Analysis of fibronectin-binding protein D(FbpD), a putative peptidoglycan hydrolase in Clostridium perfringens

N. Matsunaga, K. Mrimoto, R. Kazaore, Y. Hitsumoto, S. Katayama

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

Institut Pasteur

Paris, France

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

Clostridium perfringens is a gram positive, rod-shaped, anaerobic bacterium
known to cause gas gangrene. To infect a host, C. perfringens invades through a
wound or surgical injury, ultimately leading to the development gas gangrene in
untreated infections. This infection is considered to be the binding of C.
perfringens cells to extracellular matrix proteins (ECM), including fibronectin (Fn).
Our study reported that C. perfringens on cells surface expressed that two
proteins, FbpC (CPE0625) and FbpD (CPE0630), bound to Fn [1 ]. However,
both FbpC and FbpD were not main Fn-binding proteins, because both fbpC
and/or fbpD deficient strains similarly bound to Fn compared with their parents'
strain [2]. Bioinformatic analysis using Pfam predicted that FbpD contains signal
peptides, 10 putative cell wall-binding repeats, and NlpC/P60 domain,
characteristic of peptidoglycan hydrolases. We therefore focused on role of FbpD
in peptidoglycan hydrolase, performed pull-down assay, SOS-PAGE reduction
assay (zymography), turbidity reduction assay, observation of bacterial
morphology and western blotting assay. The pull-down assay confirmed FbpD's
association with the cell wall. Zymography assay and turbidity reduction assay
demonstrated that FbpD exhibits peptigoglycan hydrolase activity. Zymography
of recombinant FbpD (rFbpD) using several bacterial cell wall components from
C. perfringens, Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis.
rFbpD has sufficient lytic activity in C. perfringens and S. aureus, but not M.
luteus and B. subtilis. These findings suggest that FbpD cleaves the
peptidoglycan between glycine and D-alanine. Furthermore, observation of
bacterial morphology showed that FbpD involve in maintaining normal
morphology. Western blotting confirmed that FbpD is constitutively expressed on
the bacterial surface regardless of growth phase. Our findings suggest that FbpD
functions as a peptidoglycan hydrolase and may contribute to C. perfringens cell
wall remodeling and maintenance.
1. Katayama S, et al. lnt J Anal Bio-Sci. 2015. 3: 1-9
2. Aono R, et la. Anaerobe 2023. 83 doi: 10.1016/j.anaerobe.2023.102769.