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Activation of purified proMP2 Xa by bovine Factor Xa. (A) Purified recombinant proMP2 Xa (350 ng) was incubated with Factor Xa (200 ng) at 37uC for 1 h, and the mixtures were separated by 10% SDS-PAGE followed by immunoblot analysis using Anti-His antibodies (diluted 1:1000). The sizes and positions of molecular weight standards are indicated on the right. Circle, arrow, and diamond indicated proMP2 Xa , catalytic domain of proMP2 Xa , and non-specific cleavage of proMP2 Xa , respectively. (B) Amidase activity assay of activated MP2 Xa using IEARpNA as a substrate, as described under ''Materials and methods''. The bars represent mean 6 S.D. (n = 3). Bars labeled with different letters (a, b, and c) are significantly different (analysis of oneway ANOVA followed by Newman-Keuls test, P,0.05). doi:10.1371/journal.pone.0079533.g002
Source publication
In arthropods, melanization plays a major role in the innate immune response to encapsulate and kill the invasive organisms. It is mediated by a serine protease cascade and is regulated by serpins. The identification of the molecular components of melanization and the regulation of those components are still unclear in Drosophila melanogaster, alth...
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... possessed clip domain as well as a catalytic triad with the characteristics of active serine proteases. A phylogenetic analysis was then performed based on the deduced amino acid sequences of these 26 selected Drosophila sequences and others known to be involved in activation of melanization reaction in other species. As shown in Fig. S1 and Fig. S2, many of the clades in the phylogeny have low support. One clade, with a bootstrap value of 34, includes Drosophila MP2 (CG3066), An. gambiae CLIPB9 and H. diomphalia PPAF1. Another clade, with a bootstrap value of 52, groups Drosophila CG9737 with M. sexta PAP-2 and -3, and with B. mori PPAE. All of the proteases that cluster with ...
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... detect activation of proMP2 Xa by Factor Xa, we performed immunoblot analysis and amidase activity assays (Fig. 2). Incuba- Figure 1. SDS-PAGE analysis of recombinant proMP2 Xa , proMP2, Spn27A and PPO1. The purified recombinant proMP2 Xa (0.7 mg), proMP2 (0.99 mg) and Spn27A (2.6 mg) were treated with SDS sample buffer containing b-mercaptoethanol and separated by 10% SDS-PAGE followed by coomassie brilliant blue staining. Recombinant PPO1 (1.3 ...
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... PPO1 (1.3 mg) was separated by 7.5% SDS-PAGE. The sizes and positions of the molecular mass standards are indicated on the right. doi:10.1371/journal.pone.0079533.g001 tion of purified proMP2 Xa with Factor Xa resulted in decreased intensity of the 49-kDa zymogen band and appearance of a 36- kDa band corresponding to the catalytic domain ( Fig. 2A), as expected for activation. This result indicates that Factor Xa effectively cleaved proMP2 Xa . In addition to the 36-kDa band, a band with apparent molecular weight of ,30 kDa was detected by anti-His antibodies. This 30-kDa band might be due to the non- expected cleavage of proMP2 Xa by Factor Xa, as this band also appeared in the ...
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... confirm the activation of proMP2 Xa by Factor Xa, we assayed for amidase activity of MP2 Xa using the colorimetric substrate IEARpNA. A significant increase in activity (above that of Factor Xa alone) was observed in the presence of MP2 Xa that was activated by Factor Xa (Fig. 2B), thus confirming activation of proMP2 Xa ...
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... that Drosophila is especially appropriate for such continued studies, because of the wide range of genetic and molecular genetic tools available in that system. In combination with the expression of recombinant proteins for biochemical studies, these genetic and molecular genetic tools will allow deeper understanding of PPO activation cascade in (Fig. S1, Fig. S2 and Ref [47]). Among them, in our phylogenetic tree MP2 is clustered with An. gambiae CLIPB9 and H. diomphalia PPAF1, which are both known to function in melanization ( Fig. S1 and Ref [24,25]). MP2 has been suggested to be involved in PPO activation because overexpression of activated MP2 resulted in constitutive melanization in ...
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... absolutely conserved cysteines in clip domain are bold-italicized and numbered. The paired numbers (1-1, 2-2, 3-3) indicate the intramolecular disulfide linkage. Another two cysteines with numbers indicate the disulfide linkage between clip domain and catalytic domain, which remains two domains connected after cleavage at the activation site. ...
