No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
PoreForming Peptide of Pathogenic Entamoeba histolytica
Abstract
A polypeptide that causes pore formation in target-cell membranes is implicated in the potent cytolytic activity of pathogenic Entamoeba histolytica. Pore-forming material was purified to apparent homogeneity by a multistep procedure, and its analysis by NaDodSO4/PAGE revealed one peptide of 4-5 kDa under nonreducing or under reducing conditions. Pore-forming activity was measured by depolarization of liposome membrane potential and was found to be optimally expressed at low pH. Active material preferentially inserted into negatively charged lipid vesicles. Treatment of purified amoeba peptide in solution or bound to liposomes with glutaraldehyde revealed oligomers upon NaDodSO4/PAGE, suggesting functionally relevant peptide-peptide interactions. The NH2-terminal amino acid sequence of the amoeba peptide was determined by protein sequencing and revealed a structural similarity to melittin, the membranolytic peptide of bee venom.
... Amoebiasis produced by the protozoan parasite Entamoeba histolytica represents an important health problem especially in developing countries [1]. The mechanisms by which the parasites exert the tissue damage has been widely studied [2], and several molecular analyses have been done to understand the molecular basis of hostparasite interactions [3][4][5]. It is well known that the virulence of amoeba involved sequential steps, including adherence [6], contactdependent cytolysis [7] and phagocytosis [8]. ...
... It is well known that the virulence of amoeba involved sequential steps, including adherence [6], contactdependent cytolysis [7] and phagocytosis [8]. Factors involved in these processes, such as Gal/GalNac lectin subunits [9] amoebapores [4] and cysteine proteinases [5] have been described and several in vitro studies have demonstrated the induction of contact dependent-apoptosis in E. histolytica infection [10]. In vivo, Yan and Stanley [11], demonstrated that the use of pan-caspase inhibitor (Z-VADfmk) to treat mice, protect animals from amoebic liver abscess (ALA), showing a direct involvement of apoptosis in E. histolytica infection. ...
Aim: In the present study, we exploited DNA microarray-based transcriptome analysis and showed overall changes in gene expression in vivo of amoebic trophozoites that interact with animal soluble factors using an intraperitoneal dialysis bag model to elucidate putative molecular pathways and genes involved in this interaction. Study Design: We exploited DNA microarray-based transcriptome analysis. Results: An analysis from a network including the interactions of up-regulated genes and their neighbors revealed the presence of 11 functionally related modules. Six of the modules obtained were related to endoplasmic reticulum (ER) functions, such as degradation, stress, proteasome-ubiquitination, phosphorylation, lipid metabolism, and protein sorting. Furthermore, major transcriptional changes displayed by the parasite at the beginning of interaction were attributed to the response to the host defense. These data are consistent with the notion that the concerted expression of genes necessary for survival such as increment in protein synthesis, cytoskeleton rearrangement, vesicular traffic and genes involved in cell death including calcium imbalance and the ER signals associated with protein degradation (ERAD) is an overall landscape during the in vivo interaction between the amoebic trophozoites and animal soluble factors, and suggest that the ER stress is one of the main pathways leading to programmed cell death in E. histolytica. Conclusion: The present findings on the global transcriptional changes displayed by the parasite at the early stages of interaction with host environments in peritoneal implantation indicate that a substantial proportion of concerted changes in gene expression in amoebic trophozoites are attributable to the parasite’s response for cell death signals due to ER stress. A detailed knowledge of the underlying molecular mechanism might suggest the efficient elimination of this parasite by promoting their death pathways.
... The amebapore of E. histolytica is a channel-forming peptide of 77 amino acid residues, which has now been purified; the protein has been sequenced, and the respective genes have been cloned (68,69). Three isoforms, amebapores A, B, and C, are present at a ratio of 35:10:1, respectively, with the genes showing 35 to 57% deduced amino acid sequence identity. ...
... In addition to a collagenolytic activity, EDGs seem to contain at least 25 polypeptides with acidic pIs (6.06 to 6.59), 9 gelatinase activities, actin, small molecules including inorganic phosphate (P i ) and pyrophosphate (PP i ), and several ions. Six of these polypeptides (108,106,104,97,68, and 59 kDa) and two protease activities of 40 and 85 kDa have been claimed to be detected in EDGs but not in total-trophozoite extracts (76). Whether some of the abovementioned components represent contamination with cytoplasmic molecules not found in EDGs remains to be established. ...
In spite of a wealth of knowledge on the biochemistry and cellular and molecular biology of Entamoeba histolytica, little has been done to apply these advances to our understanding of the lesions observed in patients with intestinal amebiasis. In this review, the pathological and histological findings in acute amebic colitis are related to the molecular mechanisms of E. histolytica pathogenicity described to date. Infection of the human colon by E. histolytica produces focal ulceration of the intestinal mucosa, resulting in dysentery (diarrhea with blood and mucus). Although a complete picture has not yet been achieved, the basic mechanisms involved in the production of focal lytic lesions include complex multifactorial processes in which lectins facilitate adhesion, proteases degrade extracellular matrix components, porins help nourish the parasite and may also kill incoming polymorphonuclear leukocytes and macrophages, and motility is used by the parasite to invade deeper layers of the colon. In addition, E. histolytica has developed mechanisms to modulate the immune response during acute infection. Nevertheless, much still needs to be unraveled to understand how this microscopic parasite has earned its well-deserved histolytic name.
... Functionally, SAPLIPs fulfill various biological functions, but the members of this family have in common that they interact with lipids and membranes (Munford et al., 1995;Bruhn, 2005;Kolter et al., 2005). SAPLIPS with antimicrobial activity can be found in phylogenetically diverse organisms ranging from protozoans to mammals (Leippe et al., 1991;Andersson et al., 1995;Peña et al., 1997). ...
... Pore-forming activity using the liposome depolarisation assay (Leippe et al., 1991) and permeabilization of cytoplasmic membranes of live bacteria using the fluorescent dye SYTOX Green (Herbst et al., 2002) were measured as described previously. Briefly, in the liposome-depolarization assay a valinomycin-induced diffusion potential across the membrane of liposomes prepared from asolectin, a crude mixture of soy bean phospholipids, resulted in quenching of the enclosed fluorescent dye. ...
Due to their archaic life style and microbivor behavior, amoebae may represent a source of antimicrobial peptides and proteins. The amoebic protozoon Dictyostelium discoideum has been a model organism in cell biology for decades and has recently also been used for research on host-pathogen interactions and the evolution of innate immunity. In the genome of D. discoideum, genes can be identified that potentially allow the synthesis of a variety of antimicrobial proteins. However, at the protein level only very few antimicrobial proteins have been characterized that may interact directly with bacteria and help in fighting infection of D. discoideum with potential pathogens. Here, we focus on a large group of gene products that structurally belong to the saposin-like protein (SAPLIP) family and which members we named provisionally Apls (amoebapore-like peptides) according to their similarity to a comprehensively studied antimicrobial and cytotoxic pore-forming protein of the protozoan parasite Entamoeba histolytica. We focused on AplD because it is the only Apl gene that is reported to be primarily transcribed further during the multicellular stages such as the mobile slug stage. Upon knock-out (KO) of the gene, aplD− slugs became highly vulnerable to virulent Klebsiella pneumoniae. AplD− slugs harbored bacterial clumps in their interior and were unable to slough off the pathogen in their slime sheath. Re-expression of AplD in aplD− slugs rescued the susceptibility toward K. pneumoniae. The purified recombinant protein rAplD formed pores in liposomes and was also capable of permeabilizing the membrane of live Bacillus megaterium. We propose that the multifarious Apl family of D. discoideum comprises antimicrobial effector polypeptides that are instrumental to interact with bacteria and their phospholipid membranes. The variety of its members would allow a complementary and synergistic action against a variety of microbes, which the amoeba encounters in its environment.
... Caenopores are antimicrobial peptides from C. elegans with similarity to amoebapores from Entamoeba histolytica, naegleriapores from Naegleria fowleri, and mammalian NK-lysin and granulysin (Leippe et al., 1991;Leippe, 1995Leippe, , 1999Banyai and Patthy, 1998;Herbst et al., 2002). The caenopores are encoded by 28 spp genes, several of which are involved in responses to various pathogens (see review by Bogaerts et al., 2010;Roeder et al., 2010;Hoeckendorf et al., 2012). ...
... The pore-forming activity of all three has been confirmed using liposomes, and in B. megaterium, Saccharomyces cerevisiae, and Dictyostelium discoideum for SPP-1 and SPP-12 Hoeckendorf et al., 2012). Similar to amoebapores, SPP-1, 5, and 12 are more active at acidic pH (Leippe et al., 1991;Hoeckendorf et al., 2012). ...
