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Yersinia pestis, the causative agent of plague, is a highly lethal pathogen transmitted by the bite of infected fleas. Once ingested by a flea, Y. pestis establish a replicative niche in the gut and produce a biofilm that promotes foregut colonization and transmission. The rat flea Xenopsylla cheopis is an important vector to several zoonotic bacterial pathogens including Y. pestis. Some fleas naturally clear themselves of infection; however, the physiological and immunological mechanisms by which this occurs are largely uncharacterized. To address this, RNA was extracted, sequenced, and distinct transcript profiles were assembled de novo from X. cheopis digestive tracts isolated from fleas that were either: 1) not fed for 5 days; 2) fed sterile blood; or 3) fed blood containing ~5x108 CFU/ml Y. pestis KIM6+. Analysis and comparison of the transcript profiles resulted in identification of 23 annotated (and 11 unknown or uncharacterized) digestive tract transcripts that comprise the early transcriptional response of the rat flea gut to infection with Y. pestis. The data indicate that production of antimicrobial peptides regulated by the immune-deficiency pathway (IMD) is the primary flea immune response to infection with Y. pestis. The remaining infection-responsive transcripts, not obviously associated with the immune response, were involved in at least one of 3 physiological themes: 1) alterations to chemosensation and gut peristalsis; 2) modification of digestion and metabolism; and 3) production of chitin-binding proteins (peritrophins). Despite producing several peritrophin transcripts shortly after feeding, including a subset that were infection-responsive, no thick peritrophic membrane was detectable by histochemistry or electron microscopy of rat flea guts for the first 24 hours following blood-feeding. Here we discuss the physiological implications of rat flea infection-responsive transcripts, the function of X. cheopis peritrophins, and the mechanisms by which Y. pestis may be cleared from the flea gut., Author summary The goal of this study was to characterize the transcriptional response of the digestive tract of the rat flea, Xenopsylla cheopis, to infection with Yersinia pestis, the causative agent of plague. This flea is generally considered the most prevalent and efficient vector of Y. pestis. Because most pathogens transmitted by fleas, including Y. pestis, reside in the insect digestive tract prior to transmission, the transcriptional program induced in the gut epithelium likely influences bacterial colonization of the flea. To determine the specific response to infection, RNA profiles were generated from fleas that were either unfed, fed sterile blood, or fed blood containing Y. pestis. Comparative analyses of the transcriptomes resulted in identification of 34 infection-responsive transcripts. The functions of these differentially regulated genes indicate that infection of fleas with Y. pestis induces a limited immune response and potentially alters the insect’s behavior, metabolism, and other aspects of its physiology. Based on these data, we describe potential mechanisms fleas use to eliminate bacteria and the corresponding strategies Y. pestis uses to resist elimination. These findings may be helpful for developing targeted strategies to make fleas resistant to microbial infection and thereby reduce the incidence of diseases they spread.

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection., Author summary Protozoans of the Plasmodium genre infect red blood cells and cause malaria in humans and various other mammalian species. Estimated malaria cases are at more than 200 million, with 450,000 deaths per year, being cerebral malaria a serious complication that accounts for the majority of deaths. Neutrophils are cells that participate in host defense against pathogens. These cells use various mechanisms to kill invading microrganisms, including the release of webs of DNA, called neutrophil extracellular traps (NETs). These NETs can help control infections but can also induce tissue damage and their role in malaria and the mechanisms of NET production during malaria infection are starting to be understood. Here we show that infected red blood cells produce a cytokine, macrophage migration inhibitory factor (MIF) that stimulates neutrophils to release NETs. These NETs function to limit Plasmodium dissemination and, thus, digestion of NETs with DNAse treatment causes increased parasitemia and accelerated death in an experimental model of cerebral malaria. Our study uncovers the mechanism by which infected red blood cells stimulate neutrophils to release NETs and suggest an important participation of this process in malaria control.

