3-Hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin)-induced 28-kDa interleukin-1ß interferes with mature IL-1ß signaling.

Multiple clinical trials have shown that the 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors known as statins have anti-inflammatory effects. However, the underlying molecular mechanism remains unclear. The proinflammatory cytokine interleukin-1ß (IL-1ß) is synthesized as a non-active precursor. The 31-kDa pro-IL-1ß is processed into the 17-kDa active form by caspase-1-activating inflammasomes. Here, we report a novel signaling pathway induced by statins, which leads to processing of pro-IL-1ß into an intermediate 28-kDa form. This statin-induced IL-1ß processing is independent of caspase-1- activating inflammasomes. The 28-kDa form of IL-1ß cannot activate interleukin-1 receptor-1 (IL1R1) to signal inflammatory responses. Instead, it interferes with mature IL-1ß signaling through IL-1R1 and therefore may dampen inflammatory responses initiated by mature IL-1ß. These results may provide new clues to explain the anti-inflammatory effects of statins.

A rapid matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for single-plasmid tracking in an outbreak of carbapenem-resistant Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the bla(KPC) carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An ∼ 11,109-Da MS peak corresponding to a gene product of the bla(KPC) pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed K. pneumoniae outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of bla(KPC)-negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the bla(KPC) Tn4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other bla(KPC) Tn4401-containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak.

Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus.

BACKGROUND: Sand fly saliva plays an important role in blood feeding and Leishmania transmission as it was shown to increase parasite virulence. On the other hand, immunity to salivary components impedes the establishment of infection. Therefore, it is most desirable to gain a deeper insight into the composition of saliva in sand fly species which serve as vectors of various forms of leishmaniases. In the present work, we focused on Phlebotomus (Adlerius) arabicus, which was recently shown to transmit Leishmania tropica, the causative agent of cutaneous leishmaniasis in Israel. RESULTS: A cDNA library from salivary glands of P. arabicus females was constructed and transcripts were sequenced and analyzed. The most abundant protein families identified were SP15-like proteins, ParSP25-like proteins, D7-related proteins, yellow-related proteins, PpSP32-like proteins, antigen 5-related proteins, and 34 kDa-like proteins. Sequences coding for apyrases, hyaluronidase and other putative secreted enzymes were also represented, including endonuclease, phospholipase, pyrophosphatase, amylase and trehalase. Mass spectrometry analysis confirmed the presence of 20 proteins predicted to be secreted in the salivary proteome. Humoral response of mice bitten by P. arabicus to salivary antigens was assessed and many salivary proteins were determined to be antigenic. CONCLUSION: This transcriptomic analysis of P. arabicus salivary glands is the first description of salivary proteins of a sand fly in the subgenus Adlerius. Proteomic analysis of P. arabicus salivary glands produced the most comprehensive account in a single sand fly species to date. Detailed information and phylogenetic relationships of the salivary proteins are provided, expanding the knowledge base of molecules that are likely important factors of sand fly-host and sand fly-Leishmania interactions. Enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis.

Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior.

Ticks evolved various mechanisms to modulate their host's hemostatic and immune defenses. Differences in the anti-hemostatic repertoires suggest that hard and soft ticks evolved anti-hemostatic mechanisms independently, but raise questions on the conservation of salivary gland proteins in the ancestral tick lineage. To address this issue, the sialome (salivary gland secretory proteome) from the soft tick, Argas monolakensis, was determined by proteomic analysis and cDNA library construction of salivary glands from fed and unfed adult female ticks. The sialome is composed of approximately 130 secretory proteins of which the most abundant protein folds are the lipocalin, BTSP, BPTI and metalloprotease families which also comprise the most abundant proteins found in the salivary glands. Comparative analysis indicates that the major protein families are conserved in hard and soft ticks. Phylogenetic analysis shows, however, that most gene duplications are lineage specific, indicating that the protein families analyzed possibly evolved most of their functions after divergence of the two major tick families. In conclusion, the ancestral tick may have possessed a simple (few members for each family), but diverse (many different protein families) salivary gland protein domain repertoire.

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.

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

BACKGROUND: Salivary 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. RESULTS: Transcriptome 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. CONCLUSION: These 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.

