Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence. The facultative intracellular bacterial pathogen Listeria monocytogenes is the etiological agent of a severe foodborne disease. In humans it causes a variety of manifestations ranging from asymptomatic intestinal carriage and gastroenteritis to invasive and disseminated severe diseases. Septicemia, meningoencephalitis, and infection of the foetus in pregnant women are the most serious clinical features of listeriosis. Virulence is a trait that only manifests in a susceptible host, involving a highly coordinated interaction between bacterial factors and host components. This article reports the use of in vivo genome expression profiling as a powerful approach to gain a detailed understanding of the Listeria responses and the molecular cross-talk taking place in infected mice. We showed that, during infection, L. monocytogenes shifts the expression of its entire genome to promote virulence, subverting host defenses and adapting to host conditions. This first analysis of L. monocytogenes gene expression in vivo significantly enhances our understanding of the means by which intracellular pathogens promote infection.

Listeria monocytogenes is an intracellular Gram-positive pathogen and the etiological agent of listeriosis, a human food-borne disease potentially fatal for certain risk groups. The virulence of L. monocytogenes is supported by a highly complex and coordinated intracellular life cycle that comprises several crucial steps: host cell adhesion and invasion, intracellular multiplication and motility, and intercellular spread. The completion of each stage is dependent on the orchestrated activity of specialized bacterial factors, in turn tightly controlled by a specific set of regulators. Some virulence factors and modulators also assume an important role in bacterial resistance and evasion to host defense mechanisms. In the last years, the advent of genomics promoted an increasingly prolific identification and functional characterization of new Listeria virulence factors. In this review, we summarize the current knowledge on nearly 50 molecules deployed by L. monocytogenes to promote its cell infection cycle.

Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for L-rhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection. Listeria monocytogenes is a foodborne bacterial pathogen that preferentially infects immunocompromised hosts, eliciting a severe and often lethal disease. In humans, clinical manifestations range from asymptomatic intestinal carriage and gastroenteritis to harsher systemic states of the disease such as sepsis, meningitis or encephalitis, and fetal infections. The surface of L. monocytogenes is decorated with wall teichoic acids (WTAs), a class of carbohydrate-based polymers that contributes to cell surface-related events with implications in physiological processes, such as bacterial division or resistance to antimicrobial peptides (AMPs). The addition of other molecules to the backbone of WTAs modulates their chemical properties and consequently their functionality. In this context, we studied the role of WTA tailoring mechanisms in L. monocytogenes, whose WTAs are strictly decorated with monosaccharides. For the first time, we link WTA glycosylation with AMP resistance by showing that the decoration of L. monocytogenes WTAs with l-rhamnose confers resistance to host defense peptides. We suggest that this resistance is based on changes in the permeability of the cell wall that delay its crossing by AMPs and therefore promote the protection of the bacterial membrane integrity. Importantly, we also demonstrate the significance of this WTA modification in L. monocytogenes virulence.

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them are located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work “behind the frontline”, either supporting virulence effectors or ensuring the survival of the bacterium within its host.

To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps. Recently, we performed a genome-wide analysis of Listeria monocytogenes gene expression after growth in the presence of antibiotics, identifying genes that are up-regulated upon antibiotic treatment. One of them, lmo0762 , is a homolog of hflX , which encodes a heat shock protein that rescues stalled ribosomes by separating their two subunits. To our knowledge, ribosome splitting has never been described as an antibiotic resistance mechanism. We thus investigated the role of lmo0762 in antibiotic resistance. First, we demonstrated that lmo0762 is an antibiotic resistance gene that confers protection against lincomycin and erythromycin, and that we renamed hflXr ( hflX resistance). We show that hflXr expression is regulated by a transcription attenuation mechanism relying on the presence of alternative RNA structures and a small ORF encoding a 14 amino acid peptide containing the RLR motif, characteristic of macrolide resistance genes. We also provide evidence that HflXr is involved in ribosome recycling in presence of antibiotics. Interestingly, L. monocytogenes possesses another copy of hflX , lmo1296 , that is not involved in antibiotic resistance. Phylogenetic analysis shows several events of hflXr duplication in prokaryotes and widespread presence of hflXr in Firmicutes. Overall, this study reveals the Listeria hflXr as the founding member of a family of antibiotic resistance genes. The resistance conferred by this gene is probably of importance in the environment and within microbial communities.

Pathogenic bacteria can target host cell organelles to take control of key cellular processes and promote their intracellular survival, growth, and persistence. Mitochondria are essential, highly dynamic organelles with pivotal roles in a wide variety of cell functions. Mitochondrial dynamics and function are intimately linked. Our previous research showed that Listeria monocytogenes infection impairs mitochondrial function and triggers fission of the mitochondrial network at an early infection stage, in a process that is independent of the presence of the main mitochondrial fission protein Drp1. Here, we analyzed how mitochondrial proteins change in response to L. monocytogenes infection and found that infection raises the levels of Mic10, a mitochondrial inner membrane protein involved in formation of cristae. We show that Mic10 is important for L. monocytogenes -dependent mitochondrial fission and infection of host cells. Our findings thus offer new insight into the mechanisms used by L. monocytogenes to hijack mitochondria to optimize host infection.