There is currently insufficient data to determine the full spectrum of antimicrobial peptides (AMPs) that nematodes produce. Defensins, nemapores, cecropins, and caenacins/neuropeptide-like proteins have been identified, but none of these is produced universally by all nematode species, and no single species produces all AMP types. Therefore, it seems unlikely that there is a core set of AMPs that can be considered "archetypal" for nematodes. Additional information is also needed from under-represented Ecdysozoan and Lophotrochozoan taxa to clarify the evolution of AMPs. To avoid generalizations that may later prove inaccurate, caution should be used when choosing "representative" sequences or taxa, and analyses should be interpreted conservatively when limited information is available.
... The assortment of E. histolytica and E. dispar has been diagnosed by this method (Clark and Diamond 2002). Numerous assays, such as indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), indirect immunofluorescence assay (IFA), enzyme -linked (Leippe et al. 1991; Cysteine proteinase ...
Sexual intercourse can be a major transmission process for major parasitic diseases, including Trichomoniasis, Amoebiasis, Giardiasis, and Scabies. The oral-genital and oral-anal exchanges predispose male homosexuals to infection with these pathogens. These
parasites can cause acute and chronic disorders, including abdominal symptoms like watery diarrhea, colitis, weight loss, and vomiting. Majority of the hosts infected with these diseases are asymptomatic but proper protocols of diagnosis and treatment are necessary for the control of these diseases. Anti-parasitic drugs are used against all these parasitic diseases but due to development of resistance, attention is diverted towards vaccination and use of medicines derived from plants. Vaccination against infectious diseases, plant derivative products, and probiotics are effective approaches against these pathogens. The omics studies (Transcriptomic, genomics, proteomics) are
considered the best and authentic alternative approaches for controlling the parasitic diseases in the modern age.
... Entamoeba histolytica stimulates the chemotaxis of human polymorphonuclear neutrophils (PMNs) (Salata et al., 1989). It uses a pore-forming polypeptide, which is structurally similar to melittin (the membranolytic peptide of bee venom), to make amebic pores on the host cells; thereby causing host cell lysis (Leippe et al., 1991). It was estimated that one E. histolytica trophozoite can kill up to 3000 neutrophils (Guerrant et al., 1988). ...
For the establishment of a successful infection, i.e., long-term parasitism and a complete life cycle, parasites use various diverse mechanisms and factors, which they may be inherently bestowed with, or may acquire from the natural vector biting the host at the infection prelude, or may take over from the infecting host, to outmaneuver, evade, overcome, and/or suppress the host immunity, both innately and adaptively. This narrative review summarizes the up-to-date strategies exploited by a number of representative human parasites (protozoa and helminths) to counteract the target host immune defense. The revisited information should be useful for designing diagnostics and therapeutics as well as vaccines against the respective parasitic infections.
... Increased capsule size can protect C. neoformans against amoeba phagocytosis (46). Moreover, amoebae are known to produce antimicrobial pore-forming peptides (70), and it is conceivable that mutation of OPT1 could reduce their importance and protect C. neoformans. By reviewing the published sequences of 387 clinical and environments strains (71), we found that 6 of 287 clinical isolates had high-impact potential loss-of-function mutations in OPT1, but there were no OPT1 mutations in the 100 environmental isolates. ...
Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.IMPORTANCECryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae, like macrophages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive.
... Entamoeba histolytica amoebapore is a pore-forming protein of the saposin-like protein (SAPLIP) family that is involved in the degradation of bacterial (Andr€ a et al. 2003) and possibly host cell membranes (Leippe et al. 1991;Bujanover et al. 2003). We found a gene of the SAPLIP family in the M. balamuthi genome that resembles the E. histolytica amoebapore gene ( fig. 6 and supplementary table S11, Supplementary Material online). ...
The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living M. balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).
... They are a family of three 77-amino acid peptides capable of forming pores in lipid bilayers, causing ion channels disturbance and subsequent death of intestinal epithelial cells, hepatocytes, and host defense cells. They are found within cytoplasmic granules of E. histolytica and may exocytose to the target cell membrane on contact [80] . In a review published early in this century, it was reported that necrotized intestinal epithelial cells are capable of releasing IL-1α, stimulating cytokine production from non-infected cells. ...
Parasites developed several strategies for their survival and host tissue invasion. Helminths express potent molecules mainly for immunomodulation, which is why they stay in their hosts for years. Helminths display several mechanisms not only to evade host immune response(s), but also to preserve the host for as long as they could live. In contrast, protozoa evolve several policies primarily for pathogenesis, and invasion. Therefore, variable clinical manifestations are reported in protozoal diseases. Both symptomatic and asymptomatic cases are commonly observed in amoebiasis, giardiasis, trichomoniasis, cryptosporidiosis and toxoplasmosis, while mild, moderate, and severe cases occur in malaria, leishmaniasis, African sleeping sickness and Chagas’ disease. This was primarily attributed to strains variability and to a lesser extent, to host immune response(s). With recent evolutionary technology in molecular parasitology and bioinformatics, several molecules are established as virulence factors. These factors encourage researchers and scientists to develop novel drug targets and/or vaccine candidates. The present review aims to highlight, and review virulence strategies adapted by parasites to invade host tissue, enhance its replication and spread, as well as other processes for immunomodulation or immunoevasion of host immune response(s).
... SAPLIP, which includes human granulysins and cytoplasmic granules of Entamoeba histolytica, are known for their membrane-binding and membrane disruption activities [92,93]. This suggests that NK-lysins are ancient, highly conserved antimicrobial proteins that serve as an important defense mechanism for many hosts [94]. ...
Aquaculture’s rapid growth and commercialization has been accompanied by increased spread of fish-related diseases that threaten aquaculture’s production capabilities. Diseases caused by Gram-negative bacteria such as, Aeromonas hydrophila and Flavobacterium columnare, are particularly problematic and are responsible for tremendous losses in fish. These bacterial fish pathogens exist not only in a free-living state but also occur readily as biofilms. Biofilms are microbial communities incased in a self-produced organic matrix and are much more resistant to conventional antimicrobial agents than their free-living counterparts. The prevalence of these pathogenic bacteria as biofilms in the aquaculture setting has encouraged the exploration of alternative anti-biofilm agents. The innate immune system of fish and other organisms produce peptides that are effective in combatting a wide range of pathogens. These peptides are collectively known as antimicrobial peptides (AMPs) and they may prove to be useful anti-biofilm agents due to their broad-spectrum capabilities. These peptides have been characterized among many different fish species and their effect on different Gram-negative bacteria has been evaluated. However, there exists a gap of knowledge in their anti-biofilm capabilities. The current study has assessed the activity of different teleost AMPs against Aeromonas hydrophila, Aeromonas veronii, Flavobacterium columnare and Escherichia coli. The NK-lysins (NK-1, NK-2, etc.) inhibited the growth of E. coli and A. veronii and were particularly effective against biofilm formation in these two pathogens. The NK-lysins tended to show an expected dose response in that higher concentrations showed greater inhibition of E. coli and A. veronii. The Piscidins inhibited E. coli and F. columnare total growth and showed effectiveness against their respective biofilm formation. Unlike the NK-lysins, the Piscidins inhibited biofilm formation of E. coli and F. columnare at the lowest tested concentration. The positive control melittin demonstrated significant total and biofilm inhibition towards all four bacteria; some of the AMPs in this test showed results comparable to melittin. These results suggest that several of the AMPs might serve as effective therapeutic agents against bacteria in aquaculture settings due to their ability to inhibit both free-living and biofilm-encased bacteria and are worthy of further study. Future studies should examine mechanisms of action regarding biofilm inhibition, while also observing activity under more natural conditions.
... They are a family of three 77-amino acid peptides capable of forming pores in lipid bilayers, causing ion channels disturbance and subsequent death of intestinal epithelial cells, hepatocytes, and host defense cells. They are found within cytoplasmic granules of E. histolytica and may exocytose to the target cell membrane on contact [80] . In a review published early in this century, it was reported that necrotized intestinal epithelial cells are capable of releasing IL-1α, stimulating cytokine production from non-infected cells. ...
... Hence, multiple factors might be involved in host cell lysis. Another means of killing host cells is the activation of the apoptotic pathway (12). Following the death of cells, amoeba phagocytoses the dead cell mass. ...
Protein kinases are known to regulate several cellular processes like metabolism, motility and endocytosis through phosphorylation of specific target proteins which forms a communication system relaying extracellular signals to intracellular milieu for an adaptive response. One of the protozoan parasite Entamoeba histolytica, which causes amoebiasis and is one of the prominent reason for causing diarrhoea in infants of developing countries, where it remains the third leading cause of deaths in infants(1). The genome of this parasite codes for 331 putative protein kinases which accounts for 3.7% of the proteome. The kinome of the parasite is composed of several conserved and as well as kinase with unusual domain architecture. About one-third of kinome codes for transmembrane kinases (TMK) which is proposed to help the parasite to sense and adapt to the gut environment which is constantly changing. Many kinases are known to be involved in virulence but, the kinome of this important parasite is unexplored. In this review, we present an overview of E. histolytica kinases and their role in amoebic biology understood till now.