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis—Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine., Author summary Blood-feeding is key to sand flies reproductive success and an important link in Leishmania spp. transmission. While the sand flies attempt to modify the bite site to enhance blood feeding success, the host’s ability to react to injury becomes compromised and could facilitate pathogen invasion. In our model, several proteins found in the saliva of the New World sand fly Lutzomyia longipalpis contribute to enhance Leishmania major pathogenesis. Among these secreted salivary molecules, endonucleases (Lundep) and hyaluronidases (LuloHya) increase parasite virulence by destroying the neutrophil traps and disrupting the integrity of the extracellular matrix, respectively, leading to the diffusion of other salivary components; allowing the Leishmania parasite to evade the host immune response and to cause an infection. Immunization against these molecules significantly reduces L. major infection in mice. These vaccine candidates are intended to disrupt a very early step in the Leishmania transmission event, the internalization of parasites in neutrophils (without being killed) and spread from the inoculation site. The work presented here highlights the relevance of vector-based vaccine development to disrupt vector-borne diseases transmission.

Only adult female mosquitoes feed on blood, while both genders take sugar meals. Accordingly, several compounds associated with blood feeding (i.e. vasodilators, anti-clotting, anti-platelets) are found only in female glands, while enzymes associated with sugar feeding or antimicrobials (such as lysozyme) are found in the glands of both sexes. We performed de novo assembly of reads from adult Aedes aegypti female and male salivary gland libraries (285 and 90 million reads, respectively). By mapping back the reads to the assembled contigs, plus mapping the reads from a publicly available Ae. aegypti library from adult whole bodies, we identified 360 transcripts (including splice variants and alleles) overexpressed tenfold or more in the glands when compared to whole bodies. Moreover, among these, 207 were overexpressed fivefold or more in female vs. male salivary glands, 85 were near equally expressed and 68 were overexpressed in male glands. We call in particular the attention to C-type lectins, angiopoietins, female-specific Antigen 5, the 9.7 kDa, 12-14 kDa, 23.5 kDa, 62/34 kDa, 4.2 kDa, proline-rich peptide, SG8, 8.7 kDa family and SGS fragments: these polypeptides are all of unknown function, but due to their overexpression in female salivary glands and putative secretory nature they are expected to affect host physiology. We have also found many transposons (some of which novel) and several endogenous viral transcripts (probably acquired by horizontal transfer) which are overexpressed in the salivary glands and may play some role in tissue-specific gene regulation or represent a mechanism of virus interference. This work contributes to a near definitive catalog of male and female salivary gland transcripts from Ae. aegypti, which will help to direct further studies aiming at the functional characterization of the many transcripts with unknown function and the understanding of their role in vector-host interaction and pathogen transmission.

Ixodes ricinus is the main tick vector of the microbes that cause Lyme disease and tick-borne encephalitis in Europe. Pathogens transmitted by ticks have to overcome innate immunity barriers present in tick tissues, including midgut, salivary glands epithelia and the hemocoel. Molecularly, invertebrate immunity is initiated when pathogen recognition molecules trigger serum or cellular signalling cascades leading to the production of antimicrobials, pathogen opsonization and phagocytosis. We presently aimed at identifying hemocyte transcripts from semi-engorged female I. ricinus ticks by mass sequencing a hemocyte cDNA library and annotating immune-related transcripts based on their hemocyte abundance as well as their ubiquitous distribution.De novo assembly of 926,596 pyrosequence reads plus 49,328,982 Illumina reads (148 nt length) from a hemocyte library, together with over 189 million Illumina reads from salivary gland and midgut libraries, generated 15,716 extracted coding sequences (CDS); these are displayed in an annotated hyperlinked spreadsheet format. Read mapping allowed the identification and annotation of tissue-enriched transcripts. A total of 327 transcripts were found significantly over expressed in the hemocyte libraries, including those coding for scavenger receptors, antimicrobial peptides, pathogen recognition proteins, proteases and protease inhibitors. Vitellogenin and lipid metabolism transcription enrichment suggests fat body components. We additionally annotated ubiquitously distributed transcripts associated with immune function, including immune-associated signal transduction proteins and transcription factors, including the STAT transcription factor.This is the first systems biology approach to describe the genes expressed in the haemocytes of this neglected disease vector. A total of 2,860 coding sequences were deposited to GenBank, increasing to 27,547 the number so far deposited by our previous transcriptome studies that serves as a discovery platform for studies with I. ricinus biochemistry and physiology.