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Citations
... A Drosophila model pathway, obtained from a study on the homologous recognition of innate signalling pathways [36], was used as a reference for the genetic components of the Toll/IMD and JAK/STAT pathways. The eicosanoid biosynthetic pathway used a model pathway for insects from a study of eicosanoid signalling in insect immunology by Kim and Stanley [37], while the melanization pathway used a model for the activation of melanization in Drosophila when compared with Manduca sexta and Tenebrio molitor [38]. The apoptosis pathway was based on a cell death model described for Drosophila [39], and RNA interference used the model pathway described for eukaryotes in a study of innate and adaptive resistance to RNAi [40]. ...
The tomato spotted wilt virus (TSWV) is a member of the Tospoviridae family and has an negative/ambisense single-stranded RNA genome. Frankliniella occidentalis and F. intonsa are known to be dominant pests in Capsicum annuum (hot pepper) and can cause damage to the plant either directly by feeding, or indirectly by transmitting TSWV in a persistent and propagative manner, resulting in serious economic damage. This study compared the immune responses of two different thrips species against TSWV infection by transcriptome analysis, which then allowed the assessment of antiviral responses using RNA interference (RNAi). Both adult thrips shared about 90 % of the transcripts in non-viruliferous conditions. Most signal components of the immune pathways were shared by these two thrips species, and their expression levels fluctuated differentially in response to TSWV infection at early immature stages. The functional assays using RNAi treatments indicated that the Toll and JAK/STAT pathways were associated with the antiviral responses, but the IMD pathway was not. The upregulation of dorsal switch protein one supported its physiological role in recognizing TSWV infection and triggering the eicosanoid biosynthetic pathway, which mediates melanization and apoptosis in thrips. In addition, the signal components of the RNAi pathways fluctuated highly after TSWV infection. Individual RNAi treatments specific to the antiviral signalling and response components led to significant increases in the TSWV amount in the thrips, causing virus-induced mortality. These findings suggest that immune signalling pathways leading to antiviral responses are operating in the thrips to regulate TSWV litres to prevent a fatal viral overload. This study also indicates the differential antiviral responses between the TSWV-transmitting F. occidentalis and F. intonsa .
... The upstream cascade members that induce Toll activation also participate in melanization response (6). Although there is no direct evidence, it has been proposed that the PRR-Toll cascade likely diverges downstream of Psh and Hayan to activate Sp7 (6), which then activates PPO1 (20,21) to combat invading microbes with PO-generated reactive compounds. Note that psh and Hayan arose from a recent gene duplication and alternative splicing can produce Hayan transcripts similar to psh mRNA (6). ...
... We conducted immunoblot analyses using anti-V5 antibody to investigate the activation mechanisms of Sp7, SPE, and their paralogs cSP5, cSP11, MP1, and Ser7. Recombinant proSp7 migrated as a doublet with apparent molecular masses of 48 and 52 kDa (Fig. 1B), likely due to differences in glycosylation as its sequence contains 10 predicted glycosylation sites (21). After incubation with Grass-activated Psh, Hayan-PA, or Hayan-PB, the proSp7 doublet was fully converted to a doublet around 35 kDa (Fig. 4, A and B), consistent with the expected size of the C-terminal protease domain after cleavage activation. ...
... A significant increase in activity was observed from Sp7 that was activated by Psh, Hayan-PA, or Hayan-PB (Fig. 4E), thus confirming activation of proSp7. Collectively, these experiments demonstrate that the active Psh, Hayan-PA, and Hayan-PB generated by Grass, subtilisin, or Pr1A are active against proSp7, generating active Sp7, a PPO1 activating enzyme (20,21). We then tested whether active Psh, Hayan-PA, and Hayan-PB were able to process procSP5 and procSP11 ( fig. ...
Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial peptides, respectively, to kill pathogens. These two processes are mediated by phenoloxidase (PO) and Spätzle (Spz) through an extracellular network of serine proteases. While some proteases have been identified in Drosophila melanogaster in genetic studies, the exact order of proteolytic activation events remains controversial. Here, we reconstituted the serine protease framework in Drosophila by biochemical methods. This system comprises 10 proteases, i.e., ModSP, cSP48, Grass, Psh, Hayan-PA, Hayan-PB, Sp7, MP1, SPE and Ser7, which form cascade pathways that recognize microbial molecular patterns and virulence factors, and generate PO1, PO2, and Spz from their precursors. Furthermore, the serpin Necrotic negatively regulates the immune response progression by inhibiting ModSP and Grass. The biochemical approach, when combined with genetic analysis, is crucial for addressing problems that long stand in this important research field.
