AUTHOR=Israeli Ma’ayan , Elia Uri , Rotem Shahar , Cohen Hila , Tidhar Avital , Bercovich-Kinori Adi , Cohen Ofer , Chitlaru Theodor 

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

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.00255

DOI=10.3389/fmicb.2019.00255

ISSN=1664-302X

ABSTRACT=<p>Anthrax is a lethal disease caused by the Gram-positive spore-producing bacterium <italic>Bacillus anthracis</italic>. We previously demonstrated that disruption of <italic>htrA</italic> gene, encoding the chaperone/protease HtrA<sub>BA</sub> (High Temperature Requirement A of <italic>B. anthracis</italic>) results in significant virulence attenuation, despite unaffected ability of Δ<italic>htrA</italic> strains (in which the <italic>htrA</italic> gene was deleted) to synthesize the key anthrax virulence factors: the exotoxins and capsule. <italic>B. anthracis</italic> Δ<italic>htrA</italic> 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 <italic>htrA</italic> gene was suggested to reflect the susceptibility of Δ<italic>htrA</italic> mutated strains to stress insults encountered in the host indicating that HtrA<sub>BA</sub> represents an important <italic>B. anthracis</italic> pathogenesis determinant. As all HtrA serine proteases, HtrA<sub>BA</sub> 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<sub>BA</sub>. By inspecting the phenotype exhibited by Δ<italic>htrA</italic> strains <italic>trans</italic>-complemented with either a wild-type, truncated (ΔPDZ), or non-proteolytic form (mutated in the catalytic serine residue) of HtrA<sub>BA</sub>, as well as strains exhibiting modified chromosomal alleles, it is shown that (i) the proteolytic activity of HtrA<sub>BA</sub> is essential for its N-terminal autolysis and subsequent release into the extracellular <italic>milieu</italic>, 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<sub>BA</sub> 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<sub>BA</sub> in mediating up-regulation of the extracellular protease NprA under starvation stress, and finally (iv) in a murine model of anthrax, the HtrA<sub>BA</sub> PDZ domain, was dispensable for manifestation of <italic>B. anthracis</italic> virulence. The unexpected dispensability of the PDZ domain may represent a unique characteristic of HtrA<sub>BA</sub> amongst bacterial serine proteases of the HtrA family.</p>