Distinct Contribution of the HtrA Protease and PDZ Domains to Its Function in Stress Resilience and Virulence of Bacillus anthracis

Anthrax is a lethal disease caused by the Gram-positive spore-producing bacterium Bacillus anthracis. We previously demonstrated that disruption of htrA gene, encoding the chaperone/protease HtrABA (High Temperature Requirement A of B. anthracis) results in significant virulence attenuation, despite unaffected ability of ΔhtrA strains (in which the htrA gene was deleted) to synthesize the key anthrax virulence factors: the exotoxins and capsule. B. anthracis ΔhtrA strains exhibited increased sensitivity to stress regimens as well as silencing of the secreted starvation-associated Neutral Protease A (NprA) and down-modulation of the bacterial S-layer. The virulence attenuation associated with disruption of the htrA gene was suggested to reflect the susceptibility of ΔhtrA mutated strains to stress insults encountered in the host indicating that HtrABA represents an important B. anthracis pathogenesis determinant. As all HtrA serine proteases, HtrABA exhibits a protease catalytic domain and a PDZ domain. In the present study we interrogated the relative impact of the proteolytic activity (mediated by the protease domain) and the PDZ domain (presumably necessary for the chaperone activity and/or interaction with substrates) on manifestation of phenotypic characteristics mediated by HtrABA. By inspecting the phenotype exhibited by ΔhtrA strains trans-complemented with either a wild-type, truncated (ΔPDZ), or non-proteolytic form (mutated in the catalytic serine residue) of HtrABA, as well as strains exhibiting modified chromosomal alleles, it is shown that (i) the proteolytic activity of HtrABA is essential for its N-terminal autolysis and subsequent release into the extracellular milieu, while the PDZ domain was dispensable for this process, (ii) the PDZ domain appeared to be dispensable for most of the functions related to stress resilience as well as involvement of HtrABA in assembly of the bacterial S-layer, (iii) conversely, the proteolytic activity but not the PDZ domain, appeared to be dispensable for the role of HtrABA in mediating up-regulation of the extracellular protease NprA under starvation stress, and finally (iv) in a murine model of anthrax, the HtrABA PDZ domain, was dispensable for manifestation of B. anthracis virulence. The unexpected dispensability of the PDZ domain may represent a unique characteristic of HtrABA amongst bacterial serine proteases of the HtrA family

Supplementary Method Data

In the publication "Genome Sequence of Two Novel Virulent Clinical Strains of Burkholderia pseudomallei Isolated from Acute Melioidosis Cases Imported to Israel from India and Thailand" to which the online resource pertains, we report the genomes of two novel clinical isolates (MWH2021 and MST2022) of Burkholderia pseudomallei identified in two distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The publication includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of other Burkholderia pseudomallei strains documented in the NCBI database. Inspection of the genetic data established the presence or absence of loci encoding for several important virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or latent melioidosis established that both strains belong to the highly virulent class of Burkholderia pseudomalleii.</p

Lethal Dose 50% (LD50) values and virulence determinants

In the publication "Genome Sequence of Two Novel Virulent Clinical Strains of Burkholderia pseudomallei Isolated from Acute Melioidosis Cases Imported to Israel from India and Thailand" to which the online resource pertains, we report the genomes of two novel clinical isolates (MWH2021 and MST2022) of Burkholderia pseudomallei identified in two distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The publication includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of other Burkholderia pseudomallei strains documented in the NCBI database. Inspection of the genetic data established the presence or absence of loci encoding for several important virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or latent melioidosis established that both strains belong to the highly virulent class of Burkholderia pseudomalleii.</p

Differential Proteomic Analysis of the Bacillus anthracis Secretome: Distinct Plasmid and Chromosome CO(2)-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities

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

A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules