LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development.

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 A1; 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.

Stabilin-1 plays a protective role against Listeria monocytogenes infection through the regulation of cytokine and chemokine production and immune cell recruitment.

Scavenger receptors are part of a complex surveillance system expressed by host cells to efficiently orchestrate innate immune response against bacterial infections. Stabilin-1 (STAB-1) is a scavenger receptor involved in cell trafficking, inflammation, and cancer; however, its role in infection remains to be elucidated. Listeria monocytogenes (Lm) is a major intracellular human food-borne pathogen causing severe infections in susceptible hosts. Using a mouse model of infection, we demonstrate here that STAB-1 controls Lm-induced cytokine and chemokine production and immune cell accumulation in Lm-infected organs. We show that STAB-1 also regulates the recruitment of myeloid cells in response to Lm infection and contributes to clear circulating bacteria. In addition, whereas STAB-1 appears to promote bacterial uptake by macrophages, infection by pathogenic Listeria induces the down regulation of STAB-1 expression and its delocalization from the host cell membrane.We propose STAB-1 as a new SR involved in the control of Lm infection through the regulation of host defense mechanisms, a process that would be targeted by bacterial virulence factors to promote infection.

Listeria monocytogenes Wall Teichoic Acid Glycosylation Promotes Surface Anchoring of Virulence Factors, Resistance to Antimicrobial Peptides, and Decreased Susceptibility to Antibiotics.

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.

Virulence gene repression promotes Listeria monocytogenes systemic infection.

The capacity of bacterial pathogens to infect their hosts depends on the tight spatiotemporal regulation of virulence genes. The Listeria monocytogenes (Lm) metal efflux pump repressor CadC is highly expressed during late infection stages, modulating lipoprotein processing and host immune response. Here we investigate the potential of CadC as broad repressor of virulence genes. We show that CadC represses the expression of the bile salt hydrolase impairing Lm resistance to bile. During late infection, in absence of CadC-dependent repression, the constitutive bile salt hydrolase expression induces the overexpression of the cholic acid efflux pump MdrT that is unfavorable to Lm virulence. We establish the CadC regulon and show that CadC represses additional virulence factors activated by σB during colonization of the intestinal lumen. CadC is thus a general repressor that promotes Lm virulence by down-regulating, at late infection stages, genes required for survival in the gastrointestinal tract. This demonstrates for the first time how bacterial pathogens can repurpose regulators to spatiotemporally repress virulence genes and optimize their infectious capacity.

Scavenger Receptors: Promiscuous Players during Microbial Pathogenesis.

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.

MouR controls the expression of the Listeria monocytogenes Agr system and mediates virulence.

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.

Listeria monocytogenes CadC Regulates Cadmium Efflux and Fine-tunes Lipoprotein Localization to Escape the Host Immune Response and Promote 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.

L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane.

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 induces host DNA damage and delays the host cell cycle to promote infection.

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.

PCR-based screening of targeted mutants for the fast and simultaneous identification of bacterial virulence factors.

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.