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

Abstract

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 Δ htrA strains (in which the htrA gene was deleted) to synthesize the key anthrax virulence factors: the exotoxins and capsule. B. anthracis Δ htrA 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 Δ htrA 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 Δ htrA 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.

The proteolytic activity of Listeria monocytogenes HtrA

“…Multimers of LmHtrA could be detected at protein amounts higher than 1.25 μg protein, which were more pronounced in comparison to HpHtrA multimers. Additionally, a truncated and active version of recombinant HtrA (short HtrA, sHtrA) was detected, which has been also observed for autoprocessed HtrA from other bacterial species previously [18] (Fig. 3a, upper panel).…”

“…This appears to be in contrast to HtrA proteases expressed by Gram-positive bacteria. In Bacillus anthracis , N-terminal autoprocessing has been demonstrated as a requirement for both, secretion and activity [18]. Although it is not known whether truncated sHtrA from L. monocytogenes is the result of N-terminal or C-terminal cleavage, truncated LmHtrA is still caseinolytically active suggesting that L. monocytogenes also secretes HtrA into the environment as an active protease.…”

Importance of twoPDZdomains for the proteolytic and chaperone activities ofHelicobacter pyloriserine proteaseHtrA

“…HtrAs are known to exhibit an additional chaperone‐like activity, which is independent of the proteolytic activity (reviewed in Skorko‐Glonek et al, 2013). In some cases (DegQ Ec , or Bacillus anthracis HtrA), the presence of PDZ domains is required for the efficient chaperone activity (Israeli et al, 2019; Malet et al, 2012). To the contrary, DegP Ec deprived of both PDZ domains retains its chaperone activity (Iwanczyk et al, 2007; Jiang et al, 2008; Jomaa et al, 2007).…”

Transcriptome Sequencing Data of Bacillus anthracis Vollum Δ htrA and Its Parental Strain, Isolated under Oxidative Stress

“…Virulence-attenuating htrA gene disruption was implemented for the development of a live spore vaccine ( 5 – 7 ). Recently, we suggested that HtrA acts as both a protease/chaperone and a pleiotropic factor of gene expression ( 8 ). Here, we present transcriptome sequencing (RNA-seq) data sets describing the effect of htrA gene disruption on the global gene expression of B. anthracis in the presence/absence of H 2 O 2 .…”