... Hence, multiple factors might be involved in host cell lysis. Another means of killing host cells is the activation of the apoptotic pathway (12). Following the death of cells, amoeba phagocytoses the dead cell mass. ...
Protein kinases are known to regulate several cellular processes like metabolism, motility and endocytosis through phosphorylation of specific target proteins which forms a communication system relaying extracellular signals to intracellular milieu for an adaptive response. One of the protozoan parasite Entamoeba histolytica, which causes amoebiasis and is one of the prominent reason for causing diarrhoea in infants of developing countries, where it remains the third leading cause of deaths in infants(1). The genome of this parasite codes for 331 putative protein kinases which accounts for 3.7% of the proteome. The kinome of the parasite is composed of several conserved and as well as kinase with unusual domain architecture. About one-third of kinome codes for transmembrane kinases (TMK) which is proposed to help the parasite to sense and adapt to the gut environment which is constantly changing. Many kinases are known to be involved in virulence but, the kinome of this important parasite is unexplored. In this review, we present an overview of E. histolytica kinases and their role in amoebic biology understood till now.
... The mechanism for membrane permeabilization is not known. The homologous, saposin-like amoebapore A protein from Entamoeba histolytica, which kills phagocytosed bacteria, dimerizes to expose a His residue near its C terminus that is believed to be important for membrane intercalation (117,118). Gel filtration experiments suggest that higher-order oligomers (hexamers?) might oligomerize in the membrane. ...
Immune cells use a variety of membrane-disrupting proteins [complement, perforin, perforin-2, granulysin, gasdermins, mixed lineage kinase domain–like pseudokinase (MLKL)] to induce different kinds of death of microbes and host cells, some of which cause inflammation. After activation by proteolytic cleavage or phosphorylation, these proteins oligomerize, bind to membrane lipids, and disrupt membrane integrity. These membrane disruptors play a critical role in both innate and adaptive immunity. Here we review our current knowledge of the functions, specificity, activation, and regulation of membrane-disrupting immune proteins and what is known about the mechanisms behind membrane damage, the structure of the pores they form, how the cells expressing these lethal proteins are protected, and how cells targeted for destruction can sometimes escape death by repairing membrane damage.
Expected final online publication date for the Annual Review of Immunology, Volume 38 is April 26, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... All four amoebapore isoforms were highly expressed. Although the constitutive expression of amoebapores has been reported in axenically grown E. histolytica [37,38], their high expression is intriguing as their known function is to lyse bacteria or other surrounding cells by membrane insertion following contact of target cell with the trophozoite. This cytotoxic activity would not be required in axenically grown cells, and possibly indicates that these pore forming peptides might have other constitutive functions. ...
Background
Promoter motifs in Entamoeba histolytica were earlier analysed using microarray data with lower dynamic range of gene expression. Additionally, previous transcriptomic studies did not provide information on the nature of highly transcribed genes, and downstream promoter motifs important for gene expression. To address these issues we generated RNA-Seq data and identified the high and low expressing genes, especially with respect to virulence potential. We analysed sequences both upstream and downstream of start site for important motifs.
Results
We used RNA-Seq data to classify genes according to expression levels, which ranged six orders of magnitude. Data were validated by reporter gene expression. Virulence-related genes (except AIG1) were amongst the highly expressed, while some kinases and BspA family genes were poorly expressed. We looked for conserved motifs in sequences upstream and downstream of the initiation codon. Following enrichment by AME we found seven motifs significantly enriched in high expression- and three in low expression-classes. Two of these motifs (M4 and M6) were located downstream of AUG, were exclusively enriched in high expression class, and were mostly found in ribosomal protein, and translation-related genes. Motif deletion resulted in drastic down regulation of reporter gene expression, showing functional relevance. Distribution of core promoter motifs (TATA, GAAC, and Inr) in all genes revealed that genes with downstream motifs were not preferentially associated with TATA-less promoters. We looked at gene expression changes in cells subjected to growth stress by serum starvation, and experimentally validated the data. Genes showing maximum up regulation belonged to the low or medium expression class, and included genes in signalling pathways, lipid metabolism, DNA repair, Myb transcription factors, BspA, and heat shock. Genes showing maximum down regulation belonged to the high or medium expression class. They included genes for signalling factors, actin, Ariel family, and ribosome biogenesis factors.
Conclusion
Our analysis has added important new information about the E. histolytica transcriptome. We report for the first time two downstream motifs required for gene expression, which could be used for over expression of E. histolytica genes. Most of the virulence-related genes in this parasite are highly expressed in culture.
Electronic supplementary material
The online version of this article (10.1186/s12864-019-5570-z) contains supplementary material, which is available to authorized users.
... The cytopathic effect on fibroblast monolayers as a result of supernatants of clinical E. histolytica strains is completely inhibited by Z-Phe-Arg-CH 2 F, a specific, irreversible cysteine proteinase inhibitor that is not toxic to host cells (84). In vitro cell lysis caused by E. histolytica is a more complex process, requiring attachment via the galactose-inhibitable lectin (70) and lysis by the amebapore (51). ...
Amebiasis is a major cause of morbidity and mortality throughout the tropical world. Entamoeba histolytica is now recognized as a separate species from the morphologically identical E. dispar, which cannot invade. Cysteine proteinases are a key virulence factor of E. histolytica and play a role in intestinal invasion by degrading the extracellular matrix and circumventing the host immune response through cleavage of secretory immunoglobulin A (sIgA), IgG, and activation of complement. Cysteine proteinases are encoded by at least seven genes, several of which are found in E. histolytica but not E. dispar. A number of new animal models, including the formation of liver abscesses in SCID mice and intestinal infection in human intestinal xenografts, have proven useful to confirm the critical role of cysteine proteinases in invasion. Detailed structural analysis of cysteine proteinases should provide further insights into their biochemical function and may facilitate the design of specific inhibitors which could be used as potential chemotherapeutic agents in the future.
... It has been seen that the processes that are dependent on ion channels contribute to the excystation and metacystic development of the parasite inside the host, thus playing a critical role in pathogenesis [56]. The pore-forming toxin proteins help in pore formation and the penetration of the parasite in the epithelial cell membrane of the host intestine [57], and they are insecticidal in nature [36]. These results imply a crucial role played by the different type of channels in parasite biology, suggesting that developing drugs for these parasite-specific channels may be a novel approach for a therapeutic strategy. ...
Entamoeba histolytica is an invasive, pathogenic parasite causing amoebiasis. Given that proteins involved in transmembrane (TM) transport are crucial for the adherence, invasion, and nutrition of the parasite, we conducted a genome-wide bioinformatics analysis of encoding proteins to functionally classify and characterize all the TM proteins in E. histolytica. In the present study, 692 TM proteins have been identified, of which 546 are TM transporters. For the first time, we report a set of 141 uncharacterized proteins predicted as TM transporters. The percentage of TM proteins was found to be lower in comparison to the free-living eukaryotes, due to the extracellular nature and functional diversification of the TM proteins. The number of multi-pass proteins is larger than the single-pass proteins; though both have their own significance in parasitism, multi-pass proteins are more extensively required as these are involved in acquiring nutrition and for ion transport, while single-pass proteins are only required at the time of inciting infection. Overall, this intestinal parasite implements multiple mechanisms for establishing infection, obtaining nutrition, and adapting itself to the new host environment. A classification of the repertoire of TM transporters in the present study augments several hints on potential methods of targeting the parasite for therapeutic benefits.
... Various studies have demonstrated that polymorphisms in this region affect gene function and increase susceptibility to various inflammatory diseases, such as chronic granulomatous disease [57][58][59][60]. In the susceptibility model of ALA, there might be polymorphisms modifying the expression of the promoter elements that regulate the expression of the mpo gene in neutrophils [61], as well as factors of virulence in amoebae, such as lectin Gal/GalNac, amoebapore and cysteine proteases [62], GalNac [63], proteasas [64][65][66], amebopore [67,68]. ...
The protozoan Entamoeba histolytica is the etiological agent of amoebiasis, which can spread to the liver and form amoebic liver abscesses. Histological studies conducted with resistant and susceptible models of amoebic liver abscesses (ALAs) have established that neutrophils are the first cells to contact invasive amoebae at the lesion site. Myeloperoxidase is the most abundant enzyme secreted by neutrophils. It uses hydrogen peroxide secreted by the same cells to oxidize chloride ions and produce hypochlorous acid, which is the most efficient microbicidal system of neutrophils. In a previous report, our group demonstrated that myeloperoxidase presents amoebicidal activity in vitro. The aim of the current contribution was to analyze in vivo the role of myeloperoxidase in a susceptible (hamsters) and resistant (Balb/c mice) animal models of ALAs. In liver samples of hamsters and mice inoculated intraportally with Entamoeba histolytica trophozoites, the number of neutrophils in ALAs was determined by enzymatic activity. The presence of myeloperoxidase was observed by staining, and its expression and activity were quantified in situ. A significant difference existed between the two animal models in the number of neutrophils and the expression and activity of myeloperoxidase, which may explain the distinct evolution of amoebic liver abscesses. Hamsters and mice were treated with an MPO inhibitor (4-aminobenzoic acid hydrazide). Hamsters treated with ABAH showed no significant differences in the percentage of lesions or in the percentage of amoebae damaged compared with the untreated hamsters. ABAH treated mice versus untreated mice showed larger abscesses and a decreased percentage of damaged amoebae in these lesion at all stages of evolution. Further studies are needed to elucidate the host and amoebic mechanisms involved in the adequate or inadequate activation and modulation of myeloperoxidase.
