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 major intracellular human foodborne bacterial pathogen. We previously revealed L. monocytogenes cadC as highly expressed during mouse infection. Here we show that L. monocytogenes CadC is a sequence-specific, DNA-binding and cadmium-dependent regulator of CadA, an efflux pump conferring cadmium resistance. CadC but not CadA is required for L. monocytogenes infection in vivo. Interestingly, CadC also directly represses lspB, a gene encoding a lipoprotein signal peptidase whose expression appears detrimental for infection. lspB overexpression promotes the release of the LpeA lipoprotein to the extracellular medium, inducing tumor necrosis factor α and interleukin 6 expression, thus impairing L. monocytogenes survival in macrophages. We propose that L. monocytogenes uses CadC to repress lspB expression during infection to avoid LpeA exposure to the host immune system, diminishing inflammatory cytokine expression and promoting intramacrophagic survival and virulence. CadC appears as the first metal efflux pump regulator repurposed during infection to fine-tune lipoprotein processing and host responses.

(2014) Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection, Cell Cycle, 13:6, 928-940,

Innate immunity is the most broadly effective host defense, being essential to clear the majority of microbial infections. Scavenger Receptors comprise a family of sensors expressed in a multitude of host cells, whose dual role during microbial pathogenesis gained importance over recent years. SRs regulate the recruitment of immune cells and control both host inflammatory response and bacterial load. In turn, pathogens have evolved different strategies to overcome immune response, avoid recognition by SRs and exploit them to favor infection. Here, we discuss the most relevant findings regarding the interplay between SRs and pathogens, discussing how these multifunctional proteins recognize a panoply of ligands and act as bacterial phagocytic receptors.

The cell wall of Listeria monocytogenes (Lm), a major intracellular foodborne bacterial pathogen, comprises a thick peptidoglycan layer that serves as a scaffold for glycopolymers such as wall teichoic acids (WTAs). WTAs contain non-essential sugar substituents whose absence prevents bacteriophage binding and impacts antigenicity, sensitivity to antimicrobials, and virulence. Here, we demonstrated, for the first time, the triple function of Lm WTA glycosylations in the following: (1) supporting the correct anchoring of major Lm virulence factors at the bacterial surface, namely Ami and InlB; (2) promoting Lm resistance to antimicrobial peptides (AMPs); and (3) decreasing Lm sensitivity to some antibiotics. We showed that while the decoration of WTAs by rhamnose in Lm serovar 1/2a and by galactose in serovar 4b are important for the surface anchoring of Ami and InlB, N-acetylglucosamine in serovar 1/2a and glucose in serovar 4b are dispensable for the surface association of InlB or InlB/Ami. We found that the absence of a single glycosylation only had a slight impact on the sensibility of Lm to AMPs and antibiotics, however the concomitant deficiency of both glycosylations (rhamnose and N-acetylglucosamine in serovar 1/2a, and galactose and glucose in serovar 4b) significantly impaired the Lm capacity to overcome the action of antimicrobials. We propose WTA glycosylation as a broad mechanism used by Lm, not only to properly anchor surface virulence factors, but also to resist AMPs and antibiotics. WTA glycosyltransferases thus emerge as promising drug targets to attenuate the virulence of bacterial pathogens, while increasing their susceptibility to host immune defenses and potentiating the action of antibiotics.

The foodborne pathogen Listeria monocytogenes (Lm) causes invasive infection in susceptible animals and humans. To survive and proliferate within hosts, this facultative intracellular pathogen tightly coordinates the expression of a complex regulatory network that controls the expression of virulence factors. Here, we identified and characterized MouR, a novel virulence regulator of Lm. Through RNA-seq transcriptomic analysis, we determined the MouR regulon and demonstrated how MouR positively controls the expression of the Agr quorum sensing system (agrBDCA) of Lm. The MouR three-dimensional structure revealed a dimeric DNA-binding transcription factor belonging to the VanR class of the GntR superfamily of regulatory proteins. We also showed that by directly binding to the agr promoter region, MouR ultimately modulates chitinase activity and biofilm formation. Importantly, we demonstrated by in vitro cell invasion assays and in vivo mice infections the role of MouR in Lm virulence.

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2 -deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2 -deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire. Abbreviations : AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A 1 ; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type.

Understanding the strategies used by pathogens to infect, survive, and proliferate in their hosts requires the identification of virulence factors. We developed PCR-based screening of targeted mutants to facilitate quick, simultaneous detection of multiple novel bacterial virulence genes. Based on direct PCR screening of pooled targeted mutants, this approach provides a fast and sensitive measure of virulence attenuation while significantly reducing the number of animals and time required. We demonstrate that the careful design of gene-specific primers allows the direct relative quantification of mixed mutants in infected mouse organs. Indeed, we show that the band intensity of the PCR product is directly related to the quantity of the corresponding strain in a pool of mutants. We applied the PCR-based screening of targeted mutants to the murine model of listeriosis and revealed new genes required for full pathogenicity of Listeria monocytogenes, a facultative human intracellular pathogen. PCR-based screening is a simple, useful, and fast technique to test pools of targeted bacterial mutants in vivo, without the requirements for a rigorous purification step, complicated PCR set-up, or special equipment. This approach can be adapted to other bacterial systems, constituting a significant advance in the field of infection biology.