Summary: We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1⁺, pXO2⁺), or in the ΔVollum (pXO1^‐, pXO2^‐, nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation‐associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10⁸ spores, in the guinea pig model of anthrax), as compared with the parental wild‐type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans‐complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.

Monoclonal antibodies represent an important avenue for COVID-19 therapy and are routinely used for rapid and accessible diagnosis of SARS-CoV-2 infection. The recent emergence of SARS-CoV-2 genetic variants emphasized the need to enlarge the repertoire of antibodies that target diverse epitopes, the combination of which may improve immune-diagnostics, augment the efficiency of the immunotherapy and prevent selection of escape-mutants. Antigen-specific controlled immunization of experimental animals may elicit antibody repertoires that significantly differ from those generated in the context of the immune response mounted in the course of disease. Accordingly, rabbits were immunized by several recombinant antigens representing distinct domains of the viral spike protein and monoclonal antibodies were isolated from single cells obtained by cell sorting. Characterization of a panel of successfully isolated anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies demonstrated that they exhibit high specificity and affinity profiles. Anti-RBD antibodies revealing significant neutralizing potency against SARS-CoV-2 in vitro were found to target at least three distinct epitopes. Epitope mapping established that two of these antibodies recognized a novel epitope located on the surface of the RBD. We suggest that the antibodies isolated in this study are useful for designing SARS-CoV-2 diagnosis and therapy approaches.

A search for bacterium-specific biomarkers in peripheral blood following infection withBacillus anthraciswas carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of theB. anthracissecretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column. Besides the toxin component protective antigen, the following three secreted proteins were detected in the circulation of infected animals: the chaperone and protease HtrA (BA3660), an NlpC/P60 endopeptidase (BA1952), and a protein of unknown function harboring two SH3 (Src homology 3) domains (BA0796). The three proteins could be detected in plasma samples from infected animals exhibiting 10³to 10⁵CFU/ml blood and also in standard blood cultures at 3 to 6 h post-bacterial inoculation at a bacteremic level as low as 10³CFU/ml. Furthermore, the three biomarkers appear to be present only in the secretome ofB. anthracis, not in those of the related pathogensB. thuringiensisandB. cereus. To the best of our knowledge, this is the first report of direct detection ofB. anthracis-specific proteins, other than the toxin components, in the circulation of infected animals.

Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological cause of anthrax, a rare lethal disease of animals and humans. Development of anthrax countermeasures has gained increasing attention owing to the potential use of B. anthracis spores as a bioterror weapon. The various forms of infection by B. anthracis are characterized both by toxemia and septicemia, both of which are the result of spore entry into the host followed by their germination into rapidly multiplying, toxin-producing bacilli. Following the publication of the bacterial genome, proteomic studies were carried out to determine the protein composition of the spore and identify exposed vegetative (membrane-located or secreted) proteins. These studies included comparison of strains differing in their virulence, cultured under different conditions and, in some cases, were complemented by serological inspection, which addressed expression during infection of proteomically identified proteins and their immunogenicity. The proteomic approach emerged as a valuable strategy for the generation of a pool of potential B. anthracis protein targets for further evaluation in detection, diagnostics, therapy and prophylaxis, and contributed to the elucidation of some aspects of the pathogenesis of the disease.

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 HtrA_BA (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 HtrA_BA represents an important B. anthracis pathogenesis determinant. As all HtrA serine proteases, HtrA_BA 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 HtrA_BA. 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 HtrA_BA, as well as strains exhibiting modified chromosomal alleles, it is shown that (i) the proteolytic activity of HtrA_BA 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 HtrA_BA 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 HtrA_BA in mediating up-regulation of the extracellular protease NprA under starvation stress, and finally (iv) in a murine model of anthrax, the HtrA_BA PDZ domain, was dispensable for manifestation of B. anthracis virulence. The unexpected dispensability of the PDZ domain may represent a unique characteristic of HtrA_BA amongst bacterial serine proteases of the HtrA family.