Type VI secretion systems (T6SS) are macromolecular machines produced by many Gram-negative bacteria that play important roles in interbacterial competition. The range of bacteria targeted by the A. baumannii T6SS, and the specific toxins required for killing bacteria, are currently unknown and are the focus of this study.
Initially we inactivated a core component of the T6SS (TssM). Interbacterial competition assays showed that the wild-type A. baumannii AB307-0294 strain was able to efficiently kill E. coli Dh10β, Acinetobacter baylii and the A. baumannii strain ATCC19606. In contrast, the tssM mutant showed a significant reduction in the ability to kill all three strains; wild-type levels of killing were restored by complementation with an intact tssM gene. Bioinformatic analysis of the AB307-0294 genome identified three putative vgrG genes, each located upstream of a putative toxin and immunity gene pair. We independently inactivated each of the three vgrG genes and showed all mutants were able to kill A. baylyii as efficiently as the parent strain. We then cloned two of the putative toxin genes (rhs1 and rhs2) independently into the arabinose inducible E. coli expression vector pBAD30. E. coli containing pBAD30::rhs1 or pBAD30::rhs2 when grown on solid medium when expression of the putative toxin genes was repressed by addition of glucose but could not grow when expression was induced by addition of arabinose. Bioinformatic analyses indicated that all sequenced A. baumannii strains encode genes for T6SS expression and contain between 2 and 4 vgrG genes; however specific toxin/immunity genes show significant strain variation.
A. baumannii AB307-0294 displays T6SS-specific intra-species and inter-species killing. The T6SS associated rhs1 and rhs2 genes encode proteins which are lethal to E. coli. We are currently assessing the precise functional mechanisms by which these toxins act and are delivered to target cells.