectors of Leishmaniasis are in some cases only permissive to the complete development of the species of Leishmania that they transmit in nature. The parasite–sand fly interactions that control this specificity are related to differences in the ability of the parasite to inhibit or to resist killing by proteolytic enzymes released into the mid-gut soon after blood feeding, and/or to maintain infection in the mid-gut during excretion of the digested blood meal. In each case, surface expressed or released phosphoglycan-containing molecules appear to promote parasite survival. The evidence that the surface lipophosphoglycan (LPG) mediates promastigote attachment to the mid-gut epithelium so as to prevent their loss during blood-meal excretion is especially strong based on the comparison of development in sand flies using LPG-deficient mutants. LPG displays interspecies polymorphisms in their phosphoglycan domains that in most cases can fully account for species-specific vector competence.
We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (Imcpa) has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective meta-cyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPhe-ValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.
This report describes the construction of a DNA cassette for integration into a genomic small sub-unit rRNA locus of Leishmania mexicana by homologous recombination. Reporter genes encoding β-galactosidase or green fluorescent protein and the gene conferring hygromycin resistance were integrated downstream of a RNA polymerase I-driven rRNA promoter. To ensure high expression of the marker proteins in the intracellular, amastigote stage, transgene coding sequences were followed by the intergenic region of the L. mexicana cysteine proteinase B 2.8 gene which provides processing signals required for high level expression in this life-cycle stage. Integration of the DNA cassette was also efficiently obtained in L. major. We show that either β-galactosidase or the green fluorescent protein were abundantly, stably and uniformly expressed in promastigotes and amastigotes of both Leishmania sp. The transgenic lines allow parasite detection at high sensitivity in the tissues of infected mice and will be useful to follow infections in macrophages in culture and in animal hosts.
Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be compared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.
A new method has been developed for detecting cystatins and other cysteine proteinase inhibitors. The method, which involves protein separation by SDS-PAGE followed by a cysteine proteinase overlay step, is more sensitive than previously reported techniques: as little as 1 ng of recombinant human cystatin C can be detected and cysteine proteinase inhibitors could also be detected in complex protein mixtures such as bovine foetal serum. The method has been used to show, for the first time, cysteine proteinase inhibitors in lysates of a range of parasitic protozoa (Trypanosoma brucei, Leishmania mexicana mexicana, Toxoplasma gondii and Tritrichomonas foetus) and to confirm that one occurs in the free-living ciliate Tetrahymena pyriformis. Cystatin-like inhibitory activity was also demonstrated in boiled lysates of L. mexicana mexicana using conventional assays methods.
The multiple cysteine proteinases characteristic of the amastigote forms of Leishmania mexicana mexicana have been shown to be of three types. The groups of enzymes are distinguished by their substrate specificities and physical properties and have been purified free from other proteinases and most other proteins. One group (A) comprises at least four enzymes that bind to Con A. The cysteine proteinases comprising the other two groups (B and C) were separated by ion exchange chromatography. These last two groups of enzymes show different specificities towards a range of peptidyl aminomethylcoumarins. Notably, the two group C proteinases are more active towards compounds with a basic amino acid in the P1 position, whereas the three enzymes of group B are as active towards substrates with tyrosine in this position. All the cysteine proteinases show preference for compounds with bulky amino acids at positions P2 and P3 and all are equally susceptible to a range of inhibitors characteristically active against cysteine proteinases. Other proteinases present in amastigotes of L. m. mexicana were shown to differ significantly from the cysteine proteinases with respect to their substrate preferences and susceptibility to inhibitors.
Leishmania mexicana mexicana (M379) amastigotes were found to contain much higher activities than cultured promastigotes of five putative lysosomal enzymes: cysteine proteinase; arylsulfatase (EC 3.1.6.1); beta-glucuronidase (EC 3.2.1.31); DNase (EC 3.1.22.1), and RNase (EC 3.1.27.1). The release profiles of the first three of these enzymes from digitonin-permeabilized amastigotes suggests that they are located within organelles. Cytochemical staining for cysteine proteinase, using gold labeled antibodies and arylsulfatase, showed that both were present in large organelles previously named "megasomes." Comparative studies with L. mexicana amazonensis (LV78), L. donovani donovani (LV9), and L. major (LV39) revealed that L. mexicana amazonensis was similar to L. mexicana mexicana in possessing both high amastigote cysteine proteinase activity and large numbers of megasome organelles in amastigotes, whereas the other two species lacked both these features. The results suggest that the presence of numerous lysosome-like organelles in the amastigote is a characteristic of the L. mexicana group of parasites.
