In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

Abstract

The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein.SleL is a spore cortex lytic enzyme composed of three conserved domains: two N-terminal LysM domains and a C-terminal glycosyl hydrolase family 18 domain. Derivatives of SleL containing both, one or no LysM domains were purified and characterized. SleL is incapable of digesting intact cortical PG of either decoated spores or purified spore sacculi. However, SleL derivatives can hydrolyse fragmented PG substrates containing muramic-d-lactam recognition determinants. The muropeptides that result from SleL hydrolysis are the products of N-acetylglucosaminidase activity. These muropeptide products are small and readily released from the cortex matrix. Loss of the LysM domain(s) decreases both PG binding and hydrolysis activity but these domains do not appear to determine specificity for muramic-d-lactam. When the SleL derivatives are expressed in vivo, those proteins lacking one or both LysM domains do not associate with the spore. Instead, these proteins remain in the mother cell and are apparently degraded. SleL with both LysM domains localizes to the coat or cortex of the endospore. The information revealed by elucidating the role of SleL and its domains in B. anthracis sporulation and germination is important in designing new spore decontamination methods. By exploiting germination-specific lytic enzymes, eradication techniques may be greatly simplified.

Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

“…Isolated RNAs were verified as being free from genomic DNA by conducting PCRs with the same ypeB-specific primers (data not shown). (28)(29)(30). Strikingly, the OD 600 losses and colony-forming ability of B. megaterium sleB cwlJ sleL spores complemented with a plasmid-borne copy of ypeB (GC126) were observed to be largely commensurate with values obtained with sleB cwlJ and sleB cwlJ sleL spores (Table 1 and Fig.…”

Activity and Regulation of Various Forms of CwlJ, SleB, and YpeB Proteins in Degrading Cortex Peptidoglycan of Spores of Bacillus Species In Vitro and during Spore Germination

“…However, the presence of sleL in B. megaterium cwlJ sleB or B. subtilis cwlJ sleB spores resulted in more-rapid cortex hydrolysis and DPA release due to the added SleB C and to SleB M 's more rapid DPA release from decoated B. megaterium cwlJ sleB spores. These results are consistent with SleL playing an auxiliary role in cortex PG hydrolysis, although there are several caveats to this conclusion, as follows: (i) it appears possible that SleL is lost or inactivated when B. subtilis spores are incubated in 0.25 N NaOH, similar to the NaOH concentration used in spore decoating, as production of likely SleL products during spore germination is greatly reduced (42); (ii) the great majority, but perhaps not all, of the SleL in B. anthracis spores is located in spores' outer layers, perhaps the coat/cortex boundary, where it would presumably be removed by decoating procedures, and a decoating treatment removes most SleL from B. cereus spores (8,(24)(25)(26)41). Thus, it is possible that SleL is either inactive or not present in the decoated B. subtilis spores used in the current work for analysis of the activity of SleB forms.…”

“…Perhaps YpeB prevents premature or misplaced activation of SleB and proteolysis of YpeB during germination results in SleB activation. The second complicating factor is that spores of Bacillus species contain a third CLE, SleL (initially called YaaH), which hydrolyzes fragmented PG substrates containing MAL (24)(25)(26). While SleL alone cannot initiate cortex PG hydrolysis in vivo, perhaps SleL can facilitate cortex hydrolysis initiated by SleB and/or CwlJ sufficiently to increase the rate of spore germination.…”

Role of YpeB in Cortex Hydrolysis during Germination of Bacillus anthracis Spores

“…Identical roles for SleB and CwlJ have been demonstrated in Bacillus subtilis (26)(27)(28) and Bacillus megaterium (29). CwlJ2, a homolog of CwlJ1, appears to play a minor role at best during B. anthracis spore germination, while SleL further breaks down PG fragments first generated by SleB and/or CwlJ1 (20,(22)(23)(24)(25).…”

“…These enzymes exhibit specificity for PG containing the muramic-␦-lactam modification, ensuring that only the cortex PG is broken down (17)(18)(19)(20)(21). Bacillus anthracis contains the four GSLEs SleB, CwlJ1, CwlJ2, and SleL; SleB and CwlJ1 are partially redundant enzymes responsible for the majority of cortex hydrolysis (22)(23)(24)(25).…”