Pan-genome insights into type VI secretion systems and their functional repertoires in Enterobacter

The Enterobacter genus contains 23 species that include common nosocomial pathogens capable of causing a wide variety of infections. We obtained all available Enterobacter genomes and retained 4,805 high-quality genomes after quality control. Genome sequencing analysis of Enterobacter species revealed the presence of type VI secretion systems (T6SS) in these bacteria, but systematic analysis and comparison of these systems among different species are limited. We found that these bacteria code for three distinct types of T6SS, each with a unique set of diverse predicted effectors. Whereas at least 14 effectors are found in each strain, the number of immunity proteins is considerably fewer. By demonstrating a correlation between the abundance of known T6SS-associated proteins and the presence of T6SS, we proposed a comparative genomics model to evaluate the correlation between unknown T6SS-associated ortholog proteins and T6SSs. Among the homologous groups most strongly associated with T6SS, we potentially identified several effectors. It is conceivable that our methodology could be scaled to survey additional bacterial genera for novel T6SS effectors, thereby providing fresh perspectives and directions for subsequent biological experiments.IMPORTANCEEnterobacter species are important human pathogens that can cause severe conditions like pneumonia, urinary tract infections, and bacteremia. As Gram-negative bacteria, they frequently carry diverse T6SS loci, which are often associated with bacterial virulence and are also one of the important causes of bacterial infection. T6SS effectors play a critical role in interbacterial competition and virulence during infection. VgrG proteins are essential T6SS components that form the spike structure and mediate effector delivery, making them critical for bacterial competition and virulence. However, systematic studies on their distribution and function remain limited. Here, we analyzed all available high-quality Enterobacter genomes and revealed that T6SS diversity is shaped by both species' evolution and horizontal gene transfer (HGT). We proved that it is feasible to measure the biological relevance of unknown functional proteins to the T6SS through statistical analyses. This high-throughput approach provides a new perspective for future research on T6SS functionality, especially in Enterobacter.