Anopheles salivary gland proteomes from major malaria vectors.

BACKGROUND: Antibody responses against Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires (sialomes) from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined. RESULTS: In silico analysis of secreted salivary gland protein sequences retrieved from an NCBInr database of six Anopheles species belonging to the Cellia subgenus (An. gambiae, An. arabiensis, An. stephensi and An. funestus) and Nyssorhynchus subgenus (An. albimanus and An. darlingi) displayed a higher degree of similarity compared to salivary proteins from closely related Anopheles species. Additionally, computational hierarchical clustering allowed identification of genus-, subgenus- and species-specific salivary proteins. Proteomic and immunoblot analyses performed on salivary gland extracts from four Anopheles species (An. gambiae, An. arabiensis, An. stephensi and An. albimanus) indicated that heterogeneity of the salivary proteome and antigenic proteins was lower among closely related anopheline species and increased with phylogenetic distance. CONCLUSION: This is the first report on the diversity of the salivary protein repertoire among species from the Anopheles genus at the protein level. This work demonstrates that a molecular diversity is exhibited among salivary proteins from closely related species despite their common pharmacological activities. The involvement of these proteins as antigenic candidates for genus-, subgenus- or species-specific immunological evaluation of individual exposure to Anopheles bites is discussed.

Intrafamilial circulation of Tropheryma whipplei, France.

Tropheryma whipplei, which causes Whipple disease, has been detected in 4% of fecal samples from the general adult population of France. To identify T. whipplei within families, we conducted serologic and molecular studies, including genotyping, on saliva, feces, and serum from 74 relatives of 13 patients with classic Whipple disease, 5 with localized chronic T. whipplei infection, and 3 carriers. Seroprevalence was determined by Western blot and compared with 300 persons from the general population. We detected T. whipplei in 24 (38%) of 64 fecal samples and 7 (10%) of 70 saliva samples from relatives but found no difference between persons related by genetics and marriage. The same circulating genotype occurred significantly more often in families than in other persons. Seroprevalence was higher among relatives (23 [77%] of 30) than in the general population (143 [48%] of 300). The high prevalence of T. whipplei within families suggests intrafamilial circulation.

Rapid and cost-effective identification of Bartonella species using mass spectrometry.

Bacteria of the genus Bartonella are emerging zoonotic bacteria recognized in a variety of human diseases. Due to their poor chemical reactivity, these fastidious bacteria are poorly characterized using routine phenotypic laboratory tests. Identification is usually achieved using molecular techniques that are time-consuming, expensive and technically demanding. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a new technique for bacterial species identification. This study evaluated the use of MALDI-TOF MS for rapid genus and species identification of Bartonella species. Reference strains representing 17 recognized Bartonella species were studied. For each species, MS spectra for four colonies were analysed. The consensus spectrum obtained for each species was unique among spectra obtained for 2843 bacteria within the Bruker database, including 109 alphaproteobacteria. Thirty-nine additional blind-coded Bartonella strains were correctly identified at the species level, including 36 with a significant score. Altogether, these data demonstrate that MS is an accurate and reproducible tool for rapid and inexpensive identification of Bartonella species.

Common occurrence of antibacterial agents in human intestinal microbiota.

Laboratory experiments have revealed many active mechanisms by which bacteria can inhibit the growth of other organisms. Bacteriocins are a diverse group of natural ribosomally synthesized antimicrobial peptides produced by a wide range of bacteria and which seem to play an important role in mediating competition within bacterial communities. In this study, we have identified and established the structural classification of putative bacteriocins encoded by 317 microbial genomes in the human intestine. On the basis of homologies to available bacteriocin sequences, mainly from lactic acid bacteria, we report the widespread occurrence of bacteriocins across the gut microbiota: 175 bacteriocins were found to be encoded in Firmicutes, 79 in Proteobacteria, 34 in Bacteroidetes, and 25 in Actinobacteria. Bacteriocins from gut bacteria displayed wide differences among phyla with regard to class distribution, net positive charge, hydrophobicity and secondary structure, but the α-helix was the most abundant structure. The peptide structures and physiochemical properties of bacteriocins produced by the most abundant bacteria in the gut, the Firmicutes and the Bacteroidetes, seem to ensure low antibiotic activity and participate in permanent intestinal host defense against the proliferation of harmful bacteria. Meanwhile, the potentially harmful bacteria, including the Proteobacteria, displayed highly effective bacteriocins, probably supporting the virulent character of diseases. These findings highlight the eventual role played by bacteriocins in gut microbial competition and their potential place in antibiotic therapy.

A DNA microarray for the versatile diagnosis of infectious diarrhea.

Several bacteria, viruses, and parasites cause diarrhea as coinfecting pathogens. We designed a DNA microarray comprising 60-bp probes spotted 194 times for the multiplex detection of 33 enteropathogenic bacteria and seven enteropathogenic viruses, and the archaeon Methanobrevibacter smithii was used as an internal positive control. Nine pathogen-free stool specimens were used as negative controls. One of these control specimens was further spiked with Salmonella enterica as a positive control. The microarray was then tested with 40 pathological stool specimens, comprising S. enterica (n = 30), Campylobacter jejuni (n = 4), pathogenic Escherichia coli (n = 2), and adenovirus (n = 4). M. smithii was detected in 47/49 (95.9%) specimens, no pathogen was detected in negative controls and S. enterica was identified in the S. enterica-spiked positive control. The overall specificity was 100% and the overall sensitivity was 97.5% because one S. enterica sample was missed by the microarray. The multiplexed detection of C. jejuni spiked into an adenovirus-positive stool sample gave positive results, with fluorescence values of 14.3 and 9.1, respectively. These data indicate that using the protocol developed in this article, the DNA array allows for the multiplexed detection of some enteropathogens in stool samples.