The Bacillus anthracis SleL (YaaH) Protein Is an N -Acetylglucosaminidase Involved in Spore Cortex Depolymerization

Abstract

Bacillus anthracis spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for B. anthracis spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of sleL (yaaH), which encodes a cortex-lytic enzyme. The inactivation of sleL does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and N-acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an N-acetylglucosaminidase. When sleL is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the sleL-encoded N-acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.

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

“…3). These muropeptides are tetrasaccharide-tetrapeptide and tetrasaccharide-alanine products, respectively, that result from N-acetylglucosaminidase digestion (Dowd et al, 2008;Lambert & Popham, 2008). Digestion with excess SleB produces different muropeptide products (Heffron et al, 2011).…”

“…When one enzyme was used alone there were no detectable muropeptides produced. However, when PG sacculi were partially broken down with SleB and then further digested with SleL, Nacetylglucosaminidase products G6 (tetrasaccharide-tetrapeptide) and G7 (tetrasaccharide-alanine) (Dowd et al, 2008;Lambert & Popham, 2008) were evident. .…”

“…SleL is an N-acetylglucosaminidase that breaks b-1,4 glycosidic bonds found in cortical PG (Chen et al, 2000;Lambert & Popham, 2008). Previous studies show that sleL is expressed under the control of s E , which is active in the mother cell during early stages of sporulation (Chen et al, 2000;Kodama et al, 1999).…”

“…A second subset of GSLEs termed cortical fragment lytic enzymes (CFLEs) advance the germination cascade (Makino & Moriyama, 2002). One such protein, SleL, further digests the cortex PG, and small muropeptide fragments are released from the spore (Heffron et al, 2010;Lambert & Popham, 2008). Cortex hydrolysis allows for complete core rehydration and loss of spore-associated resistance characteristics.…”

“…Germination has been studied in an array of Gram-positive spore-forming species including Clostridium perfringens (Chen et al, 1997;Miyata et al, 1995), Bacillus thuringiensis (Hu et al, 2007), Bacillus subtilis (Ishikawa et al, 1998;Kodama et al, 1999;Moriyama et al, 1996a), Bacillus megaterium (Foster & Johnstone, 1987;Setlow et al, 2009), Bacillus cereus (Chen et al, 2000;Makino et al, 1994;Moriyama et al, 1996b) and B. anthracis (Heffron et al, 2009(Heffron et al, , 2010Lambert & Popham, 2008;Liu et al, 2004). The B. anthracis germination cascade begins when nutrient germinants contact receptors within the spore.…”