Characterization of a lysyl aminopeptidase activity associated with phosphoglucose isomerase of Vibrio vulnificus.

Phosphoglucose isomerase (PGI) is a multifunctional enzyme involved in glycolysis and gluconeogenesis and, in mammalian cells, functions as neuroleukin, autocrine motility factor (AMF), and differentiation and maturation factor (MF). We isolated and characterized PGI with a novel lysyl aminopeptidase (LysAP) activity (PGI-LysAP) from Vibrio vulnificus. Mass spectrometry revealed that PGI-LysAP is a heterodimer consisting of 23.4- and 60.8-kDa subunits. Only the heterodimer displayed LysAP activity. PGI-LysAP has a pI around 6.0 and high specificity toward the synthetic, fluorogenic substrate l-lysyl-7-amino-4-methylcoumarin. LysAP activity is optimal at pH 8.0, is 64% higher at 37 degrees C than at 21 degrees C, does not directly correlate with virulence, and is strongly inhibited by serine protease and metalloprotease inhibitors. PGI-LysAP was also identified in Vibrio parahaemolyticus and V. cholerae, but was absent from non-Vibrio human pathogens. Sequencing of the pgi gene revealed 1653 bp coding for a 550-amino-acid protein. Cloned and expressed PGI formed a homodimer with isomerase activity, but not LysAP activity. The finding of LysAP activity associated with heterodimeric PGI should foster a broad search for putative substrates in an effort to elucidate the role of PGI-LysAP in bacteria and its roles in the pathophysiology of diseases.

Bitis gabonica (Gaboon viper) snake venom gland: toward a catalog for the full-length transcripts (cDNA) and proteins.

The venom gland of the snake Bitis gabonica (Gaboon viper) was used for the first time to construct a unidirectional cDNA phage library followed by high-throughput sequencing and bioinformatic analysis. Hundreds of cDNAs were obtained and clustered into contigs. We found mostly novel full-length cDNA coding for metalloproteases (P-II and P-III classes), Lys49-phospholipase A2, serine proteases with essential mutations in the active site, Kunitz protease inhibitors, several C-type lectins, bradykinin-potentiating peptide, vascular endothelial growth factor, nucleotidases and nucleases, nerve growth factor, and L-amino acid oxidases. Two new members of the recently described short coding region family of disintegrin, displaying RGD and MLD motifs are reported. In addition, we have identified for the first time a cytokine-like molecule and a multi-Kunitz protease inhibitor in snake venoms. The CLUSTAL alignment and the unrooted cladograms for selected families of B. gabonica venom proteins are also presented. A significant number of sequences were devoid of database matches, suggesting that their biologic function remains to be identified. This paper also reports the N-terminus of the 15 most abundant venom proteins and the sequences matching their corresponding transcripts. The electronic version of this manuscript, available on request, contains spreadsheets with hyperlinks to FASTA-formatted files for each contig and the best match to the GenBank and Conserved Domain Databases, in addition to CLUSTAL alignments of each contig. We have thus generated a comprehensive catalog of the B. gabonica venom gland, containing for each secreted protein: (i) the predicted molecular weight, (ii) the predicted isoelectric point, (iii) the accession number, and (iv) the putative function. The role of these molecules is discussed in the context of the envenomation caused by the Gaboon viper.

An insight into the salivary transcriptome and proteome of the adult female mosquito Culex pipiens quinquefasciatus.

