One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator.

Unlabelled: VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF (30 kDa), and the shorter VirF (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF and VirF and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF is responsible for activation of the virulence system, VirF negatively autoregulates virF expression itself. Since VirF modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA.

Importance: Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF and VirF, that are functionally distinct. The major form, VirF, activates the genes necessary for virulence, whereas the minor VirF, which shares the C-terminal two-thirds of VirF, negatively autoregulates virF expression itself. VirF is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis.