One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator Maria Letizia Di Martino, a Cédric Romilly, b E. Gerhart H. Wagner, b Bianca Colonna, a Gianni Prosseda a Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Biology and Biotechnology C. Darwin, Sapienza Università di Roma, Rome, Italy a ; Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Uppsala, Sweden b ABSTRACT 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 (30 kDa), and the shorter VirF 21 (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF 30 and VirF 21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF 21 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 21 . The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF 30 is responsible for activation of the virulence system, VirF 21 negatively autoregulates virF expression itself. Since VirF 21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the viru- lence 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 pro- gram 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 30 and VirF 21 , that are functionally distinct. The major form, VirF 30 , activates the genes necessary for virulence, whereas the minor VirF 21 , which shares the C-terminal two-thirds of VirF 30 , negatively autoregulates virF expression itself. VirF 21 is transcribed from a newly identified gene-internal promoter and, more- over, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the reg- ulation of the Shigella invasive process and may help development of new therapies for shigellosis. Received 8 October 2016 Accepted 11 October 2016 Published 8 November 2016 Citation Di Martino ML, Romilly C, Wagner EGH, Colonna B, Prosseda G. 2016. One gene and two proteins: a leaderless mRNA supports the translation of a shorter form of the Shigella VirF regulator. mBio 7(6):e01860-16. doi:10.1128/mBio.01860-16. Editor Bonnie Bassler, Princeton University Copyright © 2016 Di Martino et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Gianni Prosseda, gianni.prosseda@uniroma1.it, or E. Gerhart H. Wagner, gerhart.wagner@icm.uu.se. This article is a direct contribution from a Fellow of the American Academy of Microbiology. External solicited reviewers: Carmen Buchrieser, Pasteur Institute, Paris, France; Charles Dorman, University of Dublin, Trinity College. S higella spp. are highly adapted human pathogens that cause bacillary dysentery (1). The sophisticated infectious strat- egy of Shigella depends on the capacity to invade, disrupt, and cause inflammatory destruction of the intestinal epithelial bar- rier (2, 3). Activation of the invasive program is exceptionally complex and involves many signals affecting gene regulation at different levels. A key factor is VirF, an AraC-like transcription factor (TF) whose expression is activated as Shigella bacteria sense entry into the host environment (4, 5). In a cascade model, VirF triggers activation of the virB and icsA genes. IcsA affects bacterial intracellular spreading, and VirB promotes ex- pression of several virulence genes, including those encoding a type III secretion system (T3SS), its effectors, and the last reg- ulator of the cascade, MxiE (6, 7). Interestingly, MxiE, another AraC-like TF, appears to rely on high-level transcriptional slip- page to generate its reading frame from two separate open reading frames (ORFs) (8). The genes virF, icsA, virB, and those controlled by VirB are located on a virulence plasmid (pINV) and are silenced outside the human host (9). At low temperatures, the nucleoid-associated protein H-NS represses transcription of the virulence genes (5). In a temperature- dependent manner, H-NS interacts with two sites within the virF promoter, spaced by an intrinsically curved DNA region, to prevent access of RNA polymerase (5, 10, 11). At a permis- sive temperature (37°C), reduced DNA curvature counteracts H-NS binding (4, 12) and unmasks a binding site for the nu- cleoid protein FIS to activate virF transcription (13). VirF then relieves H-NS-mediated repression of virB and icsA and di- rectly stimulates transcription (14, 15). By binding upstream of the virF promoter between two H-NS sites, VirB also counter- acts H-NS-dependent repression of virF transcription (16). Ex- pression of virF is further modulated by integration host factor RESEARCH ARTICLE crossmark November/December 2016 Volume 7 Issue 6 e01860-16 ® mbio.asm.org 1 Downloaded from https://journals.asm.org/journal/mbio on 15 February 2022 by 3.236.103.33.