Mechanisms of Bacillus anthracis spore cortex degradation.

Abstract

Bacillus anthracis exists in two morphologically distinct states, the spore and the vegetative cell. The deadly animal and human disease anthrax results from B. anthracis spores entering a suitable host and transitioning to a toxin producing vegetative cell through germination. The spore cortex is a modified peptidoglycan structure that is critical for spore stability and dormancy and must be hydrolyzed during germination to allow vegetative-cell outgrowth. In Bacillus anthracis, this process was thought to involve the enzymatic action of a group of proteins called germination-specific lytic enzymes (GSLEs). I have shown that the GSLEs SleB, CwlJ1 and CwlJ2 are all involved in cortex hydrolysis and that a second pathway, host-lysozyme induced germination, is capable of degrading the cortex in vivo. Traditional nutrient based germination requires either SleB or CwlJ1, both in vitro and in vivo. In the absence of these two primary GSLEs, CwlJ2 provides minimal cortex hydrolyzing activity and germination rates are severely decreased. Mutant spores lacking all three GSLEs (ΔsleB/cwlJ1/cwlJ2) are unable to complete germination in nutrient rich media in vitro. Although ΔsleB/cwlJ1/cwlJ2 spores are incapable of germinating in response to nutrients, these mutants are able to germinate at a low rate when incubated in serum, whole blood, or a solution containing purified lysozyme. Spores lacking SleB and CwlJ1 as well as ΔsleB/cwlJ1/cwlJ2 spores are highly attenuated in a mouse model of inhalation anthrax. Host lysozyme in the lungs causes germination of ΔsleB/cwlJ1/cwlJ2 spores in vivo. Due to this host factor dependent germination, ΔsleB/cwlJ1/cwlJ2 spores remain virulent at high infectious doses. When lysozyme M-/- knockout mice were challenged with ΔsleB/cwlJ1/cwlJ2 spores, the mutant was significantly more attenuated compared to a wild-type mouse infection. Also, serum and bone marrow-derived macrophages from lysozyme deficient mice were unable to induce germination of ΔsleB/cwlJ1/cwlJ2 spores. Cortex hydrolysis is a critical barrier in the process of spore germination. This work provides a more complete understanding of this process for B. anthracis spores both in vitro and in vivo.