Mark Garfield

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.

Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis

BackgroundImmune 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.ResultsTwo 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.ConclusionThis 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.

In Vitro Proteolytic Processing of the MD145 Norovirus ORF1 Nonstructural Polyprotein Yields Stable Precursors and Products Similar to Those Detected in Calicivirus-Infected Cells

ABSTRACT 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 (Q330/G331, Q696/G697, E875/G876, E1008/A1009, and E1189/G1190) 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.

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.

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.

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

BackgroundSand 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.ResultsA 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.ConclusionThis 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.

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

ABSTRACT 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 blaKPC 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 blaKPC 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 blaKPC -negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the blaKPC Tn4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other blaKPC 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.

3-Hydroxyl-3-methylglutaryl Coenzyme A (HMG-CoA) Reductase Inhibitor (Statin)-induced 28-kDa Interleukin-1β Interferes with Mature IL-1β Signaling

Background: HMG-CoA reductase inhibitors (statins) have anti-inflammatory effects, the molecular mechanisms of which remain unclear. Results: Statin treatment of macrophages induces secretion of a 28-kDa form of IL-1β that interferes with mature IL-1β signaling. Conclusion: Statins may dampen inflammation through induction of an anti-inflammatory form of IL-1β. Significance: These observations may provide clues toward elucidating the in vivo anti-inflammatory effects of statins. 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-1β (IL-1β) is synthesized as a non-active precursor. The 31-kDa pro-IL-1β 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-1β into an intermediate 28-kDa form. This statin-induced IL-1β processing is independent of caspase-1- activating inflammasomes. The 28-kDa form of IL-1β cannot activate interleukin-1 receptor-1 (IL1R1) to signal inflammatory responses. Instead, it interferes with mature IL-1β signaling through IL-1R1 and therefore may dampen inflammatory responses initiated by mature IL-1β. These results may provide new clues to explain the anti-inflammatory effects of statins.