Distribution and diversity of type VI secretion system clusters in Enterobacter bugandensis and Enterobacter cloacae

Gram-negative bacteria use type VI secretion systems (T6SSs) to antagonize neighbouring cells. Although primarily involved in bacterial competition, the T6SS is also implicated in pathogenesis, biofilm formation and ion scavenging. Enterobacter species belong to the ESKAPE pathogens, and while their antibiotic resistance has been well studied, less is known about their patho-genesis. Here, we investigated the distribution and diversity of T6SS components in isolates of two clinically relevant Entero-bacter species, E. cloacae and E. bugandensis. T6SS clusters are grouped into four types (T6SSi-T6SSiv), of which type i can be further divided into six subtypes (i1, i2, i3, i4a, i4b, i5). Analysis of a curated dataset of 31 strains demonstrated that most of them encode T6SS clusters belonging to the T6SSi type. All T6SS-positive strains possessed a conserved i3 cluster, and many harboured one or two additional i2 clusters. These clusters were less conserved, and some strains displayed evidence of dele-tion. We focused on a pathogenic E. bugandensis clinical isolate for comprehensive in silico effector prediction, with comparative analyses across the 31 isolates. Several new effector candidates were identified, including an evolved VgrG with a metal-lopeptidase domain and a Tse6- like protein. Additional effectors included an anti-eukaryotic catalase (KatN), M23 peptidase, PAAR and VgrG proteins. Our findings highlight the diversity of EnterobacterT6SSs and reveal new putative effectors that may be important for the interaction of these species with neighbouring cells and their environment.