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Universität Tübingen (2006)



Peptidoglycan (PG) hydrolases or autolysins are a group of enzymes which catalyze the degradation of bacterial cell wall at specific sites. Staphylococcus aureus produces two major PG hydrolases: major autolysin (Atl) and Aaa, a autolysin/ adhesin protein. The major autolysins of Staphylococcus aureus (AtlA) and of Staphylococcus epidermidis (AtlE) are well-studied enzymes. But little is known about the Aaa protein. To analyse the possible role of these PG hydrolases we constructed the atlA and aaa deletion mutants in S. aureus. SA?atlA formed large cell clusters and was biofilm-negative owing to a deficiency in adherence to the indwelling device surface. In electron micrographs, the mutant cells were distinguished by a rough outer cell surface. A high proportion of abnormally formed multicells that were septated but not separated from each other were observed, which suggested hampered cell separation. Both atlA and atlE complemented the mutant. The atl gene product is a bifunctional protein that has an N-terminal N-acetyl L-alanine amidase (Ami) domain, three internal repeat domains (R1, 2, 3) and a C- terminal endo-ß-N-acetylglucosaminidase (GL) domain which undergo proteolytic processing to generate the two extracellular lytic enzymes (62 kDa Ami-R1, 2 and 51 kDa R3-GL) found in the culture broth of S. aureus. In the mature protein repeats R1 and R2 are located at the C-terminal portion of the amidase (Ami-R1, 2) and repeat R3 is located at N-terminal portion of the glucosaminidase (R3-GL). To study the role of the repetitive sequences of atlE, we expressed in Escherichia coli the amidase domain encoded by the gene, carrying no repeat regions (amiE) or two repeat regions (amiE-R1, 2), or the three repeat regions alone (R1, 2, 3) as N-terminal His-tag fusion proteins. Only slight differences in the cell wall lytic activity between AmiE and AmiE-R1, 2 were observed. The repetitive sequences have a good binding affinity to isolated peptidoglycan and might contribute to the targeting of the amidase to the substrate. AmiE and AmiE-R1, 2 have a broad substrate specificity as shown by similar activities with peptidoglycan (PG) lacking wall teichoic acid, O-acetylation, or both. Since the amidase activity of AtlA and AtlE has not been proved biochemically, we used purified AmiE-R to determine the exact PG cleavage site. We provide the first evidence that the amidase indeed cleaves the amide bond between N-acetyl muramic acid and L-alanine. SA?aaa mutant did not differ from the wild type in its colony morphology, growth rate, cell cluster and biofilm formation, suggesting that Aaa does not play a vital role in cell separation or, more probably, that the function of Aaa in cell separation may have been taken over by the major autolysin Atl, which seemed to be more strongly expressed in the aaa mutant than in the wild type. Autolysin/adhesin protein Aaa is a 35 kDa protein containing two direct LysM (lysine motif) repeats at the N-terminal and catalytic domain in the C-terminus. The C-terminal catalytic domain of Aaa is homologous to the CHAP (cysteine, histidine-dependent amidohydrolases/ peptidases) domain. This domain is often found in PG hydrolysing enzymes. This work also established a purification method for isolation of soluble defined staphylococcal peptidoglycan fragments (PGs). Compared to earlier methods, which were based on using insoluble purified peptidoglycan, we standardize the purification method soluble PGs using HPLC. Commercially available mutanolysin and lysostaphin can be used to cleave PG structures. Apart from these two lytic enzymes, we biochemically characterized the amidase, which provides an alternative tool for PG analysis. Mutanolysin, lysostaphin and amidase can be used individually or together to isolate muropeptides, stem peptides or sugar residues for studying host cell signaling activities.

Cite this Research Publication

Dr. Raja Biswas and , “Characterization of Staphylococcus aureus peptidoglycan hydrolases and isolation of defined peptidoglycan structures”, Universität Tübingen, 2006.

NIRF 2017