The germination-specific lytic enzymes SleB, CwlJ1, and CwlJ2 each contribute to Bacillus anthracis spore germination and virulence

Abstract

The bacterial spore cortex is critical for spore stability and dormancy and must be hydrolyzed by germination-specific lytic enzymes (GSLEs), which allows complete germination and vegetative cell outgrowth. We created in-frame deletions of three genes that encode GSLEs that have been shown to be active in Bacillus anthracis germination: sleB, cwlJ1, and cwlJ2. Phenotypic analysis of individual null mutations showed that the removal of any one of these genes was not sufficient to disrupt spore germination in nutrient-rich media. This finding indicates that these genes have partially redundant functions. Double and triple deletions of these genes resulted in more significant defects. Although a small subset of DeltasleB DeltacwlJ1 spores germinate with wild-type kinetics, for the overall population there is a 3-order-of-magnitude decrease in the colony-forming efficiency compared with wild-type spores. DeltasleB DeltacwlJ1 DeltacwlJ2 spores are unable to complete germination in nutrient-rich conditions in vitro. Both DeltasleB DeltacwlJ1 and DeltasleB DeltacwlJ1 DeltacwlJ2 spores are significantly attenuated, but are not completely devoid of virulence, in a mouse model of inhalation anthrax. Although unable to germinate in standard nutrient-rich media, spores lacking SleB, CwlJ1, and CwlJ2 are able to germinate in whole blood and serum in vitro, which may explain the persistent low levels of virulence observed in mouse infections. This work contributes to our understanding of GSLE activation and function during germination. This information may result in identification of useful therapeutic targets for the disease anthrax, as well as provide insights into ways to induce the breakdown of the protective cortex layer, facilitating easier decontamination of resistant spores.

FIG. 1.

In vitro germination of spores in BHI. The loss of heat resistance (A and C) and the decrease in the OD₆₀₀ (B and D) were measured as markers of spore germination. A 60% decrease in the OD₆₀₀ represents approximately 100% germination (23). Spores were incubated in BHI at 37°C for 0 to 30 min. In all panels the symbols indicate the means for two or three independent experiments, and the error bars indicate standard errors. WT, wild type.

FIG. 2.

Colony-forming efficiency of GSLE mutants. The colony-forming efficiency was measured by visually determining the number of spores/ml (solid bars) of a given stock with an improved Neubauer hemocytometer and comparing the value obtained to the number of CFU/ml (open bars) for the same stock when it was plated on BHI. The data are representative of typical results. WT, wild type.

FIG. 3.

Survival curves for GSLE mutants in mice. DBA/2J mice (eight mice per group) were challenged by intratracheal infection with wild-type (WT) and mutant spores at a dose of 1.5 × 10⁶ spores/mouse. The mice were monitored for 14 days, and then all surviving mice were euthanized. The survival curves for the ΔsleB ΔcwlJ1 (C) and ΔsleB ΔcwlJ1 ΔcwlJ2 (D) mutants were found to be significantly different from those for the wild type (P = 0.0001 and P = 0.0005, respectively, as determined by a log rank [Mantel-Cox] test). Although not meeting the significance threshold, the survival curve for the ΔsleB ΔcwlJ2 mutant (B) did show a trend away from the survival curve for the wild type (P = 0.058). The survival curve for the ΔcwlJ1 ΔcwlJ2 mutant was not significantly different from that of the wild type (A).

FIG. 4.

In vitro germination of ΔsleB ΔcwlJ1 ΔcwlJ2 spores in blood. The colony-forming efficiency of ΔsleB ΔcwlJ1 ΔcwlJ2 spores in blood and serum was examined. Spore dilutions were quantified visually with an improved Neubauer hemocytometer (spores/ml) and compared to the numbers of CFU/ml when spores were allowed to germinate in phosphate-buffered saline (PBS) (pH 7.5), BHI, whole bovine blood, bovine serum, or heat-treated bovine serum for 10 min before they were plated on BHI plates and incubated at 37°C overnight.