Phylogenetic Analysis Indicates That Evasin-Like Proteins of Ixodid Ticks Fall Into Three Distinct Classes.

Chemokines are structurally related proteins that activate leucocyte migration in response to injury or infection. Tick saliva contains chemokine-binding proteins or evasins which likely neutralize host chemokine function and inflammation. Biochemical characterisation of 50 evasins from Ixodes, Amblyomma and Rhipicephalus shows that they fall into two functional classes, A and B, with exclusive binding to either CC- or CXC- chemokines, respectively. Class A evasins, EVA1 and EVA4 have a four-disulfide-bonded core, whereas the class B evasin EVA3 has a three-disulfide-bonded "knottin" structure. All 29 class B evasins have six cysteine residues conserved with EVA3, arrangement of which defines a Cys6-motif. Nineteen of 21 class A evasins have eight cysteine residues conserved with EVA1/EVA4, the arrangement of which defines a Cys8-motif. Two class A evasins from Ixodes (IRI01, IHO01) have less than eight cysteines. Many evasin-like proteins have been identified in tick salivary transcriptomes, but their phylogenetic relationship with respect to biochemically characterized evasins is not clear. Here, using BLAST searches of tick transcriptomes with biochemically characterized evasins, we identify 292 class A and 157 class B evasins and evasin-like proteins from Prostriate (Ixodes), and Metastriate (Amblyomma, Dermacentor, Hyalomma, Rhipicephalus) ticks. Phylogenetic analysis shows that class A evasins/evasin-like proteins segregate into two classes, A1 and A2. Class A1 members are exclusive to Metastriate ticks and typically have a Cys8-motif and include EVA1 and EVA4. Class A2 members are exclusive to Prostriate ticks, lack the Cys8-motif, and include IHO01 and IRI01. Class B evasins/evasin-like proteins are present in both Prostriate and Metastriate lineages, typically have a Cys6-motif, and include EVA3. Most evasins/evasin-like proteins in Metastriate ticks belong to class A1, whereas in Prostriate species they are predominantly class B. In keeping with this, the majority of biochemically characterized Metastriate evasins bind CC-chemokines, whereas the majority of Prostriate evasins bind CXC-chemokines. While the origin of the structurally dissimilar classes A1 and A2 is yet unresolved, these results suggest that class B evasin-like proteins arose before the divergence of Prostriate and Metastriate lineages and likely functioned to neutralize CXC-chemokines and support blood feeding.

The salivary transcriptome of Limnobdella mexicana (Annelida: Clitellata: Praobdellidae) and orthology determination of major leech anticoagulants.

Bloodfeeding requires several adaptations that allow the parasite to feed efficiently. Leeches and other hematophagous animals have developed different mechanisms to inhibit hemostasis, one of the main barriers imposed by their hosts. Limnobdella mexicana is a member of the leech family Praobdellidae, a family of host generalists known for their preference to attach on mucosal membranes of mammals, such as those in nasopharyngeal cavities, bladders and ocular orbits. Previous studies have hypothesized a positive relationship between diversity of anticoagulants and diversity of hosts in bloodfeeding leeches. However, orthology determination of putative anticoagulants and the lack of standardization of sequencing effort and method hinder comparisons between publicly available transcriptomes generated in different laboratories. In the present study, we examine the first transcriptome of a praobdellid leech and identify 15 putative anticoagulants using a phylogeny-based inference approach, amino-acid conservation, Pfam domains and BLAST searches. Our phylogenetic analyses suggest that the ancestral leech was able to inhibit factor Xa and that some hirudins that have been reported in previous studies on leech anticoagulants may not be orthologous with the archetypal hirudin.

Human salivary proteins and their peptidomimetics: Values of function, early diagnosis, and therapeutic potential in combating dental caries.

Saliva contains a large number of proteins that play various crucial roles to maintain the oral health and tooth integrity. This oral fluid is proposed to be one of the most important host factors, serving as a special medium for monitoring aspects of microorganisms, diet and host susceptibility involved in the caries process. Extensive salivary proteomic and peptidomic studies have resulted in considerable advances in the field of biomarkers discovery for dental caries. These salivary biomarkers may be exploited for the prediction, diagnosis, prognosis and treatment of dental caries, many of which could also provide the potential templates for bioactive peptides used for the biomimetic management of dental caries, rather than repairing caries lesions with artificial materials. A comprehensive understanding of the biological function of salivary proteins as well as their derived biomimetic peptides with promising potential against dental caries has been long awaited. This review overviewed a collection of current literature and addressed the majority of different functions of salivary proteins and peptides with their potential as functional biomarkers for caries risk assessment and clinical prospects for the anti-caries application.

