Transcriptional regulator MarT negatively regulates MarT-regulated motility gene I, a new gene involved in invasion and virulence of Salmonella enterica

The speciation of <i>Salmonella</i> occurred by acquisition of genomic islands from other bacterial species and continued to diverge into subspecies and serovars with diferent range of host. <i>S. enterica</i> serovar Typhimurium (STM) is a generalist pathogen infecting hosts that include birds, mice, and humans, whilst <i>S. enterica</i> serovar Typhi (STY) is a restricted-host pathogen, infecting only humans. Despite their ranges of hosts, STM and STY possess 97-98% identity. Gain of genes by horizontal transference and loss of genes by mutations, are believed essential for differentiation of <i>Salmonella</i>. <i>Salmonella</i> pathogenicity island 3 (SPI-3) is an example combining these two processes. SPI-3 encodes <i>misL</i> and <i>marT</i>, among other genes. In STM, <i>misL</i> is required for gut colonization. Furthermore, protein MarT, positively regulates expression of misL by binding to <i>misL</i>-promoter. On the other hand, in SPI-3 of STY, <i>marT</i> and <i>misL</i> are pseudogenes. Interestingly, the gene t3766 (gene involved in resistance to H₂O₂) is present only in STY and is negatively regulated when <i>marT</i> _STM is heterologously expressed in STY. Based on the view that MarT might regulate genes implicated in virulence, this work searched for new genes regulated by MarT. <i>In silico</i> searches for possible MarT target genes were performed, and 4 genes were selected for further analysis as they contained at least 2 copies of the consensus MarT-binding sequence in their promoters. Mutating <i>marT</i> in STM or heterologously expressing <i>marT</i> _STM in STY confirmed that MarT negatively regulates ORF STY1408 or STM14_2003, its homologue in STM. STY1408 encodes for a putative protein with homology to methyl accepting chemotaxis proteins, which participate in chemotaxis and motility. Therefore, STY1408 was named <i>mrmI</i> (MarT-regulated motility gene I). Motility assays confirmed that the product of <i>mrmI</i> modulates motility. In addition, <i>in vitro</i> infection of cells with STM and STY mutants in <i>mrmI</i> reduces association with cells at 1, 3 and 24 h post-infection. Oral infection of mice showed that a <i>mrmI</i> null mutant was defective in producing systemic disease. Therefore, we conclude that MarT regulated <i>mrmI</i>, is involved in virulence of <i>Salmonella</i>. While pseudogenization of <i>marT</i> might modulate the fitness of narrow host range STY.