A Chryseobacterium massiliae pore-forming MACPF domain protein mediates intra and interspecies competition against Bacteroides

Microbiota play crucial roles in host health, including protection against pathogens through competitive interactions between commensal and pathogenic bacteria that are mediated by direct contact or secreted factors. We previously demonstrated that Chryseobacterium massiliae, a zebrafish commensal, protects larvae against infection by Flavobacterium covae (formerly F. columnare). Here, we investigated whether interference interactions contribute to this protective effect. We found that C. massiliae culture supernatant inhibits F. covae growth and a transposon mutagenesis screen identified mutants lacking this activity. All identified mutants carried insertions in a gene encoding a protein homologous to Bacteroidales BSAP pore-forming toxins, characterized by a Membrane Attack Complex/Perforin (MACPF) domain. We showed that this protein, which we named CSAP-1 (for Chryseobacterium Secreted Antimicrobial Protein) displays bactericidal, pore-forming activity that lyses F. covae cells. Unlike BSAP proteins from Bacteroides spp., CSAP-1 displays broader antagonistic activity, targeting multiple species across the Flavobacterium and Chryseobacterium genera - thus mediating interspecies and intergenus inhibition within Bacteroidetes. Although CSAP-1 is not essential for the in vivo protective effect of C. massiliae, administration of purified CSAP-1 alone confers significant protection to zebrafish larvae against sensitive F. covae infection. This study therefore identifies CSAP-1 as the first MACPF protein from C. massiliae with broad-spectrum inhibitory activity against members of the order Flavobacteriales. These findings highlight CSAP-1 as a promising candidate for the development of novel antimicrobial strategies and warrant further mechanistic investigation.