... The caenopores belong to an ancient family of antimicrobial peptides with a saposin domain. They share similarities with the saposin-like proteins (SAPLIPs) such as the amoebapores first characterized for Entamoeba histolytica [53,54] and the mammalian NK lysin and granulysin [55,56]. In C. elegans, the caenopores or saposins (spps) form a gene family of currently 23 members. ...
Nematodes and arthropods likely form the taxon Ecdysozoa. Information on antimicrobial effectors from the model nematode Caenorhabditis elegans may thus shed light on the evolutionary origin of these defences in arthropods. This nematode species possesses an extensive armory of putative antimicrobial effector proteins, such as lysozymes, caenopores (or saposin-like proteins), defensin-like peptides, caenacins and neuropeptide-like proteins, in addition to the production of reactive oxygen species and autophagy. As C. elegans is a bacterivore that lives in microbe-rich environments, some of its effector peptides and proteins likely function in both digestion of bacterial food and pathogen elimination. In this review, we provide an overview of C. elegans immune effector proteins and mechanisms. We summarize the experimental evidence of their antimicrobial function and involvement in the response to pathogen infection. We further evaluate the microbe-induced expression of effector genes using WormExp, a recently established database for C. elegans gene expression analysis. We emphasize the need for further analysis at the protein level to demonstrate an antimicrobial activity of these molecules both in vitro and in vivo .
This article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides'.
... Studies illustrate that Eh-induced apoptosis is independent of caspase-8 or caspase-9 (Huston et al., 2000). Another study showed that Leippe et al., 1991 All three types induce pore formation in synthetic liposomes Andrä et al., 2003 Amebapore A Active at low pH 5.2. May play a role in host cell killing before ingestion Andrä et al., 2003 Amebapore B Amebapore C ...
Entamoeba histolytica (Eh) is a protozoan parasite that infects 10% of the world's population and results in 100,000 deaths/year from amebic dysentery and/or liver abscess. In most cases, this extracellular parasite colonizes the colon by high affinity binding to MUC2 mucin without disease symptoms, whereas in some cases, Eh triggers an aggressive inflammatory response upon invasion of the colonic mucosa. The specific host-parasite factors critical for disease pathogenesis are still not well characterized. From the parasite, the signature events that lead to disease progression are cysteine protease cleavage of the C-terminus of MUC2 that dissolves the mucus layer followed by Eh binding and cytotoxicity of the mucosal epithelium. The host mounts an ineffective excessive host pro-inflammatory response following contact with host cells that causes tissue damage and participates in disease pathogenesis as Eh escapes host immune clearance by mechanisms that are not completely understood. Ameba can modulate or destroy effector immune cells by inducing neutrophil apoptosis and suppressing respiratory burst or nitric oxide (NO) production from macrophages. Eh adherence to the host cells also induce multiple cytotoxic effects that can promote cell death through phagocytosis, apoptosis or by trogocytosis (ingestion of living cells) that might play critical roles in immune evasion. This review focuses on the immune evasion mechanisms that Eh uses to survive and induce disease manifestation in the host.
... Amoebapores genannt (Leippe et al., 1991;Leippe et al., 1994b). Trophozoiten, bei denen ein bestimmtes amoebapore-Gen durch antisense-Technik oder durch transkriptionelle Inaktivierung ausgeschaltet ist, verlieren ihre Virulenz (Bracha et al., 1999;Bracha et al., 2003). ...
... B. Amoebapores are a family of small proteins contained in cytoplasmic granules in the trophozoite (Leippe et al, 1991). Trophozoites are able to depolarize target cells by the insertion of these poreforming proteins that form ion channels in their lipid membranes. ...
... The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the synthetic peptide (diluted in acidified water 0.05% acetic acid) against bacterial growth were determined by a microdilution susceptibility assay in microtiter plates as previously described [18]. Permeabilization of membranes of viable bacteria and poreforming activity towards liposomes were measured as previously described in details [19], [20]. Alamethicin, cecropin P1, and magainin II were purchased as synthetic peptides from Sigma. ...
The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.
Entamoeba moshkovskii, according to recent studies, appears to exert a more significant impact on diarrhoeal infections than previously believed. The efficient identification and genetic characterization of E. moshkovskii isolates from endemic areas worldwide are crucial for understanding the impact of parasite genomes on amoebic infections. In this study, we employed a multilocus sequence typing system to characterize E. moshkovskii isolates, with the aim of assessing the role of genetic variation in the pathogenic potential of E. moshkovskii. We incorporated 3 potential genetic markers: KERP1, a protein rich in lysine and glutamic acid; amoebapore C (apc) and chitinase. Sequencing was attempted for all target loci in 68 positive E. moshkovskii samples, and successfully sequenced a total of 33 samples for all 3 loci. The analysis revealed 17 distinct genotypes, labelled M1–M17, across the tested samples when combining all loci. Notably, genotype M1 demonstrated a statistically significant association with diarrhoeal incidence within E. moshkovskii infection (P = 0.0394). This suggests that M1 may represent a pathogenic strain with the highest potential for causing diarrhoeal symptoms. Additionally, we have identified a few single-nucleotide polymorphisms in the studied loci that can be utilized as genetic markers for recognizing the most potentially pathogenic E. moshkovskii isolates. In our genetic diversity study, the apc locus demonstrated the highest Hd value and π value, indicating its pivotal role in reflecting the evolutionary history and adaptation of the E. moshkovskii population. Furthermore, analyses of linkage disequilibrium and recombination within the E. moshkovskii population suggested that the apc locus could play a crucial role in determining the virulence of E. moshkovskii.
Entamoeba histolytica is responsible for dysentery and extraintestinal disease in humans. To establish successful infection, it must generate adaptive response against stress due to host defense mechanisms. We have developed a robust proteomics workflow by combining miniaturized sample preparation, low flow‐rate chromatography, and ultra‐high sensitivity mass spectrometry, achieving increased proteome coverage, and further integrated proteomics and RNA‐seq data to decipher regulation at translational and transcriptional levels. Label‐free quantitative proteomics led to identification of 2344 proteins, an improvement over the maximum number identified in E. histolytica proteomic studies. In serum‐starved cells, 127 proteins were differentially abundant and were associated with functions including antioxidant activity, cytoskeleton, translation, catalysis, and transport. The virulence factor, Gal/GalNAc‐inhibitable lectin subunits, was significantly altered. Integration of transcriptomic and proteomic data revealed that only 30% genes were coordinately regulated at both transcriptional and translational levels. Some highly expressed transcripts did not change in protein abundance. Conversely, genes with no transcriptional change showed enhanced protein abundance, indicating post‐transcriptional regulation. This multi‐omics approach enables more refined gene expression analysis to understand the adaptive response of E. histolytica during growth stress.
A book edited by J.Joseph Marr and Miklos Müller that sketches a picture of the state of the art of the biochemical research on parasites.
α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.
Entamoeba histolytica (Eh), a parasitic protozoan and the causative agent of invasive Amoebiasis, invade the host tissue through an effective secretory pathway. There are several lines of evidence suggesting that amoebic trophozoite pore‐forming complex amoebapore and a large class of proteases enzymes including rhomboid proteases, cysteine proteases, and metalloproteases are implicated in host tissue invasion. For successful delivery of these molecules/cargos, trophozoites heavily rely on sorting machinery from the endoplasmic reticulum, Golgi to plasma membrane. Although, sole secretion machinery in E. histolytica is not characterized yet. Therefore, here our aim is to understand the properties of key molecules N‐ethylmaleimide‐sensitive fusion protein attached to protein receptors (SNAREs) in E. histolytica. SNAREs proteins are an important component of the membrane‐trafficking machinery and have been associated in a range of processes including vesicle tethering, fusion as well as specificity of vesicular transport in all eukaryotic cells. SNARE proteins are architecturally simple, categorized by the presence of one copy of a homologous coiled‐coil forming motif. However, the structural information and protein‐protein interaction study of Eh‐associated syntaxin proteins are still not known. Here, we characterize the syntaxin 1 like molecule and VAMP from Eh through physiochemical profiling, modeling, atomistic simulation, protein‐protein interaction, and docking approaches on the proteins containing SNARE and synaptobrevin domain. The modeled structures and the critical residues recognized through protein interaction and docking study may provide better structural and functional insights into these proteins and may aid in the development of newer diagnostic assays.
