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

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-δ-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-δ-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.