Genomes of trombidid mites reveal novel predicted allergens and laterally transferred genes associated with secondary metabolism.

Background: Trombidid mites have a unique life cycle in which only the larval stage is ectoparasitic. In the superfamily Trombiculoidea ("chiggers"), the larvae feed preferentially on vertebrates, including humans. Species in the genus Leptotrombidium are vectors of a potentially fatal bacterial infection, scrub typhus, that affects 1 million people annually. Moreover, chiggers can cause pruritic dermatitis (trombiculiasis) in humans and domesticated animals. In the Trombidioidea (velvet mites), the larvae feed on other arthropods and are potential biological control agents for agricultural pests. Here, we present the first trombidid mites genomes, obtained both for a chigger, Leptotrombidium deliense, and for a velvet mite, Dinothrombium tinctorium. Results: Sequencing was performed using Illumina technology. A 180 Mb draft assembly for D. tinctorium was generated from two paired-end and one mate-pair library using a single adult specimen. For L. deliense, a lower-coverage draft assembly (117 Mb) was obtained using pooled, engorged larvae with a single paired-end library. Remarkably, both genomes exhibited evidence of ancient lateral gene transfer from soil-derived bacteria or fungi. The transferred genes confer functions that are rare in animals, including terpene and carotenoid synthesis. Thirty-seven allergenic protein families were predicted in the L. deliense genome, of which nine were unique. Preliminary proteomic analyses identified several of these putative allergens in larvae. Conclusions: Trombidid mite genomes appear to be more dynamic than those of other acariform mites. A priority for future research is to determine the biological function of terpene synthesis in this taxon and its potential for exploitation in disease control.

Comparative Evolution of Sand Fly Salivary Protein Families and Implications for Biomarkers of Vector Exposure and Salivary Vaccine Candidates.

Sand fly salivary proteins that produce a specific antibody response in humans and animal reservoirs have been shown to be promising biomarkers of sand fly exposure. Furthermore, immunity to sand fly salivary proteins were shown to protect rodents and non-human primates against Leishmania infection. We are missing critical information regarding the divergence amongst sand fly salivary proteins from different sand fly vectors, a knowledge that will support the search of broad or specific salivary biomarkers of vector exposure and those for vaccines components against leishmaniasis. Here, we compare the molecular evolution of the salivary protein families in New World and Old World sand flies from 14 different sand fly vectors. We found that the protein families unique to OW sand flies are more conserved than those unique to NW sand flies regarding both sequence polymorphisms and copy number variation. In addition, the protein families unique to OW sand flies do not display as many conserved cysteine residues as the one unique to the NW group (28.5% in OW vs. 62.5% in NW). Moreover, the expression of specific protein families is restricted to the salivary glands of unique sand fly taxon. For instance, the ParSP15 family is unique to the Larroussius subgenus whereas phospholipase A2 is only expressed in member of Larroussius and Adlerius subgenera. The SP2.5-like family is only expressed in members of the Phlebotomus and Paraphlebotomus subgenera. The sequences shared between OW and NW sand flies have diverged at similar rates (38.7 and 45.3% amino acid divergence, respectively), yet differences in gene copy number were evident across protein families and sand fly species. Overall, this comparative analysis sheds light on the different modes of sand fly salivary protein family divergence. Also, it informs which protein families are unique and conserved within taxon for the choice of taxon-specific biomarkers of vector exposure, as well as those families more conserved across taxa to be used as pan-specific vaccines for leishmaniasis.

Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry.

In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite transmission during the process of blood feeding.

Comparative analysis of salivary gland transcriptomes of Phlebotomus orientalis sand flies from endemic and non-endemic foci of visceral leishmaniasis.

