HtrA is a major virulence determinant of Bacillus anthracis

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 Δ htrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation‐associated neutral protease A (NprA). VollumΔ htrA 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 × 10 8 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 htr A mutation, are restored in their corresponding trans‐complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the Δ htrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum Δ htr A is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.