... The approach combining the biochemical study of recombinant proteins can be useful for solving problems that are hard to disentangle by genetic analysis alone. For instance, bovine coagulation factor Xa activated the purified proMP2x a expressed in Drosophila S2 cells (29). The active MP2x a cleaved PPO1 from E. coli likely at the classical activation site between Arg 52 and Phe 53 (30), suggesting that MP2 is a PAP of the PPO1. ...
... The active MP2x a cleaved PPO1 from E. coli likely at the classical activation site between Arg 52 and Phe 53 (30), suggesting that MP2 is a PAP of the PPO1. Despite the fact that half of the correctly folded PPO1 was processed, no PO activity was reported in that paper (29). Based on the cofactor studies in other insects and the low PO activity generated by Drosophila MP2x a alone, we hypothesized that one or two SPHs may act as a cofactor for PPO activation in Drosophila. ...
... Drosophila MP2 acts as a PAP to directly activate PPO1 (29) and some insect PAPs also generate their own cofactors by cleaving SPHI and SPHII precursors (17, 32,35). Therefore, we activated proMP2x a by bovine factor X a and detected its catalytic domain at 34 kDa and an increase in IEARpNA hydrolysis from 1.33 U to 2.63 U ( Figure S3). ...
Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high Mr cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. In this study, we produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high Mr complexes of the cSPHs, PO at a high specific activity of 260 U/μg was generated in vitro. To complement the in vitro analysis, we measured hemolymph PO activity levels in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/μl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults.
... Serpin is cleaved at the residue site P1-P1' in the RCL when it bound to a target protease, and then forms an irreversible serpin-protease complex to inserted into the beta-sheet of the serpin protein (Reichhart et al., 2011;Guo et al., 2015). Some physiological responses are regulated by serpins in many insects, including insect immune response, insect development and melanization (Gorman et al., 2007;Reichhart et al., 2011;An et al., 2013;He et al., 2017;Yuan et al., 2017). To date, some studies indicated that increased expression of serpin might be involved in Bt toxicity and resistance by regulating the activities of trypsin and chymotrypsin. ...
... Serpins work in the inhibit the enzyme activities of trypsin and chymotrypsin in all living organisms (Kanost and Jiang, 1997), it also regulates insect immune response, insect development and melanization in many insects (Gorman et al., 2007;Reichhart et al., 2011;An et al., 2013;He et al., 2017;Yuan et al., 2017). As it reported sublethal Bt toxicity could affected the insect development , and the Bt resistance insect trade-off the development with the abilities to counter the Bt toxins (Rahman et al., 2011). ...
Insect resistance to Bacillus thuringiensis (Bt) is a critical limiting factor for applying the Bt crops. Some studies indicated that decreased protoxin activation because of lower enzymatic activities of trypsin and chymotrypsin and increased expression of serpin might involve in Bt resistance. Our previous study identified an endogenous serpin could inhibit the midgut proteases to activate Cry1Ac and reduce the insecticide activity to Helicoverpa armigera. We hypothesis that up-regulated serpin involved in resistance via inhibiting enzymatic activities of trypsin and chymotrypsin to decrease protoxin activation. Herein, we found the serpin-e gene relative expression in midgut was significantly higher in the LF30 resistant strain than that in the susceptible strain during all developmental stages. Importantly, RNAi-mediated silencing of serpin-e gene expression caused 4.46-fold mortality changes in LF30 strain, but the trypsin and chymotrypsin proteases activities were only changed 0.79-fold and 2.22-fold. In addition, although proteases activities were significantly lower in LF30 strain than that in the susceptible strain, the resistance ratios of LF30 to Cry1Ac protoxin and to activated Cry1Ac toxin were no difference. The results indicated serpins caused insect resistance to Cry1Ac protoxins partly through inhibiting the trypsin and chymotrypsin proteases activities, but it also existed other mechanisms in LF30.
... In our analysis of the Toll pathway, we found three GNBP transcripts that may play this role, as they were DE in the Gr+ bacteria treatment at 24 hpi. In other insects, GNBPs also can induce melanization in parallel with Toll pathway activation [30,33,98,99]. This occurs by the differential activation of SPs that eventually cleave Spätzle in the Toll pathway or PPO in the melanotic biochemical pathway. ...