BACKGROUND: In East Africa, Phlebotomus orientalis serves as the main vector of Leishmania donovani, the causative agent of visceral leishmaniasis (VL). Phlebotomus orientalis is present at two distant localities in Ethiopia; Addis Zemen where VL is endemic and Melka Werer where transmission of VL does not occur. To find out whether the difference in epidemiology of VL is due to distant compositions of P. orientalis saliva we established colonies from Addis Zemen and Melka Werer, analyzed and compared the transcriptomes, proteomes and enzymatic activity of the salivary glands. METHODOLOGY/PRINCIPAL FINDINGS: Two cDNA libraries were constructed from the female salivary glands of P. orientalis from Addis Zemen and Melka Werer. Clones of each P. orientalis library were randomly selected, sequenced and analyzed. In P. orientalis transcriptomes, we identified members of 13 main protein families. Phylogenetic analysis and multiple sequence alignments were performed to evaluate differences between the P. orientalis colonies and to show the relationship with other sand fly species from the subgenus Larroussius. To further compare both colonies, we investigated the humoral antigenicity and cross-reactivity of the salivary proteins and the activity of salivary apyrase and hyaluronidase. CONCLUSIONS: This is the first report of the salivary components of P. orientalis, an important vector sand fly. Our study expanded the knowledge of salivary gland compounds of sand fly species in the subgenus Larroussius. Based on the phylogenetic analysis, we showed that P. orientalis is closely related to Phlebotomus tobbi and Phlebotomus perniciosus, whereas Phlebotomus ariasi is evolutionarily more distinct species. We also demonstrated that there is no significant difference between the transcriptomes, proteomes or enzymatic properties of the salivary components of Addis Zemen (endemic area) and Melka Werer (non-endemic area) P. orientalis colonies. Thus, the different epidemiology of VL in these Ethiopian foci cannot be attributed to the salivary gland composition.

Tryptogalinin is a tick Kunitz serine protease inhibitor with a unique intrinsic disorder.

BACKGROUND: A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions. RESULTS: We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for ß-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for ß-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases). CONCLUSIONS: By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

Identification and characterization of histatin 1 salivary complexes by using mass spectrometry.

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein's function is the identification of its interaction partners. We investigated interactions among and functions of histatin 1 and the other proteins that are present in saliva by using high-throughput mass spectrometric techniques. This led to the identification of 43 proteins able to interact with histatin 1. In addition, we found that these protein-protein interactions protect complex partners from proteolysis and modulate their antifungal activity. Our data contribute significantly to characterization of the salivary interactome and to understanding the biology of salivary protein complexes.

Bioinformatics advances in saliva diagnostics.

There is a need recognized by the National Institute of Dental & Craniofacial Research and the National Cancer Institute to advance basic, translational and clinical saliva research. The goal of the Salivaomics Knowledge Base (SKB) is to create a data management system and web resource constructed to support human salivaomics research. To maximize the utility of the SKB for retrieval,integration and analysis of data, we have developed the Saliva Ontology and SDxMart. This article reviews the informatics advances in saliva diagnostics made possible by the Saliva Ontology and SDxMart.

From the salivary proteome to the OralOme: comprehensive molecular oral biology.

OBJECTIVES: There have been several efforts to identify the protein components of saliva. Some of these studies were conducted in healthy individuals and other in individuals with different oral and systemic disorders. However, a resource compiling and reviewing all of the proteins identified in proteomic studies is still lacking. The aim of this project is to develop such a resource. DESIGN: The proteins identified by proteomic studies were compiled and all information concerning them was manually curated according to "IPI History search" and UniProt. Proteins were classified according to gene ontology using PANTHER. The involvement of each protein in disease was scrutinized using DAVID and a classification into protein disease classes was performed. RESULTS: This survey of proteins in the oral cavity lead to the identification of 3397 non-redundant proteins, 605 altered in pathological conditions and 51 present only in disease. These proteins originate from different sources: 3115 from saliva, 990 from oral mucosa and 1929 from plasma. All protein sources contribute with different numbers and types of proteins to identical functions. However, each source produces specific proteins. Examples of the use of this proteomics database of saliva included the analysis of the changes in the proteome associated with periodontitis and a survey of systemic disease potential biomarkers in saliva. CONCLUSION: The database generated with this work and the information therein stands as a resource for investigators/clinicians studying the oral biology, searching for molecular disease markers, developing diagnostic and prognostic tests, and contributing to the discovery of new therapeutic agents.

Glycoprotein 340 and sialic acid in minor-gland and whole saliva of children, adolescents, and adults.

