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 ⌬sleB ⌬cwlJ1 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. ⌬sleB ⌬cwlJ1 ⌬cwlJ2 spores are unable to complete germination in nutrient-rich conditions in vitro. Both ⌬sleB ⌬cwlJ1 and ⌬sleB ⌬cwlJ1 ⌬cwlJ2 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.

In Vitro Studies of Peptidoglycan Binding and Hydrolysis by the Bacillus anthracis Germination-Specific Lytic Enzyme SleB

“…However, none of these proteins have been investigated in vitro to conclusively define their roles and limitations during germination. SleB is a prime candidate for investigation because genetic analyses have shown that it can facilitate complete spore germination independent of the other known GSLEs (14,17). This study demonstrates that purified SleB can carry out lytic transglycosylase activity on a variety of spore PG substrates.…”

“…The C-terminal domain, encompassing Lys-128 to Lys-253, belongs to a family of hydrolases (pfam07486). It is homologous to the sole putative domain of two other GSLEs in B. anthracis, CwlJ1 and CwlJ2, the former of which is capable of facilitating cortex hydrolysis independently (14,16).…”

“…anthracis has four GSLEs that participate in cortex degradation during spore germination, namely, SleB, CwlJ1, CwlJ2, and SleL (14,16). However, none of these proteins have been investigated in vitro to conclusively define their roles and limitations during germination.…”

Crystal Structure of the Catalytic Domain of the Bacillus cereus SleB Protein, Important in Cortex Peptidoglycan Degradation during Spore Germination

“…Cortex PG differs from germ cell wall PG in that in cortex PG a large fraction of the NAM residues lose their peptide and are converted to muramic-␦-lactam (MAL), and a further large fraction of NAM residues are linked only to a single L-alanine residue rather than to the usual oligopeptide; these two modifications contribute to a much lower level of peptide cross-links between the glycan strands in cortex PG (37). Hydrolysis of the cortex PG by the germinationspecific cortex-lytic enzymes (CLEs) is required for later germination events and thus for spore outgrowth (25,37,41).Studies in spores of a number of Bacillus species have shown that initiation of cortex degradation occurs by the action of either of two redundant CLEs, termed SleB and CwlJ (5,12,14,15,18,33,40). Both enzymes act only on PG containing MAL and, as a consequence, degrade only cortex PG and leave the germ cell wall PG untouched.…”

Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery

“…Previous studies have shown that in vitro lysozyme can trigger germination of bacterial spores, bypassing the GRs and directly degrading the spore cortex-lytic enzyme (Lund & Peck, 1994;Peck et al, 1992). Indeed, in a recent study with Bacillus anthracis (Giebel et al, 2009), it has been shown that spores lacking all cortex lytic enzymes are able to germinate in vitro with whole blood and serum despite their lack of germination in nutrient-rich media, and kill 25 % of the challenged mice in 4 days, suggesting that hostspecific enzymes with peptidoglycan-hydrolysing activity might be involved in spore germination and virulence in vivo. Consequently, in this study we found that the main GR proteins, GerKA and GerKC, are not required for C. perfringens spore germination in blood due to the presence of a host serum germination factor with peptidoglycan hydrolysing activity (most likely lysozyme) that triggers significant germination by directly degrading the spore's peptidoglycan cortex.…”