To obtain an insight into the salivary transcriptome and proteome (sialome) of the adult female mosquito Culex quinquefasciatus, a cDNA library was randomly sequenced, and aminoterminal information for selected proteins and peptides was obtained. cDNA sequence clusters coding for secreted proteins were further analyzed. The transcriptome revealed messages coding for several proteins of known families previously reported in the salivary glands of other blood-feeding insects as well as immune-related products such as C-type lectin, gambicin, and members of the prophenol oxidase cascade. Additionally, several transcripts coding for low-complexity proteins were found, some clearly coding for mucins. Many novel transcripts were found, including a novel endonuclease previously described in crabs and shrimps but not in insects; a hyaluronidase, not described before in mosquito salivary glands but found in venom glands and in salivary glands of sand flies and black flies; several cysteine-rich peptides with possible anticlotting function, including one similar to a previously described nematode family of anti-proteases; and a completely novel family of cysteine- and tryptophane-rich proteins (CWRC family) for which 12 full-length sequences are described. Also described are 14 additional novel proteins and peptides whose function and/or family affiliation are unknown. In total, 54 transcripts coding for full-length proteins are described. That several of these are translated into proteins was confirmed by finding the corresponding aminoterminal sequences in the SDS-PAGE/Edman degradation experiments. Electronic versions of all tables and sequences can be found at http://www.ncbi.nlm.nih.gov/projects/Mosquito/C_quinquefasciatus_sialome.

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/ .

The effects of etomidate on adrenal responsiveness and mortality in patients with septic shock.

RATIONALE: Use of etomidate in the critically ill is controversial due to its links with an inadequate response to corticotropin and potential for excess mortality. In a septic shock population, we tested the hypotheses that etomidate administration induces more non-responders to corticotropin and increases mortality and that hydrocortisone treatment decreases mortality in patients receiving etomidate. METHODS: An a-priori sub-study of the CORTICUS multi-centre, randomised, double-blind, placebo-controlled trial of hydrocortisone in septic shock. Use and timing of etomidate administration were collected. Endpoints were corticotropin response and all-cause 28-day mortality in patients receiving etomidate. MEASUREMENTS AND MAIN RESULTS: Five hundred patients were recruited, of whom 499 were analysable; 96 (19.2%) were administered etomidate within the 72 h prior to inclusion. The proportion of non-responders to corticotropin was significantly higher in patients who were given etomidate in the 72 h before trial inclusion than in other patients (61.0 vs. 44.6%, P = 0.004). Etomidate therapy was associated with a higher 28-day mortality in univariate analysis (P = 0.02) and after correction for severity of illness (42.7 vs. 30.5%; P = 0.06 and P = 0.03) in our two multi-variant models. Hydrocortisone administration did not change the mortality of patients receiving etomidate (45 vs. 40%). CONCLUSIONS: The use of bolus dose etomidate in the 72 h before study inclusion is associated with an increased incidence of inadequate response to corticotropin, but is also likely to be associated with an increase in mortality. We recommend clinicians demonstrate extreme caution in the use of etomidate in critically ill patients with septic shock.

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry.

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.

Processing map and essential cleavage sites of the nonstructural polyprotein encoded by ORF1 of the feline calicivirus genome.

Feline calicivirus (FCV) nonstructural proteins are translated as part of a large polyprotein that undergoes autocatalytic processing by the virus-encoded 3C-like proteinase. In this study, we mapped three new cleavage sites (E(46)/A(47), E(331)/D(332), and E(685)/N(686)) recognized by the virus proteinase in the N-terminal part of the open reading frame 1 (ORF1) polyprotein to complete the processing map. Taken together with two sites we identified previously (E(960)/A(961) and E(1071)/S(1072)), the FCV ORF1 polyprotein contains five cleavage sites that define the borders of six proteins with calculated molecular masses of 5.6, 32, 38.9, 30.1, 12.7, and 75.7 kDa, which we designated p5.6, p32, p39 (NTPase), p30, p13 (VPg), and p76 (Pro-Pol), respectively. Mutagenesis of the E to A in each of these cleavage sites in an infectious FCV cDNA clone was lethal for the virus, indicating that these cleavages are essential in a productive virus infection. Mutagenesis of two cleavage sites (E(1345)/T(1346) and E(1419)/G(1420)) within the 75.7-kDa Pro-Pol protein previously mapped in bacterial expression studies was not lethal.

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

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.

In vitro proteolytic processing of the MD145 norovirus ORF1 nonstructural polyprotein yields stable precursors and products similar to those detected in calicivirus-infected cells.