Glycoprotein 340 (gp-340) is a bacterial-binding glycoprotein found in major-gland and minor-gland saliva. Sialic acid, a common terminal structure of salivary glycoproteins, interacts with microorganisms and host ligands, as well as with free radicals. This study investigated the contents of gp-340 and sialic acid in minor-gland saliva and whole saliva of children (3 yr of age), adolescents (14 yr of age), and adults (20-25 yr of age). Labial-gland saliva and buccal-gland saliva were collected on filter paper, and unstimulated whole saliva was collected by draining into a tube. The relative amount of gp-340 and sialic acid was determined by ELISA and by enzyme-linked lectin assay (ELLA), respectively. In minor-gland saliva, no statistically significant differences in gp-340 and sialic acid were seen between the age-groups. Among adults, significantly lower amounts of gp-340 and sialic acid were seen in labial saliva compared with buccal saliva. In whole saliva, the amount of gp-340 was significantly lower among adults compared with children. No differences between genders were seen. Stable content of gp-340 and sialic acid in minor-gland saliva across the age-groups, and a higher content of gp-340 in the whole saliva of the youngest age-group (3-yr-olds) compared with the adult group, may reflect that those components are vital innate factors of immunity in children's saliva.

Adsorption of salivary and serum proteins, and bacterial adherence on titanium and zirconia ceramic surfaces.

OBJECTIVES: The aim of this study was to determine the pattern of salivary and serum proteins present in pellicles formed on titanium (Ti) and zirconia ceramic (ZrO(2)) surfaces, and the ability of bacterial cells to adhere to the experimental pellicles. In addition, the protein profiles and bacterial binding properties of pellicles on Ti and ZrO(2) were compared to those formed on hydroxyapatite (HA) surface. METHODS: The pellicles were formed in vitro by incubating the materials with whole saliva, serum or saliva+serum. Protein composition in each of the pellicles was investigated by SDS-PAGE and immunodetection. The adherence of radiolabeled Streptococcus mutans and Actinomyces naeslundii to uncoated surfaces and experimental pellicles was determined by means of scintillation counting. Statistical analyses were done using ANOVA and Tukey's test at significance level at P<0.05. In general, the electrophoretic analysis of the pellicles formed on HA, Ti and ZrO(2) revealed few qualitative differences of the composition of proteins of the pellicles formed on HA, Ti and ZrO(2) surfaces. Pellicle components identified included amylase, IgA, IgG, albumin, fibronectin and fibrinogen. The number of S. mutans cells adhered to uncoated Ti and ZrO(2) was significantly higher than those adhered to HA (P<0.05). In contrast, lower number of A. naeslundii cells adhered to uncoated Ti and ZrO(2) than to HA (P<0.05). However, the presence of saliva and saliva+serum pellicles greatly reduced the number of S. mutans cells bound to each of the surfaces. The data showed that Ti and ZrO(2) display similar pellicle protein composition and bacterial binding properties; however, significant differences were observed when both materials were compared to HA.

Proteome of human minor salivary gland secretion.

Recent research efforts in oral biology have resulted in elucidation of the proteomes of major human salivary secretions and whole saliva. One might hypothesize that the proteome of minor gland secretions may show significantly different characteristics when compared with the proteomes of parotid or submandibular/sublingual secretions. To test this hypothesis, we conducted the first exploration into the proteome of minor salivary gland secretion. Minor gland secretion was obtained from healthy volunteers, and its components were subjected to liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry. This led to the identification of 56 proteins, 12 of which had never been identified in any salivary secretion. The unique characteristics of the minor salivary gland secretion proteome are related to the types as well as the numbers of components present. The differences between salivary proteomes may be important with respect to specific oral functions.

An annotated catalogue of salivary gland transcripts in the adult female mosquito, Aedes aegypti.