While the discovery of antibiotics has made a huge contribution to medicine, bacteria that are resistant to many antibiotics pose new challenges to medicine. Antimicrobial peptides (AMPs), a new kind of antibiotics, have attracted people's attention because they are not prone to drug resistance. In this study, glutathione transferase (GST) was used as a fusion partner to recombinantly expressed rat lung surfactant protein B precursor (proSP-B) in E. coli pLySs. Cck-8 evaluated the cytotoxicity of the fusion protein and calculated its 50% inhibitory concentration (IC50). The purified peptides showed broad-spectrum antibacterial activity using filter paper method and MIC, and propidium iodide (PI) was used to explore the antibacterial mechanism against Staphylococcus aureus. In addition, the pEGFP-N2-proSP-B vector was constructed to explore the localization of proSP-B in CCL-149 cells. We found that proSP-B has obvious antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, and has broad-spectrum antibacterial activity. Besides, proSP-B fusion protein has low toxicity and can change the permeability of Staphylococcus aureus cell membrane to realize its antibacterial. For these reasons, proSP-B can be used as a potential natural antibacterial drug.
Protozoan parasitic infections of the gastrointestinal (GI) tract are being increasingly recognized as an important public health problem in the United States. The intestinal protozoan pathogens include both parasites that are extracellular, such as Giardia lamblia, Blastocystis hominis, and Entamoeba histolytica, and the intracellular spore-forming parasites Cryptosporidium parvum, Cyclospora cayetanensis, and Isospora belli. The AIDS epidemic has played an important role in the recognition of intracellular spore-forming parasites as important gastrointestinal protozoan pathogens. Stool microscopic examinations may be performed in clinical laboratories only if specifically requested. In some cases in which infection is suspected on clinical grounds, stool examination may not be revealing, and endoscopy may be necessary to make the diagnosis. The recent rapid development of new molecular assays, will likely facilitate more rapid laboratory diagnoses of not only protozoan, but also viral and bacterial pathogens in the near future.
Entamoeba histolytica, the only intestinal ameba species known to cause disease in humans, was long considered to exhibit distinctive microscopic features. The fact that only about 10 % of subjects had E. histolytica in their stool was ascribed to variations in the virulence of this ameba. However, E. Brumpt suggested as early as 1925 that another nonpathogenic species, E. dispar, was morphologically indistinguishable from E. histolytica. Only in 1993 was existence of these two species officially recognized, based on biochemical, immunological, and genetic data. This is the most significant event that has occurred in amebiasis research during the last few years. It is anticipated that molecular comparison of these two species will allow to identify the factors responsible for the pathogenicity of E. histolytica and therefore to define new therapeutic targets and perhaps to develop a vaccine. The presence off. histolytica in stool specimens can be demonstrated using simple ELISAs, which are already available in some countries and may lead to radical changes in the treatment of amebiasis in endemic areas.
Publisher Summary
This chapter discusses the mechanisms of invasion of a parasite into host tissue or host cells. Single cell protozoan parasites evade the host immune response by residing within host cells and enter the host cell with minimal trauma to ensure that they preserve an environment suitable for their replication and do not trigger a lethal host immune response. This intracellular invasion paradigm is also shared by a helminth parasite, Trichinella spiralis. Often an invasive larval form penetrates skin or the mucosa of the gastrointestinal tract to enter into the host.. The chapter states that the invasion phenomenon itself can be divided into three successive steps: recognition and attachment, internalization and vacuole development, vacuole maturation. Entamoeba histolytica, which is a protozoan, does not invade intra cellularly at any stage of its life cycle unlike other protozoas. The process of its invasion (extracellularly) and its properties are also elaborated in the chapter. .To complete the invasion process, helminth parasites have evolved sensory organs for finding and navigating within the host, attachment mechanisms utilizing specialized mouth structures or glue-like secretions, and hydrolytic enzymes to digest macromolecular barriers in the extracellular space
The process by which viruses destabilize endosomal membranes in an acidification dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were in corporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
Infections of the gastrointestinal tract, particularly those due to invasive enteropathogens which cause an ileo-colitis, may mimic ulcerative colitis or Crohn's disease. Increased vigilance in patients with diarrhea, particularly bloody diarrhea, and an appropriate search for an infective agent are essential before a final diagnosis of non-specific inflammatory bowel disease is made. Delay in administering an appropriate antibiotic for a colonic infection may increase morbidity, including the likelihood of developing complications. Precision in diagnosis is important to ensure that antibiotics are made available for those infections in which there is a proven role for antimicrobial chemotherapy in reducing the duration and severity of disease, and also to distinguish infective colitis from non-specific inflammatory bowel disease.
The most important feature of the pathology of human amebiasis is the greatly destructive nature of the anatomical lesions produced by the protozoan Entamoeba histolytica. Recent advances on the knowledge of biochemistry, immunology, cellular and molecular biology, and genetics of this parasite, added to the use of different in vitro, in vivo, and ex vivo models to analyze host–parasite interactions or the production of intestinal and extraintestinal amebic lesions, all have given a better perception of the mechanisms of pathogenesis in amebiasis. The present chapter is divided into three parts: First, a general review of the pathology of human amebiasis; second, a short review of the mechanisms of invasion and production of damage in the host, and third, a review of the different in vivo experimental models currently available to study the mechanisms involved in amebic infection. In reference to pathogenesis, each factor, molecule or gene, or mechanism of target cell damage is reviewed individually in other chapters of this section on “Pathogenesis and Immunity” in the present book. Therefore, the meticulous or probing aspects of the studies are mentioned by each responsible and expert group of researchers. In this review, we mention in general each of the different factors of pathogenesis in amebiasis. The contributions obtained using different techniques and methodologies of experimental models are emphasized, and the subjects that still need to be unraveled to understand how this microscopic parasite has earned its well-deserved “histolytic” name are discussed.
Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO towards pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.
Increasing resistance of pathogenic bacteria against antibiotics is a severe problem in health care. Natural antimicrobial peptides and derivatives thereof have emerged as promising candidates for “new antibiotics”. In contrast to classical antibiotics, these peptides act by direct physical destabilization of the target cell membrane. Nevertheless, they exhibit a high specificity for bacteria over mammalian cells. However, the precise mechanism of action and the molecular basis for membrane selectivity are still a matter of debate. We have designed a new peptide antibiotic (NK-2) with enhanced antimicrobial activity based on an effector protein of mammalian immune cells (NK-lysin). Here we describe the interaction of this α-helical synthetic peptide with membrane mimetic systems, designed to mimic the lipid compositions of mammalian and bacterial cytoplasmic membranes. Utilizing fluorescence and biosensor assays, we could show that on one hand, NK-2 strongly interacts with negatively charged membranes; on the other hand, NK-2 is able to discriminate, without the necessity of negative charges, between the zwitterionic phospholipids phosphatidylethanolamine (PE) and phosphatidylcholine (PC), the major constituents of the outer leaflet of the cytoplasmic membranes of bacteria and mammalian cells, respectively.
Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently
involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues
at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced
mass spectrometric procedures.
Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides,
respectively.
Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated
that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys, Cys7-Cys, Cys-Cysfor saposin B and Cys5-Cys, Cys8-Cys, Cys-Cysfor saposin C.
We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the
lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the
reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on
glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results
stress the importance of the disulfide structure for the functional properties of the saposin.
Study of effects of arginineevasopressin (AVP) and its functional analogues on the contractile vacuole of the amoeba Amoeba proteus has esished that AVP (10 µM) increases frequency of vacuole contractions. This effect is reproduced by antagonist of receptors of the V 2 type desmopressin (10 µM), but is suppressed by antagonist of receptors of the V 1 type. Apart from desmopressin, acceleration of the vacuole contraction is also caused by activator of adenylyl cyclase forskolin (25 µM). Cyclic adenosine monophosphate (cAMP, 1 µM) produces a dual effect on the vacuole contraction: it either increases its frequency like AVP, desmopressin, and forskolin, or suppresses the AVP effect without producing its own action. The nonnselective blocker of adenosine receptors aminophylline inhibits the AVPPlike effect of cAMP. This indicates either effect of cAMP on adenosine receptors or conversion of cAMP into adenosine through the action of 55ectonucleotidase. In either case, we are dealing with extracellular cAMP receptors, which have been earlier established in the amoeba Dictyostelium discoideum. Results of our experiments indicate specificity of the effect of arginineevasopressin as well as a mixed character of signal transduction from membrane areas sensitive to AVP and combining features of receptors both of the V 1 and of the V2 type.
Some amoeboid protozoans are facultative or obligate parasites in humans and bear an enormous cytotoxic potential that can result in severe destruction of host tissues and fatal diseases. Pathogenic amoebae produce soluble pore-forming polypeptides that bind to prokaryotic and eukaryotic target cell membranes and generate pores upon insertion and oligomerization. This review summerizes the current knowledge of such small protein toxins from amoebae, compares them with related proteins from other species, focuses on their three-dimensional structures, and gives insights into divergent activation mechanisms. The potential use of pore-forming toxins in biotechnology will be briefly outlined.