... , b Differentially expressed (DE) transcripts of the immune serine protease (SP) cascade and tyrosine metabolism pathway of Rhodnius prolixus in response to bacterial injection. a Diagram representing the immune SP cascade of Manduca sexta (modified from An et al.[99] and Wang et al.[34]). Putative orthologs of identified SPs and SP inhibitors (SPIs) in R. prolixus are listed next to the M. sexta proteins. ...
Background
Rhodnius prolixus is an important vector of Trypanosoma cruzi , the causal agent of Chagas disease in humans. Despite the medical importance of this and other triatomine vectors, the study of their immune responses has been limited to a few molecular pathways and processes. Insect immunity studies were first described for holometabolous insects such as Drosophila melanogaster , and it was assumed that their immune responses were conserved in all insects. However, study of the immune responses of triatomines and other hemimetabolous insects has revealed discrepancies between these and the Drosophila model.
Methods
To expand our understanding of innate immune responses of triatomines to pathogens, we injected fifth instar nymphs of R. prolixus with the Gram-negative (Gr−) bacterium Enterobacter cloacae , the Gram-positive (Gr+) bacterium Staphylococcus aureus , or phosphate-buffered saline (PBS), and evaluated transcript expression in the fat body 8 and 24 h post-injection (hpi). We analyzed the differential expression of transcripts at each time point, and across time, for each treatment.
Results
At 8 hpi, the Gr− bacteria-injected group had a large number of differentially expressed (DE) transcripts, and most of the changes in transcript expression were maintained at 24 hpi. In the Gr+ bacteria treatment, few DE transcripts were detected at 8 hpi, but a large number of transcripts were DE at 24 hpi. Unexpectedly, the PBS control also had a large number of DE transcripts at 24 hpi. Very few DE transcripts were common to the different treatments and time points, indicating a high specificity of the immune responses of R. prolixus to different pathogens. Antimicrobial peptides known to be induced by the immune deficiency pathway were induced upon Gr− bacterial infection. Many transcripts of genes from the Toll pathway that are thought to participate in responses to Gr+ bacteria and fungi were induced by both bacteria and PBS treatment. Pathogen recognition receptors and serine protease cascade transcripts were also overexpressed after Gr− bacteria and PBS injections. Gr- injection also upregulated transcripts involved in the metabolism of tyrosine, a major substrate involved in the melanotic encapsulation response to pathogens.
Conclusions
These results reveal time-dependent pathogen-specific regulation of immune responses in triatomines, and hint at strong interactions between the immune deficiency and Toll pathways.
Graphical abstract
... Currently, PPO activating enzymes that directly cleave PPO belong to the CLIPB subfamily. For instance, cSP6/cSP8 in H. armigera (25,32), PAP1/PAP2/ PAP3 in M. sexta (15,24,37), PPAE in B. mori (38), SP13/ SP105 in O. furnacalis (39, 40), and MP2 in D. melanogaster (41). The recombinant An. gambiae CLIPB9 and CLIPB10 can cleave the purified M. sexta PPO protein in vitro (42,43). ...
Melanization is an integral part of the insect defense system and is often induced by pathogen invasion. Phenoloxidases (POs) are critical enzymes that catalyze melanin formation. PO3 is associated with the antifungal response of the mosquito, Aedes aegypti, but the molecular mechanism of the prophenoloxidase-3 (PPO3) activation is unclear. Here we report that PPO3 cleavage activation is mediated by a clip-domain serine protease, CLIPB9. We purified recombinant CLIPB9 and found that it cleaved PPO3 and increased PO activity in the hemolymph. We then identified CLIPA14 (a serine protease homolog) by co-immunoprecipitation using anti-CLIPB9 antibody. After being cleaved by CLIPB9, Ae. aegypti CLIPA14 acted as a cofactor for PPO3 activation. In addition, dsRNA co-silencing of CLIPB9 and CLIPA14 genes reduced melanization after infection with the entomopathogen, Beauveria bassiana, making the adult mosquitoes more sensitive to fungal infection. These results illustrate the roles of CLIPB9 and CLIPA14 in the PPO activation pathway and revealed the complexity of the upstream serine protease network controlling melanization.
... gambiae, M. sexta and D. melanogaster sequences. Given that CLIPB9, PAP1 and MP2 are known terminal proteases in proPO activation cascades, CLIPB10 was therefore an excellent candidate for PAP function in An. gambiae (Jiang et al., 1998;Tang et al., 2006;An et al., 2013). Using recombinant protein expressed with the baculovirus expression system, we obtained active CLIPB10 protein in vitro. ...