The MD145-12 strain (GII/4) is a member of the genus Norovirus in the Caliciviridae and was detected in a patient with acute gastroenteritis in a Maryland nursing home. The open reading frame 1 (ORF1) (encoding the nonstructural polyprotein) was cloned as a consensus sequence into various expression vectors, and a proteolytic cleavage map was determined. The virus-encoded cysteine proteinase mediated at least five cleavages (Q(330)/G(331), Q(696)/G(697), E(875)/G(876), E(1008)/A(1009), and E(1189)/G(1190)) in the ORF1 polyprotein in the following order: N-terminal protein; nucleoside triphosphatase; 20-kDa protein (p20); virus protein, genome linked (VPg); proteinase (Pro); polymerase (Pol). A time course analysis of proteolytic processing of the MD145-12 ORF1 polyprotein in an in vitro coupled transcription and translation assay allowed the identification of stable precursors and final mapped cleavage products. Stable precursors included p20VPg (analogous to the 3AB of the picornaviruses) and ProPol (analogous to the 3CD of the picornaviruses). Less stable processing intermediates were identified as p20VPgProPol, p20VPgPro, and VPgPro. The MD145-12 Pro and ProPol proteins were expressed in bacteria as active forms of the proteinase and used to further characterize their substrate specificities in trans cleavage assays. The MD145-12 Pro was able to cleave its five mapped cleavage sites in trans and, in addition, could mediate trans cleavage of the Norwalk virus (GI/I) ORF1 polyprotein into a similar proteolytic processing profile. Taken together, our data establish a model for proteolytic processing in the noroviruses that is consistent with nonstructural precursors and products identified in studies of caliciviruses that replicate in cell culture systems.

Toward a catalog for the transcripts and proteins (sialome) from the salivary gland of the malaria vector Anopheles gambiae.

Hundreds of Anopheles gambiae salivary gland cDNA library clones have been sequenced. A cluster analysis based on sequence similarity at e(-60) grouped the 691 sequences into 251 different clusters that code for proteins with putative secretory, housekeeping, or unknown functions. Among the housekeeping cDNAs, we found sequences predicted to code for novel thioredoxin, tetraspanin, hemopexin, heat shock protein, and TRIO and MBF proteins. Among secreted cDNAs, we found 21 novel A. gambiae salivary sequences including those predicted to encode amylase, calreticulin, selenoprotein, mucin-like protein and 30-kDa allergen, in addition to antigen 5- and D7-related proteins, three novel salivary gland (SG)-like proteins and eight unique putative secreted proteins (Hypothetical Proteins, HP). The electronic version of this paper contains hyperlinks to FASTA-formatted files for each cluster with the best match to the nonredundant (NR) and conserved domain databases (CDD) in addition to CLUSTAL alignments of each cluster. The N terminus of 12 proteins (SG-1, SG-1-like 2, SG-6, HP 8, HP 9-like, 5' nucleotidase, 30-kDa protein, antigen 5- and four D7-related proteins) has been identified by Edman degradation of PVDF-transferred, SDS/PAGE-separated salivary gland proteins. Therefore, we contribute to the generation of a catalog of A. gambiae salivary transcripts and proteins. These data are freely available and will eventually become an invaluable tool to study the role of salivary molecules in parasite-host/vector interactions.

Exploring the sialome of the tick Ixodes scapularis.

To attempt description of the set of mRNA and protein (sialome) expressed in the salivary glands of the tick Ixodes scapularis, we randomly sequenced 735 clones of a full-length salivary gland cDNA library of this arthropod and performed Edman degradation of protein bands from salivary gland homogenates (SGH) and saliva separated by SDS-PAGE. The sequences were grouped into 410 clusters, of which 383 are not associated with known I. scapularis sequences. 15- and 17-protein bands from PAGE yielded amino-terminal information on the saliva and salivary gland gels, respectively. We attributed 19 of these sequences to translation products of the cDNA library. Full-length sequences were obtained for 87 clones. Among these protein sequences are several protease inhibitors of distinct classes, metalloproteases, novel proteins with histamine-binding domains, and several peptide families of unknown function displaying different conserved cysteine residues, many of which contain single Kunitz domains. This work provides information into the diversity of messages expressed in the salivary glands of I. scapularis, describes novel sequences that may be responsible for known biological activites, indicates further biological activities that may be present in I. scapularis saliva and identifies novel vaccine targets that may be used in Lyme disease prevention.