Evidence has been presented suggesting that during assembly of the membrane attack complex (MAC) of complement, the C5b-8 complex induces polymerization of C9. The C9 polymer was detected by sodium dodecyl sulfate (SDS) gel electrophoresis of MAC isolated from complement-lysed erythrocytes. It resembled the previously described polymerized C9 (poly C9) produced from isolated monomeric C9 by prolonged incubation at 37 degrees C in that it was resistant to dissociation by SDS and reducing agents and had an apparent molecular weight of approximately 1.1 million. The presence of poly C9 in the MAC was further supported by the expression of identical neoantigens by the MAC and poly C9 and by the high C9 content of the MAC relative to its other constituents. Isolated C8 in solution was found to have a single C9-binding site. In mixture, the two proteins formed a reversible equimolar complex that had a sedimentation coefficient of 10.5S. In contrast, a single, cell-bound C5b-8 complex was found to bind up to 12-15 C9 molecules and clusters of C5b- 8 bound 6-8 C9 molecules per C8 molecule. In either case, typical ultrastructural membrane lesions were observed, suggesting that the membrane lesion is identical with the tubular poly C9 consisting of 12-16 C9 molecules, and that the MAC can have either the composition (C5b-8)polyC9 or (CSb-8)(2)polyC9. When C9 input was restricted so that the molar C9/C8 ratio was less than or equal to 3, C9-induced aggregates of C5b-8 were observed but virtually no circular membrane lesions were found. We suggest, therefore, that C9, at low dosage, causes cross-linking of multiple C5b-8 complexes within the target membrane and that, at high dosage, C9 is polymerized by C5b-8 to form a transmembrane channel within the MAC assembly. It is primarily the C9 polymer that evokes the ultrastructural image of the MAC or of membrane lesions caused by complement.
The D enantiomers of three naturally occurring antibiotics--cecropin A, magainin 2 amide, and melittin--were synthesized. In addition, the D enantiomers of two synthetic chimeric cecropin-melittin hybrid peptides were prepared. Each D isomer was shown by circular dichroism to be a mirror image of the corresponding L isomer in several solvent mixtures. In 20% hexafluoro-2-propanol the peptides contained 43-75% alpha-helix. The all-D peptides were resistant to enzymatic degradation. The peptides produced single-channel conductances in planar lipid bilayers, and the D and L enantiomers caused equivalent amounts of electrical conductivity. All of the peptides were potent antibacterial agents against representative Gram-negative and Gram-positive species. The D and L enantiomers of each peptide pair were equally active, within experimental error. Sheep erythrocytes were lysed by both D- and L-melittin but not by either isomer of cecropin A, magainin 2 amide, or the hybrids cecropin A-(1-13)-melittin-(1-13)-NH2 or cecropin A-(1-8)-melittin-(1-18)-NH2. The infectivity of the bloodstream form of the malaria parasite Plasmodium falciparum was also inhibited by the D and L hybrids. It is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.
A membrane-bound cytolytic pore-forming protein (N-PFP) produced by the pathogenic ameboflagellate Naegleria fowleri was characterized. N-PFP was solubilized from ameba membranes by detergent and enriched 300-fold by gel filtration chromatography. When analyzed by gel electrophoresis, N-PFP migrates with a molecular mass of 66 kDa and 50-54 kDa, under reducing and non-reducing conditions, respectively. In addition to lysing erythrocytes, N-PFP is cytotoxic to several tumor cell lines tested. Its hemolytic activity is not dependent on the presence of divalent cations. N-PFP rapidly depolarizes the membrane potential of microelectrode-impaled chicken embryo myocytes, suggesting that functional channel formation may represent the mode of membrane damage. In planar bilayers, N-PFP forms ion channels with heterogeneous unit conductances ranging between 150 and 400 picosiemens in 0.1 M NaCl and that are relatively resistant to closing by high voltages. Upon heat treatment (75 degrees C, 30 min), N-PFP forms channels with unit conductances that are on average larger than those formed by untreated N-PFP. N-PFP channels are slightly more permeable to cations than to anions. Using a liposome swelling-shrinkage assay, the functional diameter of N-PFP channels is estimated to range between 3.6 and 5.2 nm. N-PFP is immunologically distinct from the PFP/perforin produced by lymphocytes, the terminal components of complement and a PFP from the ameba Entamoeba histolytica, all of which produce pores on target membranes. This protein may have a direct lytic role during target cell killing mediated by N. fowleri.
Five structurally related heptadecapeptides rich in hydrophobic amino acids have been discovered in the venom of the bumblebee Megabombus pennsylvanicus. We have named them bombolitin I (Ile-Lys-Ile-Thr-Thr-Met-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin II (Ser-Lys-Ile-Thr-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin III (Ile-Lys-Ile-Met-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin IV (Ile-Asn-Ile-Lys-Asp-Ile-Leu-Ala-Lys-Leu-Val-Lys-Val-Leu-Gly-His-Val-NH2 ), and bombolitin V (Ile-Asn-Val-Leu-Gly-Ile-Leu-Gly-Leu-Leu-Gly-Lys-Ala-Leu-Ser-His-Leu-NH2 ). Bombolitins are structurally and functionally very similar. They lyse erythrocytes and liposomes, release histamine from rat peritoneal mast cells, and stimulate phospholipase A2 from different sources. The threshold dose is 0.5-2.5 micrograms/ml depending on the peptide and the bioassay. Bombolitin V is as potent as the well-known melittin in lysing guinea pig erythrocytes (ED50 = 0.7 microgram/ml = 4 X 10(-7) M) and is 5 times more potent than mastoparan in causing mast cell degranulation, making it one of the most potent degranulating peptides discovered so far (ED50 = 2 micrograms/ml = 1.2 X 10(-6) M). The bombolitins represent a unique structural class of peptides but they have the same biological properties as melittin (from honeybees), mastoparan (wasps, hornets, and yellow jackets), and crabrolin (European hornets). This unusual circumstance (peptides with different amino acid sequences having the same biological properties) may be a manifestion of their amphiphilic nature, a property these peptides have in common.
The direct interaction between phospholipids and sarcotoxin IA, a potent bactericidal protein of Sarcophaga peregrina, was studied using authentic sarcotoxin IA, its synthetic derivatives, and various liposomes. Results showed that sarcotoxin IA interacted with liposomes constituted from acidic phospholipids, resulting in the release of glucose trapped in these liposomes. The amidated carboxyl-terminal of this protein was found to be important for this interaction. Liposomes constituted from total phospholipids of Escherichia coli became less susceptible to sarcotoxin IA with an increase in their cholesterol content. Since bacterial membranes do not contain cholesterol, this finding may partly explain the selective toxicity of sarcotoxin I to bacteria.
We describe the partial purification and characterization of a pore-forming material (PEM) from Entamoeba histolytica. The formation of ion channels by PFM was examined in three systems. (a) PFM depolarizes J774 macrophages and mouse spleen lymphocytes as measured by [3H]TPP+ uptake. (b) PFM induces rapid monovalent cation flux across the membrane of phosphatidylcholine-cholesterol vesicles. (c) PFM confers a voltage-dependent conductance to artificial planar bilayers, which is resolved as a summation of opening of individually conducting steps of 67 pS in 0.1 M KCl. Monomers of PFM are functional; however, a preferential aggregation occurs in the planar bilayer. Activity is pronase, trypsin, and heat sensitive and is stable between pH 5-8. PFM is not secreted by unstimulated amoebae but after exposure to the calcium ionophore A23187, concanavalin A, and E. coli lipopolysaccharide, 5-10% of the total cell content of PFM is released into the medium within 5-10 min. High-performance gel filtration results in an approximately 1,000-fold purification of PFM and gives an Mr of 30,000. This protein may play a role in the cytotoxicity mediated by E. histolytica.