Background Ixodes ricinus is the main tick vector of the microbes that cause Lyme disease and tick-borne encephalitis in Europe. Pathogens transmitted by ticks have to overcome innate immunity barriers present in tick tissues, including midgut, salivary glands epithelia and the hemocoel. Molecularly, invertebrate immunity is initiated when pathogen recognition molecules trigger serum or cellular signalling cascades leading to the production of antimicrobials, pathogen opsonization and phagocytosis. We presently aimed at identifying hemocyte transcripts from semi-engorged female I. ricinus ticks by mass sequencing a hemocyte cDNA library and annotating immune-related transcripts based on their hemocyte abundance as well as their ubiquitous distribution. Methodology/principal findings De novo assembly of 926,596 pyrosequence reads plus 49,328,982 Illumina reads (148 nt length) from a hemocyte library, together with over 189 million Illumina reads from salivary gland and midgut libraries, generated 15,716 extracted coding sequences (CDS); these are displayed in an annotated hyperlinked spreadsheet format. Read mapping allowed the identification and annotation of tissue-enriched transcripts. A total of 327 transcripts were found significantly over expressed in the hemocyte libraries, including those coding for scavenger receptors, antimicrobial peptides, pathogen recognition proteins, proteases and protease inhibitors. Vitellogenin and lipid metabolism transcription enrichment suggests fat body components. We additionally annotated ubiquitously distributed transcripts associated with immune function, including immune-associated signal transduction proteins and transcription factors, including the STAT transcription factor. Conclusions/significance This is the first systems biology approach to describe the genes expressed in the haemocytes of this neglected disease vector. A total of 2,860 coding sequences were deposited to GenBank, increasing to 27,547 the number so far deposited by our previous transcriptome studies that serves as a discovery platform for studies with I. ricinus biochemistry and physiology., Author Summary Ixodes ricinus transmits Lyme disease and tick-borne encephalitis in Europe. Tick-borne pathogens ingested with a blood meal persist in the tick gut and, during the next feeding stage, disseminate through the haemolymph to pass into a vertebrate host via the salivary glands. Invertebrate immunity is initiated when tick pathogen recognition molecules trigger serum or cellular signalling cascades. We aimed to identify haemocyte transcripts from semi-engorged female I. ricinus ticks by deep sequencing a haemocyte cDNA library and by annotating immune-related transcripts based on their abundance in haemocytes and their sequence divergence when compared to their arthropod homo(ortho)logues. 15,716 extracted coding sequences (CDS) were generated in total, and 327 transcripts were significantly overexpressed in haemocytes. These included those coding for scavenger receptors, antimicrobial peptides, pathogen recognition proteins, proteases, and protease inhibitors. Transcriptional enrichment of vitellogenin and lipid metabolism effectors suggests that body fat components mediate tick innate immunity. Annotation of ubiquitously distributed transcripts associated with immune function and their phylogenetic analysis provide insights into the evolution of major protein families involved in tick innate immunity.

