Van M. Pham

A Role for Insect Galectins in Parasite Survival

Insect galectins are associated with embryonic development or immunity against pathogens. Here, we show that they can be exploited by parasites for survival in their insect hosts. PpGalec, a tandem repeat galectin expressed in the midgut of the sandfly Phlebotomus papatasi, is used by Leishmania major as a receptor for mediating specific binding to the insect midgut, an event crucial for parasite survival, and accounts for species-specific vector competence for the most widely distributed form of cutaneous leishmaniasis in the Old World. In addition, these studies demonstrate the feasibility of using midgut receptors for parasite ligands as target antigens for transmission-blocking vaccines.

Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito

Anopheles stephensi is the main urban mosquito vector of malaria in the Indian subcontinent, and belongs to the same subgenus as Anopheles gambiae, the main malaria vector in Africa. Recently the genome and proteome sets of An. gambiae have been described, as well as several protein sequences expressed in its salivary glands, some of which had their expression confirmed by amino terminal sequencing. In this paper, we randomly sequenced a full-length cDNA library of An. stephensi and performed Edman degradation of polyvinylidene difluoride (PVDF)-transferred protein bands from salivary homogenates. Twelve of 13 proteins found by aminoterminal degradation were found among the cDNA clusters of the library. Thirty-three full-length novel cDNA sequences are reported, including a novel secreted galectin; the homologue of anophelin, a thrombin inhibitor; a novel trypsin/chymotrypsin inhibitor; an apyrase; a lipase; and several new members of the D7 protein family. Most of the novel proteins have no known function. Comparison of the putatively secreted and putatively housekeeping proteins of An. stephensi with An. gambiae proteins indicated that the salivary gland proteins are at a faster evolutionary pace. The possible role of these proteins in blood and sugar feeding by the mosquito is discussed. The electronic tables and supplemental material are available at http://www.ncbi.nlm.nih.gov/projects/Mosquito/A_stephensi_sialome/ .

Toward a description of the sialome of the adult female mosquito Aedes aegypti

To describe the set of mRNA and protein expressed in the salivary glands (sialome) of Aedes aegypti mosquitoes, we randomly sequenced a full-length cDNA library of this insect and performed Edman degradation of PVDF-transferred protein bands from salivary homogenates. We found 238 cDNA clusters which contained those coding for 10 of the 11 proteins found by aminoterminal degradation. All six previously described salivary proteins were found in this library. Full-length sequences of 32 novel cDNA sequences are reported, one of which is the product of a transposable element. Among the 31 novel protein sequences are 4 additional members of the D7 protein family; 4 novel members of the antigen 5 family (a protein family not reported in Aedes); a novel serpin; a novel member of the 30-kDa allergen of Ae. Aegypti; a secreted calreticulin; 2 proteins similar to mammalian angiopoietins; adenosine deaminase; purine hydrolase; lysozyme; a C-type lectin; 3 serine proteases, including one with high similarity to Bombyx prophenoloxidase activating enzyme; 2 proteins related to invertebrate immunity; and several sequences that have no significant matches to known proteins. The possible role of these proteins in blood and sugar feeding by the mosquito is discussed.

Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis, vector of Leishmania chagasi

SUMMARY Using massive cDNA sequencing, proteomics and customized computational biology approaches, we have isolated and identified the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis. Out of 550 randomly isolated clones from a full-length salivary gland cDNA library, we found 143 clusters or families of related proteins. Out of these 143 families, 35 were predicted to be secreted proteins. We confirmed, by Edman degradation of Lu. longipalpis salivary proteins, the presence of 17 proteins from this group. Full-length sequence for 35 cDNA messages for secretory proteins is reported, including an RGD-containing peptide, three members of the yellow-related family of proteins, maxadilan, a PpSP15-related protein, six members of a family of putative anticoagulants, an antigen 5-related protein, a D7-related protein, a cDNA belonging to the Cimex apyrase family of proteins, a protein homologous to a silk protein with amino acid repeats resembling extracellular matrix proteins, a 5′-nucleotidase, a peptidase, a palmitoyl-hydrolase, an endonuclease, nine novel peptides and four different groups of proteins with no homologies to any protein deposited in accessible databases. Sixteen of these proteins appear to be unique to sand flies. With this approach, we have tripled the number of isolated secretory proteins from this sand fly. Because of the relationship between the vertebrate host immune response to salivary proteins and protection to parasite infection, these proteins are promising markers for vector exposure and attractive targets for vaccine development to control Leishmania chagasi infection.

The salivary gland transcriptome of the neotropical malaria vector Anopheles darlingi reveals accelerated evolution of genes relevant to hematophagy

BackgroundMosquito saliva, consisting of a mixture of dozens of proteins affecting vertebrate hemostasis and having sugar digestive and antimicrobial properties, helps both blood and sugar meal feeding. Culicine and anopheline mosquitoes diverged ~150 MYA, and within the anophelines, the New World species diverged from those of the Old World ~95 MYA. While the sialotranscriptome (from the Greek sialo, saliva) of several species of the Cellia subgenus of Anopheles has been described thoroughly, no detailed analysis of any New World anopheline has been done to date. Here we present and analyze data from a comprehensive salivary gland (SG) transcriptome of the neotropical malaria vector Anopheles darlingi (subgenus Nyssorhynchus).ResultsA total of 2,371 clones randomly selected from an adult female An. darlingi SG cDNA library were sequenced and used to assemble a database that yielded 966 clusters of related sequences, 739 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 183 protein sequences, 114 of which code for putative secreted proteins.ConclusionComparative analysis of sialotranscriptomes of An. darlingi and An. gambiae reveals significant divergence of salivary proteins. On average, salivary proteins are only 53% identical, while housekeeping proteins are 86% identical between the two species. Furthermore, An. darlingi proteins were found that match culicine but not anopheline proteins, indicating loss or rapid evolution of these proteins in the old world Cellia subgenus. On the other hand, several well represented salivary protein families in old world anophelines are not expressed in An. darlingi.

High degree of conservancy among secreted salivary gland proteins from two geographically distant Phlebotomus duboscqi sandflies populations (Mali and Kenya)

BackgroundSalivary proteins from sandflies are potential targets for exploitation as vaccines to control Leishmania infection; in this work we tested the hypothesis that salivary proteins from geographically distant Phlebotomus duboscqi sandfly populations are highly divergent due to the pressure exerted by the host immune response. Salivary gland cDNA libraries were prepared from wild-caught P. duboscqi from Mali and recently colonised flies of the same species from Kenya.ResultsTranscriptome and proteome analysis resulted in the identification of the most abundant salivary gland-secreted proteins. Orthologues of these salivary proteins were identified by phylogenetic tree analysis. Moreover, comparative analysis between the orthologues of these two different populations resulted in a high level of protein identity, including the predicted MHC class II T-cell epitopes from all these salivary proteins.ConclusionThese data refute the hypothesis that salivary proteins from geographically distinct populations of the same Phlebotomus sandfly species are highly divergent. They also suggest the potential for using the same species-specific components in a potential vector saliva-based vaccine.

An insight into the sialome of the oriental rat flea, Xenopsylla cheopis (Rots)

BackgroundThe salivary glands of hematophagous animals contain a complex cocktail that interferes with the host hemostasis and inflammation pathways, thus increasing feeding success. Fleas represent a relatively recent group of insects that evolved hematophagy independently of other insect orders.ResultsAnalysis of the salivary transcriptome of the flea Xenopsylla cheopis, the vector of human plague, indicates that gene duplication events have led to a large expansion of a family of acidic phosphatases that are probably inactive, and to the expansion of the FS family of peptides that are unique to fleas. Several other unique polypeptides were also uncovered. Additionally, an apyrase-coding transcript of the CD39 family appears as the candidate for the salivary nucleotide hydrolysing activity in X.cheopis, the first time this family of proteins is found in any arthropod salivary transcriptome.ConclusionAnalysis of the salivary transcriptome of the flea X. cheopis revealed the unique pathways taken in the evolution of the salivary cocktail of fleas. Gene duplication events appear as an important driving force in the creation of salivary cocktails of blood feeding arthropods, as was observed with ticks and mosquitoes. Only five other flea salivary sequences exist at this time at NCBI, all from the cat flea C. felis. This work accordingly represents the only relatively extensive sialome description of any flea species. Sialotranscriptomes of additional flea genera will reveal the extent that these novel polypeptide families are common throughout the Siphonaptera.