The ninth component of complement (C9) polymerizes to tubules either spontaneously at a very slow rate (3 days at 37 degrees C) or within minutes if induced by C5b-8 as part of the membrane attack assembly (Tschopp, J., Podack, E. R., and Müller-Eberhard, H. J. (1984) J. Immunol., in press). The spontaneous formation of tubular sodium dodecyl sulfate-resistant C9 polymers was completely inhibited in the presence of 1 mM EDTA. In contrast, the presence of Cu2+, Zn2+, and Cd2+ accelerated C9 polymerization. Zn2+ was the most efficient metal ion. At a molar ratio of one Zn2+ per monomeric C9, all C9 polymerized within 2 h. At Zn2+ concentrations above 10(-3) M, tubular C9 formation was inhibited due to nonspecific aggregation of C9. It is concluded that the presence of metal ions is required for the formation of tubular poly C9 and that Zn2+ in particular catalyzes C9 polymerization.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The molecular mechanisms underlying the various effects of melittin on membranes have not been completely defined and much of the evidence described indicates that different molecular mechanisms may underlie different actions of the peptide. Ideas about the formation of transbilayer aggregates of melittin under the influence of a transbilayer potential, and for bilayer structural perturbation arising from the location of the peptide helix within the head group region of the membrane have been made based on the crystal structure of the peptide, the kinetics and concentration dependence of melittins membrane actions, together with simple ideas about the conformational properties of amphipathic helical peptides and their interactions with membranes. Physical studies of the interaction of melittin with model membranes have been useful in determining the potential of the peptide to adopt different locations, orientations and association states within membranes under different conditions, but the relationship of the results obtained to the actions of melittin in cell membranes or under the influence of a membrane potential are unclear. Experimental definition of the interaction of melittin with more complex membranes, including the erythrocyte membrane or in bilayers under the influence of a transmembrane potential, will require direct study in these membranes. Experiments employing labeled melittins for ESR, NMR or fluorescence experiments are promising both for their sensitivity (ESR and fluorescence) and the ability to focus on the peptide within the background of endogenous proteins within cell membranes. The study of melittin in model membranes has been useful for the development of methodology for determination of membrane protein structures. Despite the structural complexity of integral membrane proteins, it is interesting that in some respects their study be more straightforward, lacking as they do the elusive properties of melittin (and other structurally labile membrane peptides) which limit the possibility of defining their interaction with membranes in terms of a single conformation, location, orientation and association state within the membrane.
Protozoan parasite T. cruzi invades cells within acidic vacuoles, but shortly afterward escapes into the cytosol. Exit from the phagosome is blocked by raising the pH of acidic compartments, suggesting that a previously described acid-active hemolysin secreted by T. cruzi might be involved in the membrane disruption process. Here we show that T. cruzi supernatants are cytotoxic for nucleated cells at pH 5.5 and contain a protein reactive with antibodies against reduced and alkylated human C9 (the ninth component of complement). The C9 cross-reactive protein (TC-TOX) copurified with the cytolytic activity, and the active fractions induced conductance steps characteristic of transmembrane ion channels in planar phospholipid bilayers. Immunocytochemical studies using antibodies against purified TC-TOX showed that the protein was localized to the luminal space of parasite-containing phagosomes. We postulate that TC-TOX, when secreted into the acidic environment of the phagosome, forms pores in the membrane, which contribute to its disruption.
The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.
Molecular modeling and energy calculations have been used to study how delta-hemolysin and melittin helices may aggregate on membrane surfaces and insert through membranes to form channels. In these models adjacent antiparallel amphipathic helices form planar "raft" structures, in which one surface is hydrophobic and the other hydrophilic. Models of delta-hemolysin crystal structure were developed using these "rafts." These models are based on the unit cell constants and the crystal symmetry obtained from the preliminary crystal data. Energy calculations favor channel models of delta-hemolysin with six or eight monomers per channel.
The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.
The association of the peptide melittin with small unilamellar DMPC vesicles was studied as a function of pH. The results are discussed quantitatively assuming a water-membrane partition equilibrium. Electrostatic surface charging is taken into account as more and more of the strongly basic peptide accumulates at the bilayer/water interface. The data could be well described in terms of a Gouy-Chapman approach involving an effective interfacial charge well below the actual physical charge carried by the individual peptide molecules. The partition coefficient turned out to be pH invariant, so that one can exclude deprotonation reactions upon insertion of the peptide into the bilayer. The effective interfacial charge per associated melittin molecule decreased over a broad range of pH (pH 7 to pH above 10). Contributions of the free amino terminus and of the arginine residues could be determined by comparing with results obtained using modified melittin (N-terminally formylated and fully acetylated). The data suggest approximately equal fractional contributions of the amino terminus and the three lysines to the effective interfacial charge. The two arginines contribute less. Thus, they may be located farther away from the interface or be closely associated with counter-ions. The analysis is extended to the effect of different ionic strengths.
This review summarizes the molecular events resulting in the formation of membrane lesions by lymphocytes and by complement and our knowledge of additional sytolytic factors. It will become apparent that there are striking analogies in the morphology and function of the tubular complexes, their mode of assembly, as well as their utilization as transmembrane channels for entry of cytolytic factors.
A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.
Ion channel proteins are important for the conduction of ions across biological membranes. Recent analyses of their sequences have suggested that they are composed of bundles of alpha-helices that associate to form ion-conducting channels. To gain insight into the mechanisms by which alpha-helices can aggregate and conduct ions, three model peptides containing only leucine and serine residues were synthesized and characterized. A 21-residue peptide, H2N-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, which was designed to be a membrane-spanning amphiphilic alpha-helix, formed well-defined ion channels with ion permeability and lifetime characteristics resembling the acetylcholine receptor. In contrast, a 14-residue version of this peptide, which was too short to span the phospolipid bilayer as an alpha-helix, failed to form discrete, stable channels. A third peptide, H2N-(Leu-Ser-Leu-Leu-Leu-Ser-Leu)3-CONH2, in which one serine per heptad repeat was replaced by leucine, produced proton-selective channels. Computer graphics and energy minimization were used to create molecular models that were consistent with the observed properties of the channels.
The delta-toxin of Staphylococcus aureus has been investigated in terms of its potential to form ion channels in planar lipid bilayers formed at the tip of patch electrodes. Channel formation has been shown to occur for delta-toxin concentrations in the range 0.1 to 2.0 microM. In 0.5 M KCl, two major classes of channels were seen--'small' with conductances of 70-100 pS, and 'large' with a conductance of approx. 450 pS. Current-voltage relationships for lipid bilayers containing several delta-toxin channels revealed both voltage-dependent and independent components to channel gating. Reversal potential measurements showed the channels to be cation selective. In the presence of 3.0 M KCl, the channel gating kinetics were complex, with multiple open and closed states. The results are interpreted in terms of a model for the channel consisting of a hexameric cluster of alpha-helical delta-toxin molecules.
A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and--because there is no protein fixation step--nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Schägger and G. Von Jagow (1987) Anal. Biochem. 166, 368-379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected.
Entamoeba histolytica kills cells by contact dependent cytolysis. The mechanism underlying this process must be of rapid onset because target cells round up and show marked zeiosis within 15 min of contact. In earlier work, we identified a remarkable ion-channel forming protein which we named amoebapore, that may contribute to the amoeba-induced target cell killing. Within the amoeba it exists as part of a supramolecular aggregate together with other proteins of unknown function. In this work we report the purification of a solubilized form of the amoebapore. Amoebapore was found to exist as an apparent dimer of the previously reported protein whose molecular weight had been determined under denaturing conditions. Two isoforms of this dimer, with pI values of 6.8 and 5.3 present at a ratio of 7 to 1, were identified and purified. Both isoforms demonstrate ion-channel forming activity in planar lipid membranes. These channels show a unit conductance of 5-20 pS and remain open for less than 1 s. Upon lateral aggregation, opening becomes concerted to a greater degree with channel conductance are observed. The isolated particulate form of amoebapore depolarizes cells.
Pore-forming protein from Entamoeba histolytica forms cation-selective channels in planar bilayers. With increasing potentials, the open-state probability of these channels decreases, and channel aggregates collapse (Young, J.D.-E. and Cohn, Z.A. (1985) J. Cell. Biochem. 29, 299-308). In this communication we report the following observations: (i) incorporation of the pore in black-lipid membranes was stimulated by membrane potential, (ii) pores were rectifying, (iii) breakdown of pores resulted in a continuous spectrum of subconductance states, (iv) the open-state probability increased strongly with pH. This pattern of behaviour is similar to that of the barrel-stave aggregates (alamethicin and related toxins). We therefore conclude that the amebal pores, like those of the barrel-stave class, may consist of complexes involving variable numbers of membrane-spanning subunits.
Several proteins and polypeptides of reptilian, amphibian, insect, and microbial origin share a common cytolytic property. However, these cytolysins fulfill different objectives. They provide offensive armament in the case of toxins, but defensive systems in the case of antibacterial peptides. The sequences of several nonenzymatic cytolysins and their analogues were compared to identify the structural requirements for cytolytic activity. These cytolysins, although isolated from phylogenetically unrelated organisms, possess the common sequence features of a cationic site flanked by a hydrophobic surface. The presence of such a region apparently confers the cytolytic activity of various cytolysins. The concept of a cytolytic region is strongly supported by the existence of several natural and synthetic analogues of cytolysins and by chemical modification studies of these cytolysins. This prediction provides a new focus for cytolysin research. The understanding of this structure-function relationship should facilitate the design, synthesis, and development of better antibacterial and anticancer peptides.
In order to investigate structure-activity relationships of cecropins, model peptides that mimic certain structural features of the cecropin molecules were designed and synthesized. The conformational analysis of cecropins and the design of the model peptides were based on Chou-Fasman calculations. The peptides were synthesized by solid-phase methods and purified by reverse-phase liquid-chromatography on C18-silica columns. Their secondary structures were studied by circular dichroism measurements. Antibacterial activities against seven test organisms were determined and compared to the activities of the natural cecropins A and B. These results were discussed on the basis of structural features of the model peptides and on model mechanisms. It was concluded that high antibacterial activity for this class of compounds requires a basic helical amphipathic N-terminal segment that is connected to a hydrophobic helical C-terminal segment by a flexible non-helical hinge region.