Background Sand fly saliva plays a crucial role in establishing Leishmania infection. We identified adenosine (ADO) and adenosine monophosphate (AMP) as active pharmacologic compounds present in Phlebotomus papatasi saliva that inhibit dendritic cell (DC) functions through a PGE2/IL 10-dependent mechanism. Methodology/Principal Findings Herein, we prepared a mixture of ADO and AMP in equimolar amounts similar to those present in the salivary-gland extract (SGE) form one pair of salivary glands of P. papatasi and co-injected it with Leishmania amazonensis or L. major into mouse ears. ADO+AMP mimicked exacerbative effects of P. papatasi saliva in leishmaniasis, increasing parasite burden and cutaneous lesions. Enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect associated with IL-10 enhancement. Immunosuppressive factors COX2 and IL-10 were upregulated and failed to enhance ear lesion and parasite burden in IL 10-/- infected mice. Furthermore, nucleosides increased regulatory T cell (Treg) marker expression on CD4+CD25- cells, suggesting induction of Tregs on effector T cells (T eff). Treg induction (iTreg) was associated with nucleoside-induced tolerogenic dendritic cells (tDCs) expressing higher levels of COX2 and IL-10. In vitro generation of Tregs was more efficient in DCs treated with nucleosides. Suppressive effects of nucleosides during cutaneous leishmaniasis were mediated through an A2AR-dependent mechanism. Using BALB/c mice deficient in A2A ADO receptor (A2AR–/–), we showed that co-inoculated mice controlled infection, displaying lower parasite numbers at infection sites and reduced iTreg generation. Conclusion/Significance We have demonstrated that ADO and AMP in P. papatasi saliva mediate exacerbative effects of Leishmania infection by acting preferentially on DCs promoting a tolerogenic profile in DCs and by generating iTregs in inflammatory foci through an A2AR mechanism., Author Summary Leishmania parasites are transmitted to their vertebrate hosts by infected Phlebotomine sand flies during the blood meal of the flies. During the Leishmania transmission, the saliva is inoculated together with parasites and exhibit several pharmacological compounds that facilitate blood feeding, interfering on homeostasis and avoiding inflammation. Thus, these compounds allow the establishment of pathogen infection. We recently identified adenosine (ADO) and adenosine monophosphate (AMP) as major immunomodulatory compounds present within the Old World sand fly species Phlebotomus papatasii, which protected mice from extreme inflammatory insults. ADO limits the magnitude of immune response by displaying a potent anti-inflammatory activity. Here, we demonstrated that ADO and AMP present in Phlebotomus papatasi saliva are involved in the establishment of parasite infection. Such nucleosides act through adenosine A2A receptor (A2AR), inducing a tolerogenic profile on dendritic cells (tDC) that may generate regulatory T cells differentiation, thus leading to suppression of the immune response and parasite survival. The identification of the active salivary constituents could serve as a strategy for the development of new vaccines to control pathogen transmission.

Salivary gland homogenate (SGH) from the female mosquitoes Anopheles gambiae, An. stephensi, An. freeborni, An. dirus and An. albimanus were found to exhibit hemagglutinating (lectin) activity. Lectin activity was not found for male An. gambiae, or female Ae aegypti, Culex quinquefasciatus, Phlebotomus duboscqi, and Lutzomyia longipalpis. With respect to species-specificity, An. gambiae SGH agglutinates red blood cells (RBC) from humans, horse, sheep, goat, pig, and cow; it is less active for rats RBC, and not detectable for guinea-pigs or chicken RBC. Notably, lectin activity was inhibited by low concentrations of dextran sulfate 50-500 K, fucoidan, heparin, laminin, heparin sulfate proteoglycan, sialyl-containing glycans (e.g. 3'-sialyl Lewis X, and 6'-sialyl lactose), and gangliosides (e.g. GM3, GD1, GD1b, GTB1, GM1, GQ1B), but not by simple sugars. These results imply that molecule(s) in the salivary gland target sulfated glycans. SGH from An. gambiae was also found to promote agglutination of HL-60 cells which are rich in sialyl Lewis X, a glycan that decorates PSGL-1, the neutrophils receptor that interacts with endothelial cell P-selectin. Accordingly, SGH interferes with HL-60 cells adhesion to immobilized P-selectin. Because An. gambiae SGH expresses galectins, one member of this family (herein named Agalectin) was expressed in E. coli. Recombinant Agalectin behaves as a non-covalent homodimer. It does not display lectin activity, and does not interact with 500 candidates tested in a Glycan microarray. Gel-filtration chromatography of the SGH of An. gambiae identified a fraction with hemagglutinating activity, which was analyzed by 1D PAGE followed by in-gel tryptic digestion, and nano-LC MS/MS. This approach identified several genes which emerge as candidates for a lectin targeting sulfated glycans, the first with this selectivity to be reported in the SGH of a blood-sucking arthropod. The role of salivary molecules (sialogenins) with lectin activity is discussed in the context of inflammation, and parasite-vector-host interactions.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet., Journal Article, Research Support, N.I.H. Intramural, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Background Triatoma infestans is the main vector of Chagas disease in South America. As in all hematophagous arthropods, its saliva contains a complex cocktail that assists blood feeding by preventing platelet aggregation and blood clotting and promoting vasodilation. These salivary components can be immunologically recognized by their vector's hosts and targeted with antibodies that might disrupt blood feeding. These antibodies can be used to detect vector exposure using immunoassays. Antibodies may also contribute to the fast evolution of the salivary cocktail. Methodology Salivary gland cDNA libraries from nymphal and adult T. infestans of breeding colonies originating from different locations (Argentina, Chile, Peru and Bolivia), and cDNA libraries originating from F1 populations of Bolivia, were sequenced using Illumina technology. Coding sequences (CDS) were extracted from the assembled reads, the numbers of reads mapped to these CDS, sequences were functionally annotated and polymorphisms determined. Main findings/Significance Over five thousand CDS, mostly full length or near full length, were publicly deposited on GenBank. Transcripts that were over 10-fold overexpressed from different geographical regions, or from different developmental stages were identified. Polymorphisms were mapped to derived coding sequences, and found to vary between developmental instars and geographic origin of the biological material. This expanded sialome database from T. infestans should be of assistance in future proteomic work attempting to identify salivary proteins that might be used as epidemiological markers of vector exposure, or proteins of pharmacological interest., Author Summary Triatoma infestans is the main vector of Chagas disease in South America. As in all hematophagous arthropods, its saliva contains a complex cocktail that assists blood feeding by preventing platelet aggregation and blood clotting and promoting vasodilation. These salivary components can be immunologically recognized by their hosts and targeted with antibodies that might disrupt blood feeding. The respective antibodies can be used to detect vector exposure using immunoassays. On the other hand, antibodies may also contribute to the fast evolution of the salivary cocktail. In this work, we attempted to identify variations in the salivary proteins of T. infestans using Illumina technology that allowed identification of over five thousand proteins based on over 300 million sequences obtained from ten salivary gland libraries. This expanded sialome database from T. infestans should be of assistance in future work attempting to identify salivary proteins that might be used as epidemiological markers of vector exposure, or proteins of pharmacological interest.

Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex.

International audience; INTRODUCTION: Sand fly saliva plays an important role in both blood feeding and outcome of Leishmania infection. A cellular immune response against a Phlebotomus papatasi salivary protein was shown to protect rodents against Leishmania major infection. In humans, P. papatasi salivary proteins induce a systemic cellular immune response as well as a specific antisaliva humoral immune response, making these salivary proteins attractive targets as markers of exposure for this Leishmania vector. Surprisingly, the repertoire of salivary proteins reported for P. papatasi-a model sand fly for Leishmania-vector-host molecular interactions-is very limited compared with other sand fly species. We hypothesize that a more comprehensive study of the transcripts present in the salivary glands of P. papatasi will provide better knowledge of the repertoire of proteins of this important vector and will aid in selection of potential immunogenic proteins for humans and of those proteins that are highly conserved between different sand fly strains. METHODS AND FINDINGS: A cDNA library from P. papatasi (Tunisian strain) salivary glands was constructed, and randomly selected transcripts were sequenced and analyzed. The most abundant transcripts encoding secreted proteins were identified and compared with previously reported sequences. Importantly, we identified salivary proteins not described before in this sand fly species. CONCLUSIONS: Comparative analysis between the salivary proteins of P. papatasi from Tunisia and Israel strains shows a high level of identity, suggesting these proteins as potential common targets for markers of vector exposure or inducers of cellular immune responses in humans for different geographic areas.

BackgroundRhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.Methodology/principal findingsHere we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities.Conclusions/significanceTaken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

Background Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. Methodology/Principal Findings T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. Conclusions/Significance The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for detecting the presence of small numbers of different species of triatomines and could be developed for use as a new tool in surveillance programs, especially to corroborate vector elimination in Chagas disease vector control campaigns., Author Summary Chagas disease, caused by Trypanosoma cruzi, is a neglected disease with 20 million people at risk in Latin America. The main control strategies are based on insecticide spraying to eliminate the domestic vectors, the most effective of which is Triatoma infestans. This approach has been very successful in some areas. However, there is a constant risk of recrudescence in once-endemic regions resulting from the re-establishment of T. infestans and the invasion of other triatomine species. To detect low-level infestations of triatomines after insecticide spraying, we have developed a new epidemiological tool based on host responses against salivary antigens of T. infestans. We identified and synthesized a highly immunogenic salivary protein. This protein was used successfully to detect differences in the infestation level of T. infestans of households in Bolivia and the exposure to other triatomine species. The development of such an exposure marker to detect low-level infestation may also be a useful tool for other disease vectors.