Bacillus cereus is a widespread Gram-positive pathogenic bacterium capable of producing toxins that induce severe illness or mortality in humans and animals. Cyclic diguanylate monophosphate (c-di-GMP), a bacterial second messenger, governs critical virulence traits such as biofilm formation and motility. While its regulatory role has been extensively characterized in Gram-negative bacteria, the functional significance of c-di-GMP in bovine pathogenic B. cereus remains poorly understood. Genomic analysis of the studied strain revealed a 5,484,345 bp chromosome and a 609,501 bp plasmid, harboring six c-di-GMP metabolism-associated genes (five encoding GGDEF domain-containing proteins and one encoding an EAL domain-containing protein). Targeted gene knockout studies demonstrated that deletion of dgc1 and eal significantly impaired biofilm formation, whereas disruption of dgc3 and dgc5 reduced motility. Notably, dgc5 inactivation paradoxically enhanced biofilm development. No discernible phenotypic alterations were observed in dgc2 or dgc4 mutants. In murine infection models, eal-deficient strains accelerated host mortality. Histopathological examination confirmed organ damage induced by all mutant strains. This study provides the first systematic evidence highlighting the pivotal role of c-di-GMP metabolic genes in modulating virulence pathways of bovine pathogenic B. cereus, offering novel targets for therapeutic intervention.

To Cite This Article: Jia Z, Meng Q, Wang H, Yu M, Zhang X, Cui J, Xiong H, Zhang Y and Wang X, 2025. Functional analysis of c-di-AMP metabolic proteins in a bovine-pathogenic bacillus cereus isolate. Pak Vet J. http://dx.doi.org/10.29261/pakvetj/2025.296