An insight into the sialotranscriptome and proteome of the coarse bontlegged tick, Hyalomma marginatum rufipes.

Ticks are mites specialized in acquiring blood from vertebrates as their sole source of food and are important disease vectors to humans and animals. Among the specializations required for this peculiar diet, ticks evolved a sophisticated salivary potion that can disarm their hosts hemostasis, inflammation, and immune reactions. Previous transcriptome analysis of tick salivary proteins has revealed many new protein families indicative of fast evolution, possibly due to host immune pressure. The hard ticks (family Ixodidae) are further divided into two basal groups, of which the Metastriata have 11 genera. While salivary transcriptomes and proteomes have been described for some of these genera, no tick of the genus Hyalomma has been studied so far. The analysis of 2084 expressed sequence tags (EST) from a salivary gland cDNA library allowed an exploration of the proteome of this tick species by matching peptide ions derived from MS/MS experiments to this data set. We additionally compared these MS/MS derived peptide sequences against the proteins from the bovine host, finding many host proteins in the salivary glands of this tick. This annotated data set can assist the discovery of new targets for anti-tick vaccines as well as help to identify pharmacologically active proteins.

An insight into the sialotranscriptome of the West Nile mosquito vector, Culex tarsalis

BackgroundSaliva of adult female mosquitoes help sugar and blood feeding by providing enzymes and polypeptides that help sugar digestion, control microbial growth and counteract their vertebrate host hemostasis and inflammation. Mosquito saliva also potentiates the transmission of vector borne pathogens, including arboviruses. Culex tarsalis is a bird feeding mosquito vector of West Nile Virus closely related to C. quinquefasciatus, a mosquito relatively recently adapted to feed on humans, and the only mosquito of the genus Culex to have its sialotranscriptome so far described.ResultsA total of 1,753 clones randomly selected from an adult female C. tarsalis salivary glands (SG) cDNA library were sequenced and used to assemble a database that yielded 809 clusters of related sequences, 675 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 283 protein sequences, 80 of which code for putative secreted proteins.ConclusionComparison of the C. tarsalis sialotranscriptome with that of C. quinquefasciatus reveals accelerated evolution of salivary proteins as compared to housekeeping proteins. The average amino acid identity among salivary proteins is 70.1%, while that for housekeeping proteins is 91.2% (P < 0.05), and the codon volatility of secreted proteins is significantly higher than those of housekeeping proteins. Several protein families previously found exclusive of mosquitoes, including only in the Aedes genus have been identified in C. tarsalis. Interestingly, a protein family so far unique to C. quinquefasciatus, with 30 genes, is also found in C. tarsalis, indicating it was not a specific C. quinquefasciatus acquisition in its evolution to optimize mammal blood feeding.

An Insight into the Sialotranscriptome of Simulium nigrimanum, a Black Fly Associated with Fogo Selvagem in South America

Pemphigus foliaceus is a life threatening skin disease that is associated with autoimmunity to desmoglein, a skin protein involved in the adhesion of keratinocytes. This disease is endemic in certain areas of South America, suggesting the mediation of environmental factors triggering autoimmunity. Among the possible environmental factors, exposure to bites of black flies, in particular Simulium nigrimanum has been suggested. In this work, we describe the sialotranscriptome of adult female S. nigrimanum flies. It reveals the complexity of the salivary potion of this insect, comprised by over 70 distinct genes within over 30 protein families, including several novel families, even when compared with the previously described sialotranscriptome of the autogenous black fly, S. vittatum. The uncovering of this sialotranscriptome provides a platform for testing pemphigus patient sera against recombinant salivary proteins from S. nigrimanum and for the discovery of novel pharmacologically active compounds.