The SPI-20 and SPI-21 T6SS gene clusters from Salmonella enterica subspecies arizonae encode effector proteins that display antibacterial activity

The type VI secretion system (T6SS) is a contact-dependent multiprotein apparatus that contributes to interbacterial competition and pathogenesis in many Gram-negative bacteria. Salmonella harbors five T6SS gene clusters within the pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22, differentially distributed among serotypes. Salmonella enterica subspecies arizonae (S. arizonae) is most frequently associated with reptiles but, in some circumstances, can cause disease in mammals, including humans. Notably, although it encodes both T6SS SPI-20 and T6SS SPI-21, no report to date has demonstrated the antibacterial activity of either system. In addition, only two putative effector proteins have been previously predicted, though they have not been experimentally validated. In the present study, we demonstrate that both T6SS SPI-20 and T6SS SPI-21 contribute to interbacterial competition in S. arizonae when grown on McConkey agar plates, suggesting a role for bile in T6SS SPI-20 and T6SS SPI-21 activity. In addition, through bioinformatic analyses, interbacterial competition assays, and heterologous expression, we further characterize the antibacterial activity of a novel antibacterial E/I module (SARI₀2625/SARI₀2624, encoded in SPI-21) that, in addition to the previously identified evolved VgrG protein SARI₀2603 and VgrG2b homolog SARI₀2727, contribute to T6SS-dependent antibacterial competition in S. arizonae.