Genomic profiling and experimental validation of type VI secretion system-associated proteins in Klebsiella

Klebsiella is widely distributed in nature and recognized for its dual role as a human commensal and an opportunistic pathogen capable of causing severe infections. Among its key virulence mechanisms, the Type VI Secretion System (T6SS) plays a critical role in microbial competition, interspecies interactions, and environmental adaptation. In this study, we investigated a representative set of high-quality Klebsiella genomes and identified two major T6SS loci (T6SSkleb1 and T6SSkleb2), which exhibit distinct structural features and are predominantly found within the Klebsiella pneumoniae species complex (KpSC). Comparative genomics further revealed the diverse distribution of effector and immunity proteins and their correlation with T6SS loci. Based on this distributional bias, we developed a novel computational approach to identify protein families significantly associated with T6SS. These T6SS-associated gene clusters were implicated in mediating adhesion to host cell surfaces during urinary tract infections, as well as in metabolism and immune defense. Moreover, we identified three putative orphan effectors harboring DUF3258, DUF3751, and Sel1 domains. Overexpression of these proteins in Escherichia coli resulted in cytotoxic effects, supporting their potential as T6SS effectors. These findings establish a comprehensive framework for T6SS analysis, aiming to expand and enrich our understanding of the diversity, evolutionary trajectory, and functional implications of T6SS in Klebsiella.