Functional analyses of the cell wall-binding domain of a major autolysin in Clostridium perfringens

S. Katayama, R. Aono, M. Shiraga, N. Matsunaga, H. Nariya, H. Sekiya, E. Tamai

The 14th International Conference on the Molecular Biology and Pathogenesis of Clostridia (Clostpath 14)

Institute Pasteur

Paris, France

https://www.clostpath2025.conferences-pasteur.org/

Bacterial cells degrade and reorganize peptidoglycans during division, a process facilitated by autolysins, which are peptidoglycan-hydrolyzing enzymes. Understanding the molecular mechanism of peptidoglycan reorganization is crucial for bacterial cell division and the development of novel antimicrobial agents. Clostridium perfringens, an obligate anaerobe causing food poisoning and gas gangrene, has Acp (CPE1231) as its major autolysin, thought to be essential for cell division. Acp is a 120-kDa polypeptide with a cell wall-binding domain (containing 10 SH3b motifs, cell wall-binding repeats [CWBR]) at the N-terminus and a catalytic domain (CD) at the C-terminus, homologous to glucosaminidase [1]. The structures of the CD and the sixth CWBR have been previously reported [2, 3]. This study reports on the localization of Acp and the functional role of the CWBR. To determine how many CWBRs are needed for cell division, we constructed an Acp null mutant (HN13 ∆acp) and expression vectors to express mutated Acps with varying CWBRs or just the CD (AcpCD). Expression of these mutated Acps was confirmed by western blotting with an anti-AcpCD antibody. The amount of mutated Acp bound to cells decreased as the number of CWBRs decreased, especially for mutants with a seventh or later CWBR. The localization of wild type (CWBR1-10+CD) and the mutated Acps was analyzed by fluorescence microscopy using anti-AcpCD antibody. Wild type and most mutated Acps localized to the septa of mitotic cells, but the signals weakened in CWBR9-10+CD and disappeared entirely in CWBR10+CD and AcpCD. To assess the impact on cell division, we Gram-stained C. perfringens cells expressing different mutated Acps and observed cell length and cellular condition. Compared to the CWBR1-10+CD, the number of undivided cells was significantly increased in HN13 ∆acp expressing CWBR10+CD or AcpCD. Cell length was also increased in Acp mutants lacking the fifth and further CWBRs. These findings suggest that at least two CWBRs are required for Acp to bind to the septum and promote effective cell division.
1. Camiade, E., et al. J Bacteriol. 2010. 192:2373-2384.
2. Tamai, E., et al. FEBS Lett. 2017 591:231-239. 
3. Shan, Y., et al. Molecules 2021. 26:5716.