The Intestinal Microbiota Interferes with the microRNA Response upon Oral Listeria Infection

The intestinal tract is the largest reservoir of microbes in the human body. The intestinal microbiota is thought to be able to modulate alterations of the gut induced by enteropathogens, thereby maintaining homeostasis. Listeria monocytogenes is the agent of listeriosis, an infection transmitted to humans upon ingestion of contaminated food. Crossing of the intestinal barrier is a critical step of the infection before dissemination into deeper organs. Here, we investigated the role of the intestinal microbiota in the regulation of host protein-coding genes and microRNA (miRNA or miR) expression during Listeria infection. We first established the intestinal miRNA signatures corresponding to the 10 most highly expressed miRNAs in the murine ileum of conventional and germfree mice, noninfected and infected with Listeria. Next, we identified 6 miRNAs whose expression decreased upon Listeria infection in conventional mice. Strikingly, five of these miRNA expression variations (in miR-143, miR148a, miR-200b, miR-200c, and miR-378) were dependent on the presence of the microbiota. In addition, as is already known, protein-coding genes were highly affected by infection in both conventional and germfree mice. By crossing bioinformatically the predicted targets of the miRNAs to our whole-genome transcriptomic data, we revealed an miRNA-mRNA network that suggested miRNA-mediated global regulation during intestinal infection. Other recent studies have revealed an miRNA response to either bacterial pathogens or commensal bacteria. In contrast, our work provides an unprecedented insight into the impact of the intestinal microbiota on host transcriptional reprogramming during infection by a human pathogen. IMPORTANCE While the crucial role of miRNAs in regulating the host response to bacterial infection is increasingly recognized, the involvement of the intestinal microbiota in the regulation of miRNA expression has not been explored in detail. Here, we investigated the impact of the intestinal microbiota on the regulation of protein-coding genes and miRNA expression in a host infected by L. monocytogenes, a food-borne pathogen. We show that the microbiota interferes with the microRNA response upon oral Listeria infection and identify several protein-coding target genes whose expression correlates inversely with that of the miRNA. Further investigations of the regulatory networks involving miR-143, miR-148a, miR-200b, miR-200c, and miR-378 will provide new insights into the impact of the intestinal microbiota on the host upon bacterial infection. Received 27 August 2013 Accepted 12 November 2013 Published 10 December 2013 Citation Archambaud C, Sismeiro O, Toedling J, Soubigou G, Bécavin C, Lechat P, Lebreton A, Ciaudo C, Cossart P. 2013. The intestinal microbiota interferes with the microRNA response upon oral Listeria infection. mBio 4(6):e00707-13. doi:10.1128/mBio.00707-13. Editor Jeff Miller, UCLA School of Medicine Copyright © 2013 Archambaud et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Pascale Cossart, pascale.cossart@pasteur.fr, or Cristel Archambaud, christel.archambaud@jouy.inra. fr. Listeria monocytogenes is a Gram-positive food-borne pathogen that infects both humans and animals and has emerged as a model organism in infection biology and host-bacterium interaction studies (1). Infection starts with the ingestion of contaminated food. Then, L. monocytogenes crosses the intestinal barrier to reach the lymph and the bloodstream, allowing the bacterium to disseminate in the whole organism. To remain hidden from the innate and adaptive immune systems and to dampen the host inflammatory response, L. monocytogenes deploys an arsenal of virulence factors (2). L. monocytogenes peptidoglycan N-deacetylase and O-acetyltransferase play a role in inhibition of signaling cascades that allows Listeria to evade the host innate immune system (3–5). The L. monocytogenes surface protein ActA and the internalin InlK prevent bacterial recognition by the cell autophagic machinery (6–8). InlC, another member of the internalin family, by interacting with IKK , a subunit of the I B kinase complex critical for the phosphorylation of I B and activation of NFB, dampens the host innate response induced by Listeria infection (9). Listeriolysin O (LLO) is a secreted hemolysin. Besides its role in allowing escape from the phagosome, LLO causes T lymphocyte apoptosis and a variety of other signaling events (10, 11). Conversely, the host uses a combination of cellular and molecular mechanisms for clearance of the invader and recovery from possible damage (12). Among these, a fast reprogramming of the host transcriptome with expression of immune-related genes is thought to be crucial to mount an efficient antibacterial defense. Strikingly, the mechanisms by which both the bacterium and RESEARCH ARTICLE November/December 2013 Volume 4 Issue 6 e00707-13 ® mbio.asm. org 1 m b.asm . rg on D ecem er 7, 2013 P ubished by m b.asm . rg D ow nladed fom the host cell reprogram their transcription during infection involve RNA-mediated regulation through small noncoding RNA molecules. Research highlighting the multiple roles played by regulatory noncoding RNAs in both eukaryotes and prokaryotes has exploded during the last decade. Noncoding RNAs have emerged as major regulators of various biological processes, including virulence (13, 14). In 2009, the first Listeria whole-genome transcriptomic analysis revealed that, when Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs, relying on a sigma B-mediated activation of specific virulence genes. In contrast, in the blood, the function of the master regulator of Listeria virulence genes, PrfA, dominates transcriptional reprogramming. Remarkably, several noncoding RNAs, absent in the nonpathogenic species Listeria innocua, exhibit the same expression patterns as the L. monocytogenes virulence genes (15). Together, these data have unraveled successive and coordinated global transcriptional changes during infection. In eukaryotes, microRNAs (miRNAs or miRs) are single-stranded RNAs that are involved in several functions, such as cellular growth, apoptosis, metabolism, immunity, and cancer (16–18). miRNAs regulate gene expression by binding to partially complementary sites found in target transcripts and then promoting their translational repression and decay via deadenylation, decapping, and exonucleolysis. Various roles of miRNAs in plant and mammal infections have been reported (19–21). A specific host miRNA response involving, among others, miR-155 and miR-146, takes place during infection with Helicobacter pylori, as well as with Citrobacter rodentium and intracellular bacteria like Mycobacteria, L. monocytogenes, Francisella tularensis, and Salmonella enterica (19, 20). The intestinal translocation is the first step of L. monocytogenes infection. The intestinal tract is the largest reservoir of microbes in the human body. This microbiota provides the host with a wide range of enzymes and metabolites that are key to its physiology and represents a metabolic activity that makes it a virtual organ whose importance is increasingly acknowledged (22–24). Together with the intestinal mucosa and the lymphoid organs, the intestinal microbiota contributes to protection against gut colonization by enteropathogens (25). Strikingly, it is still largely unknown how the microbiota regulates host miRNA expression. Recent studies provided evidence that the microbiota exploits the microRNA pathway to maintain the intestinal homeostasis (26– 30). We previously identified 3 intestinal miRNAs (miR-192, miR-200b, and miR-215) which are modulated in the intestinal tissue during L. monocytogenes infection and whose levels are affected by an oral pretreatment by lactobacilli (31). Here, we undertook a comprehensive analysis of the host gene expression in the ileum of mice infected by L. monocytogenes. Using RNA-sequencing technology, we compared the miRNA profiles of conventional (CV) and germfree (GF) mice during infection and showed that the presence of the microbiota in the lumen influences miRNA expression in the intestinal tissue. Our work thus reveals the existence of an miRNA modulation through which the intestinal microbiota may affect host transcriptional reprogramming during infection by a human pathogen.