Surface-Associated Lipoproteins Link Enterococcus faecalis Virulence to Colitogenic Activity in IL-10-Deficient Mice Independent of Their Expression Levels

The commensal Enterococcus faecalis is among the most common causes of nosocomial infections. Recent findings regarding increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases and induction of colitis in IL-10-deficient (IL-10-/-) mice put a new perspective on the contribution of E. faecalis to chronic intestinal inflammation. Based on the expression of virulence-related genes in the inflammatory milieu of IL-10-/- mice using RNA-sequencing analysis, we characterized the colitogenic role of two bacterial structures that substantially impact on E. faecalis virulence by different mechanisms: the enterococcal polysaccharide antigen and cell surface-associated lipoproteins. Germ-free wild type and IL-10-/- mice were monoassociated with E. faecalis wild type OG1RF or the respective isogenic mutants for 16 weeks. Intestinal tissue and mesenteric lymph nodes (MLN) were collected to characterize tissue pathology, loss of intestinal barrier function, bacterial adhesion to intestinal epithelium and immune cell activation. Bone marrow-derived dendritic cells (BMDC) were stimulated with bacterial lysates and E. faecalis virulence was additionally investigated in three invertebrate models. Colitogenic activity of wild type E. faecalis (OG1RF score: 7.2±1.2) in monoassociated IL-10-/- mice was partially impaired in E. faecalis lacking enterococcal polysaccharide antigen (ΔepaB score: 4.7±2.3; p<0.05) and was almost completely abrogated in E. faecalis deficient for lipoproteins (Δlgt score: 2.3±2.3; p<0.0001). Consistently both E. faecalis mutants showed significantly impaired virulence in Galleria mellonella and Caenorhabditis elegans. Loss of E-cadherin in the epithelium was shown for all bacterial strains in inflamed IL-10-/- but not wild type mice. Inactivation of epaB in E. faecalis reduced microcolony and biofilm formation in vitro, altered bacterial adhesion to intestinal epithelium of germ-free Manduca sexta larvae and impaired penetration into the colonic mucus layer of IL-10-/- mice. Lipoprotein-deficient E. faecalis exhibited an impaired TLR2-mediated activation of BMDCs in vitro despite their ability to fully reactivate MLN cells as well as MLN-derived colitogenic T cells ex vivo. E. faecalis virulence factors accounting for bacterial adhesion to mucosal surfaces as well as intestinal barrier disruption partially contribute to colitogenic activity of E. faecalis. Beyond their well-known role in infections, cell surface-associated lipoproteins are essential structures for colitogenic activity of E. faecalis by mediating innate immune cell activation

Prevention of Staphylococcus aureus biofilm formation on metallic surgical implants via controlled release of gentamicin

Implant associated infections are a critical health concern following orthopaedic surgery. Sustained local delivery of antibiotics has been suggested as a means of preventing these infections. Poly(D,L-lactide) (PDLLA) is a biodegradable polymer that has been used to coat implants for the delivery of antibiotics and other bioactive molecules. While effective, these studies show that antibiotics are released in a burst profile. Here we evaluated a method for controlled release of gentamicin from implant surfaces using the palmitate alkyl salt to decrease its solubility in aqueous solution. Steel Kirschner wires (K-wires) were coated with Gentamicin-palmitate (GP)-PDLLA, gentamicin sulphate (GS)-PDLLA or vancomycin sulphate (VS)-PDLLA, and elution of antibiotics from coated K-wires investigated using HPLC/MS/MS. In contrast to burst antibiotic release from the GS-PDLLA and VS-PDLLA groups, GP was released in a slower sustained manner. Colonisation and initial attachment of Staphylococcus aureus Xen29 to gentamicin-coated K-wires was reduced by 90% when compared to the non-coated control group. However there was no statistical difference in recovery of bacteria from GS or GP groups. Bacteria recovered from VS-PDLLA coated K-wires decreased by 36%. Bioluminescence emitted by S. aureus Xen29 was also reduced over seven days in the antibiotic control groups, demonstrating that growth and biofilm development over the longer term was impaired by antibiotic-PDLLA coating. These results indicate that using alkyl salts of antibiotics may be an effective strategy for controlling the release of antibiotics from implants

Nod1 also dampens <i>S.</i> Typhimurium Δ<i>msbB</i>-induced colitis.

<p>Streptomycin-pretreated C57Bl/6, <i>Nod1^−/−</i> and DKO mice were orally infected with <i>S.</i> Typhimurium Δ<i>msbB</i> for 2 days. (A) <i>S.</i> Typhimurium colonization of ileum, cecum and colon. Dashed line: limit of detection; cfu: colony forming units. (B) Pathology scores of infected ceca. Statistical analysis: 1way ANOVA with Bonferroni's multiple comparison post-test. * <i>p</i><0.05; ** <i>p</i><0.01. (C) H&E staining of infected cecum sections. Original magnification 200×; scale bars = 100 µm.</p

Impaired bacterial clearance and exacerbated inflammation in <i>Nod2^−/−</i> mice.

<p>Streptomycin-pretreated C57Bl/6 and <i>Nod2^−/−</i> mice were orally infected with wild-type <i>S.</i> Typhimurium or the Δ<i>msbB</i> mutant for the indicated times. <i>S.</i> Typhimurium Δ<i>msbB</i> colonization of the (A) ileum, (B) cecum and (C) colon. The dashed line indicates the limit of detection. cfu: colony forming units. Statistical analysis: one-way ANOVA with Tukey's multiple comparison post-test after logarithmic transformation. (D) <i>S.</i> Typhimurium wild-type colonization 2 days post infection. (E) Pathology scores of <i>S.</i> Typhimurium infected ceca and mock-infected controls at day 2 p.i., Statistical analysis: Student's <i>t</i> test. (F) H&E staining of <i>S.</i> Typhimurium Δ<i>msbB</i> infected cecal sections and mock-infected controls at day 2 p.i. Original magnification: 40×; scale bars = 500 µm; L = lumen, M = mucosa, SM = submucosa. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001; ns: not significant.</p

Primer sequences used for quantitative real-time PCR.

