Genome-Based Bioinformatic Selection of Chromosomal<i>Bacillus anthracis</i>Putative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens

Ariel, Naomi; Zvi, Anat; Makarova, Kira S.; Chitlaru, Theodor; Elhanany, Eytan; Velan, Baruch; Cohen, Sara; Friedlander, Arthur M.; Shafferman, Avigdor

doi:10.1128/iai.71.8.4563-4579.2003

Cited by 108 publications

(101 citation statements)

References 112 publications

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“…The concept relies upon the mining of genomic sequences to find putative antigen coding genes. This technology has been successfully used for the identification of vaccine candidates against several bacterial pathogens (6,55,59,81). With the recent sequencing of the E. coli CFT073 genome, a urovirulent E. coli strain, the application of reverse vaccinology to E. coli became possible.…”

Section: Discussionmentioning

confidence: 99%

Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli

et al. 2007

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Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause a large spectrum of infections. The majority of ExPEC strains are closely related to the B2 or the D phylogenetic group. The aim of our study was to develop a protein-based vaccine against these ExPEC strains. To this end, we identified ExPEC-specific genomic regions, using a comparative genome analysis, between the nonpathogenic E. coli strain K-12 MG1655 and ExPEC strains C5 (meningitis isolate) and CFT073 (urinary tract infection isolate). The analysis of these genomic regions allowed the selection of 40 open reading frames, which are conserved among B2/D clinical isolates and encode proteins with putative outer membrane localization. These genes were cloned, and recombinant proteins were purified and assessed as vaccine candidates. After immunization of BALB/c mice, five proteins induced a significant protective immunity against a lethal challenge with a clinical E. coli strain of the B2 group. In passive immunization assays, antigen-specific antibodies afforded protection to naive mice against a lethal challenge. Three of these antigens were related to iron acquisition metabolism, an important virulence factor of the ExPEC, and two corresponded to new, uncharacterized proteins. Due to the large number of genetic differences that exists between commensal and pathogenic strains of E. coli, our results demonstrate that it is possible to identify targets that elicit protective immune responses specific to those strains. The five protective antigens could constitute the basis for a preventive subunit vaccine against diseases caused by ExPEC strains.

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Section: Discussionmentioning

confidence: 99%

Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli

et al. 2007

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“…The availability of the B. anthracis genomic DNA sequence (109,121) paved the way for high-throughput genomic, transcriptomic, and proteomic analyses of B. anthracis (6,7,8,17,25,41,50,64,78,85,121,144) in an effort to elucidate pathogenicity mechanisms by identification of novel virulence factors or in search for specific therapeutic and/or diagnostic targets. Indeed, bioinformatic surveys of the B. anthracis genome (7,8,121) suggested that proteins other than PA, LF, and EF, may participate in anthrax pathogenesis.…”

mentioning

confidence: 99%

“…Indeed, bioinformatic surveys of the B. anthracis genome (7,8,121) suggested that proteins other than PA, LF, and EF, may participate in anthrax pathogenesis. Furthermore, many B. anthracis open reading frames (ORFs) encode potentially secreted or membrane-bound proteins exhibiting homology to known virulence factors from other bacteria (7,8).…”

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confidence: 99%

Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO₂-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities

et al. 2006

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The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulence plasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomic approach. More than 400 protein spots, representing the products of 64 genes, were identified, and a unique pattern of protein relative abundance with respect to the presence of the virulence plasmids was revealed. In minimal medium under high CO 2 tension, conditions considered to simulate those encountered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carried genes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5 pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2 plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic medium not supplemented with CO 2 , the virulent and avirulent B. anthracis strains manifest very similar protein signatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of gene products attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome. Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulence determinants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification factors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO 2 -responsive chromosome-and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.Bacillus anthracis is a gram-positive spore-forming bacterium that is the etiological agent of anthrax, a lethal disease sporadically affecting humans and animals, in particular herbivores. In its most severe manifestation, B. anthracis infection is initiated by inhalation of spores, which are taken up by alveolar macrophages and germinate into fast-dividing vegetative cells which secrete toxins and virulence factors during growth (81,99). If untreated by prompt antibiotic administration, the bacteria invade the bloodstream, resulting in massive bacteremia and consequently generalized systemic failure and death. B. anthracis is considered to represent a potential biothreat agent, owing to the severity of the anthrax disease, the ease of respiratory contamination, and the perpetual environmental stability of the infective spores. The recent deliberate dissemination of B. anthracis (15) accelerated the efforts to identify new B. anthracis virulence-related determinants for the design of novel diagnostic, preventive, and/or therapeutic strategies.Fully virulent B. anthracis strains harbor two native plasmids, pXO1 and pXO2, which encode critical pathogenicity factors. The absence of either one of the two plasmids results in a pronounce...

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“…Available data demonstrate that InhA (encoded by BA0672 and BA1295 genes) can be detected in the sera of infected animals as active protein (54,55). In mice challenged with a lethal dose of Sterne spores, the protein levels of ADAMTS13 were significantly reduced on day 3, although mRNA levels remained steadily elevated until death (Ͼ50% of animals died by day 3) (Fig.…”

Section: Discussionmentioning

confidence: 94%

Degradation of Circulating von Willebrand Factor and Its Regulator ADAMTS13 Implicates Secreted Bacillus anthracis Metalloproteases in Anthrax Consumptive Coagulopathy

Chung

Popova

Jorgensen

et al. 2008

Journal of Biological Chemistry

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Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37°C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F 2 strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.Bacillus anthracis, the causative agent of anthrax, is a zoonotic, Gram-positive, spore-forming Bacillus. It is capable of causing a highly lethal systemic infection in people and experimental animals upon inhalation of the spores. The pathological mechanisms underlying high mortality of inhalation anthrax infection are complex and remain insufficiently understood. The major virulence factors, lethal toxin (LT) 2 and edema toxin (ET) produced by B. anthracis can cause death of experimental animals with symptoms resembling those of anthrax infection. Therefore, many studies have been focused on the cytotoxic effects of LT, including the cells of monocyte lineage (peripheral blood monocytes, macrophages, and dendritic cells) (1) and epithelial cells (2). It has also been reported that the LT-induced cell death could cause a massive release of the pro-inflammatory cytokines, such as the tumor necrosis factor ␣ (TNF␣), which may result in lethality (3), but recent data clearly indicate that the intoxication mediate...

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Genome-Based Bioinformatic Selection of ChromosomalBacillus anthracisPutative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens

Cited by 108 publications

References 112 publications

Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli

Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli

Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO₂-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities

Degradation of Circulating von Willebrand Factor and Its Regulator ADAMTS13 Implicates Secreted Bacillus anthracis Metalloproteases in Anthrax Consumptive Coagulopathy

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Genome-Based Bioinformatic Selection of ChromosomalBacillus anthracisPutative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens

Cited by 108 publications

References 112 publications

Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli

Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli

Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO2-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities

Degradation of Circulating von Willebrand Factor and Its Regulator ADAMTS13 Implicates Secreted Bacillus anthracis Metalloproteases in Anthrax Consumptive Coagulopathy

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Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO₂-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities