HtrA is a major virulence determinant of Bacillus anthracis

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+), pXO2(+)), or in the Delta Vollum (pXO1(-), pXO2(-), nontoxinogenic and non-capsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The Delta htrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). Vollum Delta 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 x 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 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 Delta htrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum Delta htrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.