It is well established that Leishmania mexicana amastigotes contain large amounts of cysteine proteinases in their extended lysosomes. In this study it is shown that the cell-free supernatant of homogenized lesion tissue from infected mice contains large amounts of acid proteinases. The majority of this enzymatic activity also corresponds to cysteine proteinases from L. mexicana amastigotes. Immunoelectron microscopy of mouse lesion sections suggests, that frequently amastigotes lyse and release lysosomal cysteine proteinases into the parasitophorous vacuole of infected macrophages. The cysteine proteinases are also found extracellularly in the tissue presumably as a result of macrophage rupture and appear to persist in the lesion tissue, where they may damage host cells and the extracellular matrix.
The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) encoded on a tandem array of 19 genes (lmcpb). Homozygous null mutants for lmcpb have been produced by targeted gene disruption. All life-cycle stages of the mutant can be cultured in vitro, demonstrating that the gene is not essential for growth or differentiation of the parasite. However, the mutant exhibits a marked phenotype affecting virulence-- its infectivity to macrophages is reduced by 80%. The mutants are as efficient as wild-type parasites in invading macrophages but they only survive in a small proportion of the cells. However, those parasites that successfully infect these macrophages grow normally. Despite their reduced virulence, the mutants are still able to produce subcutaneous lesions in mice, albeit at a slower rate than wild-type parasites. The product of a single copy of lmcpb re-expressed in the null mutant was enzymatically active and restored infectivity toward macrophages to wild-type levels. Double null mutants created for lmcpb and lmcpa (another cathepsin L-like cysteine proteinase) have a similar phenotype to the lmcpb null mutant, showing that LmCPa does not compensate for the loss of LmCPb.
We have tested the suitability of the green fluorescent protein (GFP) of Aequorea victoria as a marker for studies of gene expression and protein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. Transfection of a modified GFP gene containing GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FACS analysis revealed a clear quantitative separation between GFP-transfected and control parasites, with pXG-GFP+ transfectants showing fluorescence signals more than 100-fold background. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marker for FACS separations. GFP was fused to the C-terminus of the LPG1 protein, which retained its ability to restore LPG expression when expressed in the lpg- R2D2 mutant of L. donovani. The LPG1(GFP) fusion was localized to a region situated between the nucleus and kinetoplast; its pattern was similar to that of LPG2, which is known to be located in the Golgi apparatus. This is notable as LPG1 participates in the biosynthesis of the glycan core of the LPG GPI anchor, whereas protein GPI anchor biosynthesis occurs in the endoplasmic reticulum. These studies suggest that the GFP will be a broadly useful marker in Leishmania.
Synthetic peptides corresponding to the proregions of papain-like cysteine proteases have been shown to be good and selective inhibitors of their parental enzymes. The molecular basis for their selectivity, quite remarkable in some cases, is not fully understood. The recent determination of the crystal structures of three distinct papain-like cysteine protease zymogens allows detailed structural comparisons to be made. The reasons for the specificity shown by each proregion toward its cognate enzyme are explained in terms of the three-dimensional structure of the proregion and the interface between the mature enzyme and the proregion. These comparisons reveal that insertion and substitution of amino acids within the proregion cause major rearrangement of sidechains on the enzyme/proregion interface, allowing detailed surface and charge recognition.
African trypanosomes, such as Trypanosoma brucei, are protozoan parasites that are transmitted by the tsetse fly and cause sleeping sickness in humans and Nagana in cattle. Trypanosomes evade the immune responses of their hosts by varying their surface coat protein (VSG) and restricting exocytosis and endocytosis to an invagination of the plasma membrane called the flagellar pocket (FP). The FP represents only 0.5% of the cellular surface but membrane turnover here occurs at high rates [1] [2] [3]. No model has yet been proposed to account for the sequestration of membrane proteins and the rate of membrane turnover that occur in the FP. Recent data have suggested that glycans are involved in the sorting of membrane proteins in polarized cells [4] [5] [6] [7]. Here, we show that N-linked glycans containing linear poly-N-acetyllactosamine (pNAL) are only associated with proteins of the FP/endocytic pathway in T. brucei and are present only in bloodstream forms of the parasite. These glycoproteins bind to tomato lectin (TL), a property that allowed their single-step isolation. Chito-oligosaccharides that compete specifically for pNAL binding to TL also inhibited receptor-mediated uptake of several ligands. These results suggest a model in which N-linked linear pNAL acts as a sorting signal for endocytosis in trypanosomes.
The susceptibilities of the protozoan parasites Leishmania mexicana and Trypanosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 microg ml(-1) was sufficient to cause cell death within 72 h of L. mexicana promastigotes and bloodstream forms of T. brucei in vitro. The gene encoding blasticidin S deaminase (BSD) was therefore incorporated into cassettes for targeting to the cysteine proteinase C locus of L. mexicana (CPC::BSD) and the tubulin locus of T. brucei (tub::RAD51-BSR). Following transfection of mutant parasites that contained other well-established selectable marker genes (HYG, NEO, BLE, PAC and SAT), clones resistant to 10 microg ml(-1) blasticidin S were shown by PCR and Southern blotting to have integrated the cassettes by homologous recombination. The results confirm that BSD can be used as a selectable marker gene for targeted chromosomal integration during genetic manipulations of trypanosomatids.
Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPB(C25G)). When CPB(C25G) was expressed in a L. mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPB(C25G) was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPB(C25G) and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA or CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.
Promastigotes of Leishmania
mexicana mutants lacking the multicopy CPB
cysteine proteinase genes (ΔCPB) are markedly
less able than wild-type parasites to infect macrophages in
vitro. ΔCPB promastigotes invade macrophages in large
numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within
macrophages in
vitro. This extreme in
vitro stage-specific difference was not mimicked in
vivo; both promastigotes and
amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than
those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in
situ using cell-permeant
peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in
vitro. In contrast,
N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%.
Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting
that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L.
mexicana encode enzymes
that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian
host.
Leishmania are a group of primitive eukaryotic trypanosomatid protozoa that are apically polarized with a flagellum at their anterior end. Surrounding the base of the flagellum is the flagellar reservoir that constitutes the site for endocytosis and exocytosis in these organisms. In the present study, we define a novel multivesicular tubular compartment involved in the intracellular trafficking of macromolecules in Leishmania. This dynamic structure appears to subtend the flagellar reservoir and extends towards the posterior end of the cell. Functional domains of several surface-expressed proteins, such as the gp63 glycosyl phosphatidyl inositol anchor and the 3'nucleotidase/nuclease transmembrane domain were fused to green fluorescent protein. These chimeric proteins were found to traffic through the secretory pathway and, while reaching their intended destinations, also accumulated within the intracellular tubular compartment. Using various compounds that are efficient fluid-phase markers used to track endocytosis in higher eukaryotes, we showed that this tubular compartment constitutes an important station in the endocytic pathway of these cells. Based on our functional observations of its role in the trafficking of expressed proteins and endocytosed markers, this compartment appears to have properties similar to endosomes of higher eukaryotes.
Leishmaniasis is caused by a wide range of parasites that are transmitted by an even wider range of sand fly vectors. The phlebotomine vectors of Leishmaniasis are in some cases only permissive to the complete development of the species of Leishmania that they transmit in nature. The parasite-sand fly interactions that control this specificity are related to differences in the ability of the parasite to inhibit or to resist killing by proteolytic enzymes released into the mid-gut soon after blood feeding, and/or to maintain infection in the mid-gut during excretion of the digested blood meal. In each case, surface expressed or released phosphoglycan-containing molecules appear to promote parasite survival. The evidence that the surface lipophosphoglycan (LPG) mediates promastigote attachment to the mid-gut epithelium so as to prevent their loss during blood-meal excretion is especially strong based on the comparison of development in sand flies using LPG-deficient mutants. LPG displays interspecies polymorphisms in their phosphoglycan domains that in most cases can fully account for species-specific vector competence.
SYPRO Orange and SYPRO Ruby staining methods, modified for use with large-format two dimensional (2-D) gels, are compared to the manufacturer's recommended protocols to determine sensitivity and reproducibility of the new methods. This study examines the critical aspects of fixation, washing, and staining to develop an optimized fluorescent staining method. It was determined that careful control of sodium dodecyl sulfate (SDS) levels and pH in the gel was critical for successful staining with SYPRO Orange. Overnight fixation in 40% ethanol/2% acetic acid/0.0005% SDS preserved protein content, eliminated ampholyte-generated staining artifacts, and had no detrimental effects on staining. Three one-hour washes in 2% acetic acid/0.0005% SDS, followed by staining with SYPRO Orange diluted 1:5,000 with washing solution for 3 or more hours, produced high sensitivity, low background images using a STORM 860 laser scanner. Gels viewed two years after staining showed no significant changes with respect to the initial protein patterns, and allowed successful mass spectrometric postgel characterization of protein spots. Protocol changes applied to SYPRO Ruby staining improved the contrast of STORM 860-generated images, but had little impact on staining sensitivity. A comparison of the cost benefits of staining with SYPRO Orange vs. SYPRO Ruby is also discussed.