<p>Primer sequences used for quantitative real-time PCR.</p

Reduced TLR4, but enhanced TLR2 reactivity of <i>S.</i> Typhimurium Δ<i>msbB</i>.

<p>HEK293 cells were transiently transfected with (A) TLR4, (B) Nod2 or (C) TLR2 and stimulated with purified LPS either of <i>S.</i> Typhimurium wild-type or <i>S.</i> Typhimurium Δ<i>msbB</i> or with heat-killed bacteria. As a negative control only medium was added. Positive controls used were muramyldipeptide (MDP) for Nod2 stimulation and the synthetic lipopeptide <i>N</i>-palmitoyl-<i>S</i>-[2,3-bis(palmitoyloxy)-(2R,<i>S</i>)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P₃CSK₄) for TLR2 stimulation. IL-8 release was measured by ELISA. Shown are representative experiments of 3 independent experiments. Statistical analysis: 1way ANOVA with Bonferroni's multiple comparison post-test. * <i>p</i><0.05; *** <i>p</i><0.001; only significant differences between the same concentrations of wild-type and Δ<i>msbB</i> are marked. D–E: Gene expression of <i>tlr2</i> and <i>tlr4</i> in the cecum of mock infected or <i>S.</i> Typhimurium Δ<i>msbB</i> infected mice 2 days post infection was measured by quantitative real-time PCR. Data were normalized to <i>gapdh</i> expression levels; Statistical analysis: 1way ANOVA with Tukey's multiple comparison post-test. No significant differences were observed.</p

<i>Salmonella enterica</i> serovar Typhimurium Δ<i>msbB</i> Triggers Exacerbated Inflammation in Nod2 Deficient Mice

<div><p>The intracellular pathogen <i>Salmonella enterica</i> serovar Typhimurium causes intestinal inflammation characterized by edema, neutrophil influx and increased pro-inflammatory cytokine expression. A major bacterial factor inducing pro-inflammatory host responses is lipopolysaccharide (LPS). <i>S.</i> Typhimurium Δ<i>msbB</i> possesses a modified lipid A, has reduced virulence in mice, and is being considered as a potential anti-cancer vaccine strain. The lack of a late myristoyl transferase, encoded by MsbB leads to attenuated TLR4 stimulation. However, whether other host receptor pathways are also altered remains unclear. Nod1 and Nod2 are cytosolic pattern recognition receptors recognizing bacterial peptidoglycan. They play important roles in the host's immune response to enteric pathogens and in immune homeostasis. Here, we investigated how deletion of <i>msbB</i> affects <i>Salmonella's</i> interaction with Nod1 and Nod2. <i>S.</i> Typhimurium Δ <i>msbB</i>-induced inflammation was significantly exacerbated in <i>Nod2</i>^−/− mice compared to C57Bl/6 mice. In addition, <i>S.</i> Typhimurium Δ<i>msbB</i> maintained robust intestinal colonization in <i>Nod2</i>^−/− mice from day 2 to day 7 p.i., whereas colonization levels significantly decreased in C57Bl/6 mice during this time. Similarly, infection of <i>Nod1</i>^−/− and <i>Nod1/Nod2</i> double-knockout mice revealed that both Nod1 and Nod2 play a protective role in <i>S.</i> Typhimurium Δ<i>msbB</i>-induced colitis. To elucidate why <i>S.</i> Typhimurium Δ<i>msbB</i>, but not wild-type <i>S.</i> Typhimurium, induced an exacerbated inflammatory response in <i>Nod2</i>^−/− mice, we used HEK293 cells which were transiently transfected with pathogen recognition receptors. Stimulation of TLR2-transfected cells with <i>S.</i> Typhimurium Δ<i>msbB</i> resulted in increased IL-8 production compared to wild-type <i>S.</i> Typhimurium. Our results indicate that <i>S.</i> Typhimurium Δ<i>msbB</i> triggers exacerbated colitis in the absence of Nod1 and/or Nod2, which is likely due to increased TLR2 stimulation. How bacteria with “genetically detoxified” LPS stimulate various innate responses has important implications for the development of safe and effective bacterial vaccines and adjuvants.</p></div

<i>S.</i> Typhimurium Δ<i>msbB</i> infection induces pro-inflammatory gene expression in C57Bl/6 and <i>Nod2^−/−</i> mice.

<p>Gene expression of <i>mcp1</i> and <i>tnfα</i>, were measured by quantitative real-time PCR. Data were normalized to <i>gapdh</i> expression levels; Statistical analysis: 1way ANOVA with Tukey's multiple comparison post-test. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001; ns: not significant.</p

Increased early influx of neutrophils and macrophages in <i>Nod2^−/−</i> mice.

<p>Streptomycin-pretreated C57Bl/6 and <i>Nod2</i>^−/− mice were orally infected <i>with S.</i> Typhimurium Δ<i>msbB</i> for 2 days and cecum sections were stained to visualize neutrophils and macrophages, respectively. (A) Cecum sections were stained for DAPI (blue), E-cadherin (epithelial cells, red) and MPO (neutrophils, green). (B) Cecum sections were stained for DAPI (blue) and CD68 (macrophages, red). Original magnification: 400×, scale bars = 50 µm. (C) Quantification of MPO+ and (D) CD68+ cells. HPF = high power field. Statistical analysis: Student's <i>t</i> test. *** <i>p</i><0.001.</p