Contributions of Four Cortex Lytic Enzymes to Germination of Bacillus anthracis Spores

Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca²⁺-dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore''s integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca²⁺-DPA-induced germination. SleL is an N-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.The Gram-positive bacterium Bacillus anthracis is the etiologic agent of cutaneous, gastrointestinal, and inhalational anthrax (24). An anthrax infection begins when the host is infected with highly resistant, quiescent B. anthracis spores (1, 24). Within the host, the spore''s sensory mechanism recognizes chemical signals, known as germinants, and triggers germination, which leads to the resumption of metabolism (36). Spores that have differentiated into vegetative cells produce a protective capsule and deadly toxins. These virulence factors allow the bacteria to evade the host''s immune system and establish an infection resulting in septicemia, toxemia, and frequently death (24). Although vegetative cells produce virulence factors that are potentially fatal, these cells cannot initiate infections and are much more susceptible to antimicrobial treatments than spores (24). Therefore, efficient triggering of spore germination may enhance current decontamination methods.Spores are highly resistant to many environmental insults because the spore core (cytoplasm) is dehydrated, dormant, and surrounded by multiple protective layers, including a modified layer of peptidoglycan (PG) known as the cortex (36). The cortex functions to maintain dormancy and heat resistance by preventing core rehydration (9). It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) sugars (Fig. ​(Fig.1).1). Peptide side chains on the NAM residues are either involved in interstrand cross-linking, cleaved to single l-alanine side chains, or fully removed with accompanying formation of muramic-δ-lactam (2, 31, 38). After germination is initiated by either nutrient or nonnutrient germinants, the cortex is depolymerized, resulting in complete core rehydration, resumption of metabolic activity, and outgrowth (33, 36).Open in a separate windowFIG. 1.Spore PG structure and hydrolysis. The central structure shows a representative spore PG strand with alternating NAG and NAM or muramic-δ-lactam (MδL) residues and with tetrapeptide or l-Ala side chains on the NAM residues. Forked arrows originate at sites of hydrolysis by the indicated enzymes and point to muropeptide products. The indicated “aG” muropeptide names are as previously published (7, 11). SleB lytic transglycosylase activity produces muropeptides terminating in anhydro-NAM. Cleavage at adjacent NAM residues produces the tetrasaccharide aG7a or aG7b, while cleavage further apart can produce octasaccharides or larger fragments. These can be further cleaved by muramidase treatment, resulting in the production of tetrasaccharide N, which terminates in NAM. The N-acetylglucosaminidase activity of SleL produces tetrasaccharides terminating in NAG, which can be further cleaved by muramidase to trisaccharides terminating in NAM.Cortex hydrolysis is driven by autolysins called germination-specific cortex lytic enzymes (GSLEs) that recognize the cortex-specific muramic-δ-lactam residues (2, 4, 21, 32). GSLEs fall into two classes: spore cortex lytic enzymes (SCLEs), which are thought to depolymerize intact cortical PG, and cortical fragment lytic enzymes (CFLEs), which further degrade partially hydrolyzed cortex (21). Both SCLEs and CFLEs have been identified in a variety of spore-forming species, including B. anthracis (11, 18, 19), Bacillus cereus (4, 20, 26), Bacillus megaterium (8, 34), Bacillus subtilis (13, 16, 25), Bacillus thuringiensis (12), and Clostridium perfringens (5, 23). Of the four GSLEs identified in B. anthracis, CwlJ1, CwlJ2, and SleB are predicted to be SCLEs (11), whereas SleL is thought to be a CFLE (18).Recently, independent studies showed that CwlJ1 and the lytic transglycosylase SleB (Fig. ​(Fig.1)1) play partially redundant roles and that either is sufficient for spore germination and outgrowth (10, 11). However, these same studies report conflicting results concerning the role of CwlJ2 during germination. Heffron et al. found no effect of CwlJ2 on the biochemistry of cortex hydrolysis or on colony-forming efficiency of spores (11). Giebel et al. reported that loss of CwlJ2 caused a minor defect in germination kinetics and that in the absence of SleB and CwlJ1, further loss of CwlJ2 had a major effect on colony forming efficiency (10). SleL in Bacillus anthracis is proposed to be an N-acetylglucosaminidase (Fig. ​(Fig.1)1) whose role is to further degrade cortex fragments resulting from SCLE hydrolysis (18). SleL is not essential for the completion of germination but does promote the release of small muropeptides to the spore''s surrounding environment (18).This study reports the effects of multiple deletion mutations affecting GSLEs on spore germination efficiency and kinetics of cortex hydrolysis. The data confirm the dominant roles played by CwlJ1 and SleB in the initiation of cortex hydrolysis and the major role of SleL in release of small cortex fragments. A minor role of CwlJ2 in nutrient-triggered germination and the contributions of CwlJ1 and CwlJ2 to Ca²⁺-dipicolinic acid (DPA)-triggered germination were revealed.