Restriction-modification (RM) systems comprise two opposing enzymatic activities: a restriction endonuclease, that targets specific DNA sequences and performs endonucleolytic cleavage, and a modification methyltransferase that renders these sequences resistant to cleavage. Studies on molecular genetics and biochemistry of RM systems have been carried out over the past four decades, laying foundations for modern molecular biology and providing important models for mechanisms of highly specific protein-DNA interactions. Although the number of known, relevant sequences 3D structures of RM proteins is growing steadily, we do not fully understand their functional diversities from an evolutionary perspective and we are not yet able to engineer new sequence specificities based on rational approaches. Recent findings on the evolution of RM systems and on their structures and mechanisms of action have led to a picture in which conserved modules with defined function are shared between different RM proteins and other enzymes involved in nucleic acid biochemistry. On the other hand, it has been realized that some of the modules have been replaced in the evolution by unrelated domains exerting similar function. The aim of this review is to give a survey on the recent progress in the field of structural phylogeny of RM enzymes with special emphasis on studies of sequence-structure-function relationships and emerging potential applications in biotechnology.
The recently described inhibitor of cysteine proteinases from Trypanosoma cruzi, chagasin, was found to have close homologs in several eukaryotes, bacteria and archaea, the first protein inhibitors of cysteine proteases in prokaryotes. These previously uncharacterized 110-130 residue-long proteins share a well-conserved sequence motif that corresponds to two adjacent beta-strands and the short loop connecting them. Chagasin-like proteins also have other conserved, mostly aromatic, residues, and share the same predicted secondary structure. These proteins adopt an all-beta fold with eight predicted beta-strands of the immunoglobulin type. The phylogenetic distribution of the chagasins generally correlates with the presence of papain-like cysteine proteases. Previous studies have uncovered similar trends in cysteine proteinase binding by two unrelated inhibitors, stefin and p41, that belong to the cystatin and thyroglobulin families, respectively. A hypothetical model of chagasin-cruzipain interaction suggests that chagasin may dock to the cruzipain active site in a similar manner with the conserved NPTTG motif of chagasin forming a loop that is similar to the wedge structures formed at the active sites of papain and cathepsin L by stefin and p41.
Leishmania species are intracellular parasites that inhabit a parasitophorous vacuole (PV) within host macrophages and engage with the host endo-membrane network to avoid clearance from the cell. Intracellular Leishmania amastigotes exhibit a high degree of proteolytic/lysosomal activity that may assist degradation of MHC class II molecules and subsequent interruption of antigen presentation. As an aid to further analysis of the endosomal/lysosomal events that could facilitate this process, we have characterised a Leishmania homologue of the late endosomal marker, Rab7, thought to be involved in the terminal steps of endocytosis and lysosomal delivery. The Leishmania major Rab7 (LmRAB7) protein is expressed throughout the life-cycle, shows 73 and 64% identity to Trypanosoma cruzi and Trypanosoma brucei Rab7s (TcRAB7 and TbRAB7), respectively, and includes a kinetoplastid-specific insertion. The recombinant protein binds GTP and polyclonal antibodies raised against this antigen recognise structures in the region of the cell between the nucleus and kinetoplast. By immunoelectron microscopy of axenic amastigotes, Leishmania mexicana Rab7 (LmexRAB7) is found juxtaposed to and overlapping membrane structures labelled for the megasomal marker, cysteine proteinase B, confirming a late-endosomal/lysosomal localisation.
The endocytic pathway of Leishmania parasites has recently come under intense research focus through the development of several markers for various compartments of this pathway. Through these studies a novel multivesicular tubule lysosome has been discovered in promastigote-stage parasites. This organelle has a highly dynamic role during parasite growth and differentiation. This review discusses recent insights into the Leishmania lysosome with respect to its organisation within the endocytic pathway, stage-specific functions, and biogenesis.
The endocytic system of kinetoplastid parasites is a highly polarized membrane network focused on the flagellar pocket localized at one end of the cell. When first characterized, the endosomal network was envisioned as a simple system for uptake of extracellular material by fluid-phase or receptor-mediated mechanisms. Subsequently, it has become clear that the kinetoplastid endosomal system has an active and vital role in avoiding the host immune system and virulence, as well as providing the basic functions to fulfil cellular nutritional requirements. In two reviews, recent advances in the definition and comprehension of kinetoplastida endocytosis are discussed and, in Trypanosoma brucei in particular as the more developed experimental system. In Part 1, the endocytic system is considered in context of the surface molecules and their potential roles in virulence.
Endocytic systems within eukaryotic cells are a diverse set of intracellular transport pathways responsible for uptake, recycling, interaction with the exocytic system and degradation of molecules. Each of these pathways requires the interaction of distinct protein components that function in macromolecule sorting, control of transport rates and in membrane biogenesis. In the second of two articles on kinetoplastida endocytosis, the endocytic system in Trypanosoma brucei is considered as a model, and the molecules that control this system and the protein components of the endocytic pathway are discussed. We also consider novel mechanisms for sorting that have been proposed to operate in trypanosomes.
Cysteine peptidase inhibitor genes (ICP) of the chagasin family have been identified in protozoan (Leishmania mexicana and Trypanosoma brucei) and bacterial (Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.