Entamoeba histolytica is the human pathogen responsible for amebiasis. This infection is characterized by an invasive enteric illness that may spread to multiple organs. The parasite E histolytica may now be cultivated in vitro, with Diamond's axenic medium [1]. In culture, this protozoan remains cytolytic to a variety of cell types, including neutrophils and macrophages (reviewed in [2]). The mechanism(s) involved in the expression of this potent cytolysis remain(s) unclear. Previous studies from several laboratories have suggested that the cell killing mediated by amoeba is dependent on intimate contact of amoeba with the target cell membrane [3–9]. Following contact, the amoeba readily ingests the target cell (Fig. 1A). However, more recent cinemicroscopic and kinetic studies [8,9] (see also Fig. 1B) indicate that the cytolysis mediated by amoeba may occur prior to phagocytosis, raising the possibility of an extracellular cytolytic event triggered by surface contact.
Previous work from this laboratory has described the properties of a highly enriched plasma membrane fraction and surface polypeptides from axenically grown E histolytica [10,11]. Studies from this laboratory on its extensive vacuolar apparatus and its role in pinocytosis and intracellular acidification have also been published [12,13]. Here, we review some recent evidence for the isolation and characterization of a pore-forming protein (PFP) extracted from this protozoan, which may be closely related to the killing of target cells mediated by pore-forming proteins of the immune system (the complement cascade as well as proteins extracted from granules of different immune cells [Young et al, in preparation]), which may thereby suggest a very similar and general mechanism of cell-mediated cytotoxicity.
When Escherichia coli was treated with sarcotoxin I, a potent bactericidal protein of Sarcophaga peregrina (fleshfly), K+ inside of the cells leaked out rapidly and the ATP pool of the cells rapidly decreased. These results suggested that the bactericidal effect of sarcotoxin I was due to its ionophore activity, and that it blocked the generation of ATP by inhibiting formation of the proton gradient essential for oxidative phosphorylation. This was confirmed by use of an uncA mutant, which was much less susceptible than the wild-type strain to sarcotoxin I under fixed ionic conditions.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
We have identified a remarkable ion-channel forming material in virulent strains of Entamoeba histolytica that may be responsible for many of the symptoms associated with amoebic dysentery. A polypeptide that we refer to as amoebapore is shed into the growth media and is also found within the amoeba in a high speed sedimentable fraction. Amoebapore has the distinctive property of spontaneously incorporating into lipid bilayers, liposomes, and cells, leading to progressive and irreversible changes in the ion conductance of the target membranes. Exposure of planar lipid bilayers to amoebapore -containing fractions under voltage clamp conditions results in an almost immediate and progressive incorporation of ion channels which continues in an irreversible manner leading to a fall in membrane impedance of up to five orders of magnitude. The ion-channel conductance is moderately cation-selective, voltage dependent, and displays a unit size of 1.6 +/- 0.2 nanoSiemens in 1 M KCl at -10 mV. In the bilayer, the amoebapore -induced conductance exhibits an in situ sensitivity to protease. Amoebapore is mainly concentrated in a fraction sedimenting at 150 000 g. It is insoluble in Triton X-100 but can be dissociated in an active state in 1% SDS. Under these conditions it has an apparent mol. wt. of 13 000 daltons.
A valinomycin-mediated K+ diffusion potential across the membrane of multilamellar liposomes is stable for longer than 30 min and can be collapsed by a nonselective channel such as gramicidin. The kinetics of the potential collapse are complex but can be qualitatively broken down into a series of processes involving (1) binding of the gramicidin to the outer membrane, (2) dimerization to form a functional channel, (3) the flow of ions through the channel, (4) the establishment of a new diffusion potential on the next bilayer within the multilamellar liposome, and (5) the dissociation of gramicidin from the outer bilayer into the adjacent internal aqueous space. These processes are then repeated, in turn, for all the internal bilayers until the K+ concentration gradient (and membrane potential) is completely dissipated. Process 5 appears to be rate limiting at high gramicidin concentrations, but ion flux, process 3, becomes slower at low gramicidin concentrations where the collapse of the K+ gradient displays voltage dependence. Of course the rates of these processes can also be manipulated by changing the composition or size of the liposome and by varying the ion concentrations. Since the diffusion potential can be conveniently monitored with a voltage-sensitive fluorescent dye, 3,3'-diethylthiodicarbocyanine iodide [diS-C2-(5)], a simple method for the detection and partial characterization of membrane pores emerges from this investigation.
Immune responses have been described for many different insect species. However, it is generally acknowledged that immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia. The expression of this multicomponent system requires de novo synthesis of RNA and proteins and its broad antibacterial activity is due to at least three independent mechanisms, the most well known of which is the insect lysozyme. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. WE recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacterial activity against Escherichia coli and several other Gram-negative bacteria. We believe that P9A and P9B plays an important part in the humoral immune responses described previously and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom which lyses both bacteria and eukaryotic cells.
The ninth component of complement C9 can undergo circular polymerization in the fluid phase and on lipid membranes. The concomitant hydrophilic-amphiphilic transition is the result of a conformational reorganization of C9 and allows insertion of poly C9 into membranes in the form of a transmembrane protein channel. The ultrastructure of poly C9 resembles that of membrane lesions caused by complement.
A reconstitution procedure for a glucose-transporting protein based on a rapid freezing of sonicated phospholipid mixtures with the solubilized protein followed by thawing and a brief sonication was described by Kasahara and Hinkle (1977, J. Biol. Chem.252, 7384–7390). In this paper we demonstrate that the liposomes prepared by this simple procedure referred to as the freezing-thawing-sonication procedure have a specific trapping volume of 10 μl H2O/mg phospholipid and a maximal trapping efficiency of 25–30% of the solution including ions, small organic molecules, or proteins. Formation of large liposomes by this technique which probably results from fusion of sonicated liposome is strongly inhibited by increasing the ionic strength of the medium, by sucrose, and by increasing the liposome concentration. The freezing-thawing-sonication procedure is effective with a crude phospholipid extract obtained from soybeans or with mixtures of phosphatidylcholine with either a negatively charged phospholipid (phosphatidylserine) or a positively charged lipid (cetyltrimethylammonium bromide) but not with pure phosphatidylcholine liposomes. Based on these findings, we suggest that water molecules have to crystallize on the charged phospholipid interface during the rapid freezing in order to allow the fusion process to take place. Liposomes prepared by the freezing-thawing-sonication procedure have a low permeability to protons, cations, and anions which makes them a useful system for studies of ion transport. We give a few examples of ionophore-mediated ion transport reactions which give rise to accumulation of protons and Ca ions inside the lipsomes.
Over the past decade or so, pore-forming proteins (PFPs) have been isolated from various immune cells and nonpathogenic bacteria. It is now becoming apparent that PFPs may also be produced by a number of parasites. Although far from definitive, the evidence currently available for the role of PFPs in the survival and pathogenesis by parasites in briefly presented by David Ojcius and John Ding-E Young.
- G Kreil
Kreil, G. (1973) FEBS Lett. 33, 241-244.
- I Rosenberg
- D Bach
- L M Loew
- C Gitler
Rosenberg, I., Bach, D., Loew, L. M. & Gitler, C. (1989) Mol.
Biochem. Parasitol. 33, 237-248.
- J D Young
- E Cohn
Young, J. D.-E. & Cohn, Z. A. (1985) J. Cell. Biochem. 29,
299-308.
- J D Young
- E Lowrey
Young, J. D.-E. & Lowrey, D. M. (1989) J. Biol. Chem. 264,
1077-1083.
- H Schagger
- G Von Jagow
Schagger, H. & von Jagow, G. (1987) Anal. Biochem. 166,
368-379.
- R A Parente
- L Nadasni
- N K Subbarao
- F C Szoka
- Jr
Parente, R. A., Nadasni, L., Subbarao, N. K. & Szoka, F. C.,
Jr. (1990) Biochemistry 29, 8713-8719.
- J D E Young
- T M Young
- L P Lu
- J C Unkeless
- Cohn
Young, J. D.-E., Young, T. M., Lu, L. P., Unkeless, J. C. &
Cohn, Z. A. (1982) J. Exp. Med. 156, 1677-1690.
- Y Nakajima
- X Qu
- S Natori
Nakajima, Y., Qu, X. & Natori, S. (1987) J. Biol. Chem. 262,
1665-1669.
- U Pick
Pick, U. (1981) Arch. Biochem. Biophys. 212, 186-194.
- D Marion
- M Zasloff
- A Bax
Marion, D., Zasloff, M. & Bax, A. (1988) FEBS Lett. 227,
216-226.
- S Stankowski
- G Schwarz
Stankowski, S. & Schwarz, G. (1990) Biochim. Biophys. Acta
1025, 164-172.