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

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

The foodborne pathogen Listeria monocytogenes (Lm) causes invasive infection in susceptible ani- mals 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 fac- tors. 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 con- trols the expression of the Agr quorum sensing sys- tem (agrBDCA) of Lm. The MouR three-dimensional structure revealed a dimeric DNA-binding transcrip- tion 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 infec- tions the role of MouR in Lm virulence.Peer reviewe

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 sigma(B) 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

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

WTA l-rhamnosylation promotes <i>Lm</i> resistance against AMPs.

<p>(<b>A</b>) Growth of <i>Lm</i> strains in BHI broth supplemented with 5% NaCl. A growth curve of wild type EGD-e in the absence of 5% NaCl was included as a control for optimal growth. (<b>B</b>) Growth of mid-exponential-phase <i>Lm</i> strains untreated (black symbols) or challenged with 50 μg/ml (gray symbols) or 1 mg/ml (white symbols) of lysozyme. Optical density of the shaking cultures was monitored spectrophotometrically at 600 nm. (<b>C</b>) Quantification of viable bacteria after treatment of mid-exponential-phase <i>Lm</i> strains (2 h, 37°C) with gallidermin (1 μg/ml), CRAMP or LL-37 (5 μg/ml). Averaged replicate values from AMP-treated samples were normalized to untreated control samples and the transformed data expressed as the percentage of surviving bacteria relative to wild type <i>Lm</i> (set at 100). Data represent mean±SD of three independent experiments. *, <i>p</i>≤0.05; ***, <i>p</i>≤0.001.</p

A functional <i>rml</i> operon is required for glycosylation of <i>Lm</i> WTAs with l-rhamnose.

<p>(<b>A</b>) Alcian blue-stained 20% polyacrylamide gel containing WTA extracts from logarithmic-phase cultures of different <i>Lm</i> strains. (<b>B–D</b>) HPAEC-PAD analyses of the sugar composition of the (B) WTA, (C) peptidoglycan and (D) cytoplasmic fractions isolated from the indicated <i>Lm</i> strains. Samples were hydrolyzed in 3 M HCl (2 h, 95°C), diluted with water and lyophilized before injection into the HPLC equipment. Standards for ribitol (Rib), l-rhamnose (Rha), glucosamine (GlcN), and muramic acid (Mur) were eluted under identical conditions to allow peak identification.</p

WTA l-rhamnosylation interferes with the <i>Lm</i> cell wall crossing by AMPs.

<p>(<b>A and B</b>) Flow cytometry analysis of <i>Lm</i> surface-exposed CRAMP levels in mid-exponential-phase <i>Lm</i> strains, following incubation (5 min) in a 5-μg/ml solution of the peptide and immunolabeling with anti-CRAMP and Alexa Fluor 488-conjugated antibodies. (A) Representative experiment showing overlaid histograms of CRAMP-treated (solid line) and untreated (dashed line) samples, with mean fluorescence intensity (MFI) values from treated samples indicated by vertical dashed lines. (B) Mean±SD of the MFI values of CRAMP-treated samples from three independent experiments. (<b>C</b>) Cell surface charge analysis of <i>Lm</i> strains deficient for WTA l-rhamnosylation as determined by cytochrome c binding assays. Mid-exponential-phase bacteria were incubated with equine cytochrome c (0.5 mg/ml), centrifuged and the supernatant was recovered for spectrophotometric quantification of the unbound protein fraction. Values from <i>Lm</i>-containing samples are expressed as the percentage of unbound cytochrome c relative to control samples lacking bacteria. Data represent the mean±SD of three independent experiments. (<b>D and E</b>) Flow cytometry analysis of total <i>Lm</i>-associated CRAMP levels in mid-exponential-phase <i>Lm</i> strains, following incubation (5 min) with a 5-μg/ml solution of fluorescently labeled peptide (5-FAM-CRAMP). (D) Representative experiment showing overlaid histograms of FAM-CRAMP-treated (solid line) and untreated (dashed line) samples, with MFI values from treated samples indicated by vertical dashed lines. (E) Mean±SD of the MFI values of 5-FAM-CRAMP-treated samples from three independent experiments. (<b>F</b>) Fluorometric quantification of the unbound CRAMP fraction in the supernatant of suspensions of mid-exponential-phase <i>Lm</i> strains, following incubation (5 min) with a 5-μg/ml solution of 5-FAM-CRAMP. Data are expressed as the percentage of unbound fluorescent peptide relative to control samples lacking bacteria, and represent the mean±SD of three independent experiments performed in triplicates. ns = not significant, <i>p</i>>0.05; **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001.</p

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

<div><p><i>Listeria monocytogenes</i> 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 <i>L</i>. <i>monocytogenes</i>, where WTA modification is achieved solely <i>via</i> glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of <i>L</i>. <i>monocytogenes</i> WTAs relies not only on the <i>rmlACBD</i> locus, which encodes the biosynthetic pathway for L-rhamnose, but also on <i>rmlT</i> 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 <i>in vitro</i> binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of <i>L</i>. <i>monocytogenes</i> delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes <i>L</i>. <i>monocytogenes</i> 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 <i>L</i>. <i>monocytogenes</i> virulence in a mouse model of infection.</p></div