Humoral immune responses in animals are often tightly controlled by regulated proteolysis. This proteolysis is exerted by extracellular protease cascades, whose activation culminates in the proteolytic cleavage of key immune proteins and enzymes. A model for such immune system regulation is the melanization reaction in insects, where the activation of prophenoxidase (proPO) leads to the rapid formation of eumelanin on the surface of foreign entities such as parasites, bacteria and fungi. ProPO activation is tightly regulated by a network of so-called clip domain serine proteases, their proteolytically inactive homologs, and their serpin inhibitors. In Anopheles gambiae, the major malaria vector in sub-Saharan Africa, manipulation of this protease network affects resistance to a wide range of microorganisms, as well as host survival. However, thus far, our understanding of the molecular make-up and regulation of the protease network in mosquitoes is limited. Here, we report the function of the clip domain serine protease CLIPB10 in this network, using a combination of genetic and biochemical assays. CLIPB10 knockdown partially reversed melanotic tumor formation induced by Serpin 2 silencing in the absence of infection. CLIPB10 was also partially required for the melanization of ookinete stages of the rodent malaria parasite Plasmodium berghei in a refractory mosquito genetic background. Recombinant serpin 2 protein, a key inhibitor of the proPO activation cascade in An. gambiae, formed a SDS-stable protein complex with activated recombinant CLIPB10, and efficiently inhibited CLIPB10 activity in vitro at a stoichiometry of 1.89:1. Recombinant activated CLIPB10 increased PO activity in Manduca sexta hemolymph ex vivo, and directly activated purified M. sexta proPO in vitro. Taken together, these data identify CLIPB10 as the second protease with prophenoloxidase-activating function in An. gambiae, in addition to the previously described CLIPB9, suggesting functional redundancy in the protease network that controls melanization. In addition, our data suggest that tissue melanization and humoral melanization of parasites are at least partially mediated by the same proteases.
... CUFF.16132). Serine proteases are implicated in several other cellular processes, including innate immune signaling-notably in Toll pathway activation [31]-and the melanization response [32]. The predicted protein for PPAI009419 shares approximately 51% identity with the Culex quinquefasciatus CLIPA15 (also known as masquerade) across its sequence. ...
Background
Leishmaniasis, caused by parasites of the genus Leishmania , is a disease that affects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control require a better understanding of the key step for transmission, namely the establishment of infection inside the fly.
Methods
The aim of this work was to identify sand fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major , L. donovani and Herpetomonas muscarum , the latter being a parasite not transmitted to humans.
Results
Of the trypanosomatids studied, only L. major was able to successfully establish an infection in the host P. papatasi . However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and they did not differ from each other. The transcriptional signatures were also indistinguishable after a non-contaminated blood meal.
Conclusions
The results imply that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.
... The melanization cascade in D. melanogaster includes the clip proteases MP1, MP2, and Hayan (13,14). MP2 is a PAP for Drosophila proPO1 (15). There is not a well-characterized linkage between the protease cascades that activate proSpätzle and proPO in D. melanogaster immune responses. ...
Significance
Phenoloxidase-catalyzed melanization and Spätzle-triggered Toll signaling are critical innate immune responses of insects that are triggered by specific proteolysis. It has been unclear whether separate protease cascades or an integrated network of serine proteases coordinates these responses. Here we present evidence that hemolymph protease HP5 acts in pathways eliciting activation of both phenoloxidase and Spätzle-1 in Manduca sexta . HP5 has a unique specificity by cleaving proHP6 at His ¹¹² , resulting in HP6 activation. CLIP proteases related to HP6 from other insect species, including Drosophila Persephone, share this activation site feature with proHP6, and therefore, HP5 orthologs in other species are candidates for key roles in regulating and integrating protease cascade pathways in innate immune responses.
... CUFF.16132). Serine proteases are implicated in several other cellular processes including innate immune signalling -notably in Toll pathway activation [ [31]] and the melanisation response [ [32]]. The predicted protein for PPAI009419 shares approx. ...
Background Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control, require a better understanding of the key step for transmission namely, the establishment of infection inside the fly.
Methods In this work we wanted to identify fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, Leishmania donovani and Herpetomonas muscarum: a parasite not transmitted to humans.
Results Of these, only L. major is able to successfully establish an infection in P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and were not different from each other. They were also indistinguishable from non-contaminated blood.
Conclusions This implies that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.
