The SPI-6 T6SS gene cluster from Salmonella Tennessee encodes a new antibacterial nuclease effector protein

The type VI secretion system (T6SS) is a contact-dependent multiprotein apparatus widely distributed in Gram-negative bacteria that contributes to interbacterial competition and pathogenesis via a contractile mechanism. In Salmonella, five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21 and SPI-22, which are differentially distributed among serotypes. One of the most studied and widely distributed T6SS corresponds to that encoded in SPI-6 (T6SS_SPI-6), which contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, there is still limited information available regarding the total number of effector proteins encoded within SPI-6 of different Salmonella enterica serotypes. In the present study, we characterized the SPI-6 T6SS gene cluster encoded in Salmonella enterica subspecies enterica serotype Tennessee (S. Tennessee), a pathogen frequently associated with foodborne gastrointestinal outbreaks. Interbacterial competition assays demonstrated that T6SS_SPI-6 of S. Tennessee displays antibacterial activity. Additionally, we performed comparative genomic and bioinformatic analyses and identified an antibacterial Effector/Immunity protein (E/I) module encoding a putative effector with DNase activity (RhsA-HNHc) and its cognate immunity protein within the variable region 3 (VR3) of the SPI-6 T6SS gene cluster. Interbacterial competition assays confirmed the antibacterial activity of this novel E/I pair. In addition, heterologous expression assays showed that induction of the RhsA-HNHc effector led to significant E. coli growth inhibition, while co-expression with its putative immunity protein fully restored bacterial growth, thus demonstrating protection against toxicity. Finally, a nuclease activity assay demonstrated that RhsA-HNHc possesses DNase activity. Altogether, this study expands the experimentally validated SPI-6 T6SS effector repertoire beyond well-studied Salmonella serotypes, providing the first functional characterization of a DNase-type Rhs effector in S. Tennessee.