Salivary Gland Transcriptomes and Proteomes of Phlebotomus tobbi and Phlebotomus sergenti, Vectors of Leishmaniasis

Phlebotomine female sand flies require a blood meal for egg development, and it is during the blood feeding that pathogens can be transmitted to a host. Leishmania parasites are among these pathogens and can cause disfiguring cutaneous or even possibly fatal visceral disease. The Leishmania parasites are deposited into the bite wound along with the sand fly saliva. The components of the saliva have many pharmacologic and immune functions important in blood feeding and disease establishment. In this article, the authors identify and investigate the protein components of saliva of two important vectors of leishmaniasis, Phlebotomus tobbi and P. sergenti, by sequencing the transcriptomes of the salivary glands. We then compared the predicted protein sequences of these salivary proteins to those of other bloodsucking insects to elucidate the similarity in composition, structure, and enzymatic activity. Finally, this descriptive analysis of P. tobbi and P. sergenti transcriptomes can aid future research in identifying molecules for epidemiologic assays and in investigating sand fly-host interactions

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

BACKGROUND:Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS:Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. CONCLUSIONS/SIGNIFICANCE:Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species

Insights into the sand fly saliva: Blood-feeding and immune interactions between sand flies, hosts, and Leishmania

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-02-16T19:11:47Z No. of bitstreams: 1 Lestinova T Insights into the sand fly saliva....pdf: 3640201 bytes, checksum: 4a9a2593c4f360001d852f24a75a23d1 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-02-16T19:20:11Z (GMT) No. of bitstreams: 1 Lestinova T Insights into the sand fly saliva....pdf: 3640201 bytes, checksum: 4a9a2593c4f360001d852f24a75a23d1 (MD5)Made available in DSpace on 2018-02-16T19:20:11Z (GMT). No. of bitstreams: 1 Lestinova T Insights into the sand fly saliva....pdf: 3640201 bytes, checksum: 4a9a2593c4f360001d852f24a75a23d1 (MD5) Previous issue date: 2017Czech Science Foundation (project 17-103083 S; https://gacr.cz/en/) - IR, MS, PV, Grant Agency of Charles University (GAUK 1642314/2014 - TL, IR, UNCE 204017/2012 - TL, MS).Charles University. Faculty of Science. Department of Parasitology. Prague, Czech RepublicCharles University. Faculty of Science. Department of Parasitology. Prague, Czech RepublicCharles University. Faculty of Science. Department of Parasitology. Prague, Czech RepublicFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilCharles University. Faculty of Science. Department of Parasitology. Prague, Czech RepublicLeishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties

Recombinant antigens from Phlebotomus perniciosus saliva as markers of canine exposure to visceral leishmaniases vector

BACKGROUND: Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs. METHODOLOGY/PRINCIPAL FINDINGS: The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate. CONCLUSIONS/SIGNIFICANCE: Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral leishmaniases for vector exposure and for estimating the risk of L. infantum transmission in dogs.This research has been funded by Instituto de Salud Carlos III ( PS09/02075 and PI1202021); Xunta de Galicia (10 PXIB 918 273PR) Fundación Mutua Madrileña. Centro de Investigacion Biomedica en Red Fisiopatología de la Obesidad y Nutrición is an ISCIII iniciative (CB06/03). CC is funded by CIBER Fisiopatología Obesidad y Nutrición (CB06/03), LLS is funded by the University Professional Development Program (FPU) of the Spanish Ministry of Education, SB-F by Xunta de Galicia. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.S

Validation of recombinant proteins.

<p>Selected recombinant proteins were validated in ELISA tests with sera of indicated domestic animals naturally-exposed to <i>Phlebotomus orientalis</i>. The analysis was based on comparison with anti-<i>Ph</i>. <i>orientalis</i> SGH IgG as a standard. The table provides cut-off values, mean values of optical density ± standard deviation of IgG levels in animals exposed to <i>Ph</i>. <i>orientalis</i>, correlation coefficients between IgG levels against SGH and a recombinant protein (ρ), positive predictive values (PPV), negative predictive values (NPV), sensitivity, and specificity. Asterisks (*) indicate significant correlations: *p<0.05, **p<0.01, ***p<0.001. Combinations with the correlation coefficient lower than 0.7 in evaluation experiments (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004553#pntd.0004553.t002" target="_blank">Table 2</a>) were excluded from the validation experiments. For each combination, the lowest cut-off value, the highest correlation coefficient, and the highest PPV, NPV, sensitivity, and specificity values are shaded grey. N.A. means not applicable.</p

Identification of <i>Phlebotomus orientalis</i> salivary antigens in dogs.

<p><i>Ph</i>. <i>orientalis</i> salivary proteins were separated under non-reducing conditions by SDS-PAGE on a 12% gel and incubated with two different pools of sera from naturally-exposed Ethiopian dogs (Et), and one pooled sera from non-exposed control dogs (neg). Each pool was a mixture of 5 individual samples. Five antigenic proteins (PorSP65, PorSP24, PorSP15, PorSP76, and PorSP67) were identified by successive proteome analysis and mass spectrometry. Molecular weights of standard (STD) are indicated. UIB means unidentified bands.</p

<i>Phlebotomus orientalis</i> salivary proteins expressed in <i>Escherichia coli</i>.

<p><i>Phlebotomus orientalis</i> salivary proteins expressed in <i>Escherichia coli</i>.</p

Specificity of recombinant proteins.

<p>Results from ELISA are presented as mean optical densities ± standard deviation of IgG antibody reaction with <i>P</i>. <i>orientalis</i> salivary gland homogenate (SGH) and three recombinant proteins (rPorSP24, rPorSP67, and rPorSP76) in mice experimentally bitten by <i>Ph</i>. <i>orientalis</i>, <i>Ph</i>. <i>papatasi</i>, or <i>Se</i>. <i>schwetzi</i>, and non-exposed control mice. Four mice per sand fly colony and four non-exposed controls were used. Asterisks (*) indicate significant differences (p<0.05, calculated with non-parametric Wilcoxon Rank-Sum Test) of IgG levels between mice bitten by <i>Ph</i>. <i>orientalis</i> and mice bitten by other sand fly species or non-bitten controls.</p

Evaluation of recombinant proteins by correlation analysis.

<p>Evaluation of recombinant proteins by correlation analysis.</p

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

<div><p>Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against <i>Leishmania</i> parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from <i>Lutzomyia longipalpis</i>. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-<i>Leishmania</i> vaccines or as an antigen to measure host exposure to sand flies.</p></div