BACKGROUND: Saliva of blood-sucking arthropods contains a cocktail of antihemostatic agents and immunomodulators that help blood feeding. Mosquitoes additionally feed on sugar meals and have specialized regions of their glands containing glycosidases and antimicrobials that might help control bacterial growth in the ingested meals. To expand our knowledge on the salivary cocktail of AEdes aegypti, a vector of dengue and yellow fevers, we analyzed a set of 4,232 expressed sequence tags from cDNA libraries of adult female mosquitoes. RESULTS: A nonredundant catalogue of 614 transcripts (573 of which are novel) is described, including 136 coding for proteins of a putative secretory nature. Additionally, a two-dimensional gel electrophoresis of salivary gland (SG) homogenates followed by tryptic digestion of selected protein bands and MS/MS analysis revealed the expression of 24 proteins. Analysis of tissue-specific transcription of a subset of these genes revealed at least 31 genes whose expression is specific or enriched in female SG, whereas 24 additional genes were expressed in female SG and in males but not in other female tissues. Most of the 55 proteins coded by these SG transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. An unexpected finding is the occurrence of four protein families specific to SG that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes. CONCLUSION: Overall, this paper contributes to the novel identification of 573 new transcripts, or near 3% of the AE. aegypti proteome assuming a 20,000-protein set, and to the best-described sialome of any blood-feeding insect.

In defense of the oral cavity: the protective role of the salivary secretions.

Saliva performs important protective roles in the oral cavity. Debate in the 1970s over the "specific" or "non-specific" action of salivary components has given way to current attempts to identify the full complement of all proteins in saliva that are now considered to act in concert. At the same time, more fundamental protective qualities of saliva water and pH control are receiving less attention. These qualities may be among saliva's most important. This presentation will review recent advances in the genomics and proteomics of saliva, as well as saliva's roles in tissue coating, alimentation, and regulation of the oral flora.

Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis.

BACKGROUND: Immune responses to sandfly saliva have been shown to protect animals against Leishmania infection. Yet very little is known about the molecular characteristics of salivary proteins from different sandflies, particularly from vectors transmitting visceral leishmaniasis, the fatal form of the disease. Further knowledge of the repertoire of these salivary proteins will give us insights into the molecular evolution of these proteins and will help us select relevant antigens for the development of a vector based anti-Leishmania vaccine. RESULTS: Two salivary gland cDNA libraries from female sandflies Phlebotomus argentipes and P. perniciosus were constructed, sequenced and proteomic analysis of the salivary proteins was performed. The majority of the sequenced transcripts from the two cDNA libraries coded for secreted proteins. In this analysis we identified transcripts coding for protein families not previously described in sandflies. A comparative sandfly salivary transcriptome analysis was performed by using these two cDNA libraries and two other sandfly salivary gland cDNA libraries from P. ariasi and Lutzomyia longipalpis, also vectors of visceral leishmaniasis. Full-length secreted proteins from each sandfly library were compared using a stand-alone version of BLAST, creating formatted protein databases of each sandfly library. Related groups of proteins from each sandfly species were combined into defined families of proteins. With this comparison, we identified families of salivary proteins common among all of the sandflies studied, proteins to be genus specific and proteins that appear to be species specific. The common proteins included apyrase, yellow-related protein, antigen-5, PpSP15 and PpSP32-related protein, a 33-kDa protein, D7-related protein, a 39- and a 16.1- kDa protein and an endonuclease-like protein. Some of these families contained multiple members, including PPSP15-like, yellow proteins and D7-related proteins suggesting gene expansion in these proteins. CONCLUSION: This comprehensive analysis allows us the identification of genus- specific proteins, species-specific proteins and, more importantly, proteins common among these different sandflies. These results give us insights into the repertoire of salivary proteins that are potential candidates for a vector-based vaccine.

Degradation of antimicrobial histatin-variant peptides in Staphylococcus aureus and Streptococcus mutans.

Histidine-free variants of salivary histatin 5 have a broad antimicrobial activity against various bacteria. In relation to a possible therapeutic application, we were interested in the susceptibility of these small peptides (14 amino acids long) to microbial proteinases and whether this affects their antimicrobial activity. Analyses by SDS-PAGE of supernatants of peptide-bacteria incubation showed a reduction in protein bands within 15 minutes' incubation, as a result of cellular internalization. Degradation products of dhvar1 and dhvar2 appeared within one hour in the supernatants of Streptococcus mutans and Staphylococcus aureus. In contrast, the variants dhvar3 and dhvar4 were more resistant to degradation under the same conditions. MALDI-TOF analyses identified cleavage of dhvar1 and dhvar2 at Glu(6). The N-terminal peptide part (1-6) of dhvar1 and 2 showed no bactericidal activity, while peptide fragment (7-14) showed a highly reduced bactericidal activity.