Corynebacterium tuberculostearicum, a human skin colonizer, induces the canonical nuclear factor-κB inflammatory signaling pathway in human skin cells.

INTRODUCTION: Corynebacterium tuberculostearicum (C. t.) is a ubiquitous bacterium that colonizes human skin. In contrast to other members of the genus Corynebacterium, such as toxigenic Corynebacterium diphtheriae or the opportunistic pathogen Corynebacterium jeikeium, several studies suggest that C. t. may play a role in skin health and disease. However, the mechanisms underlying these effects remain poorly understood. METHODS: To investigate whether C. t. induces inflammatory pathways in primary human epidermal keratinocytes (HEKs) and human cutaneous squamous carcinoma cells (SCCs), cell culture, reverse transcription-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, immunofluorescence microscopy, Western blot, chromatin immunoprecipitation-PCR, small interfering RNA knockdown and luciferase reporter expression system were used. RESULTS: Herein, we demonstrate that C. t. upregulates the messenger RNA (mRNA) and protein levels of inflammatory mediators in two human skin cell lines, HEKs and SCCs. We further show activation of the canonical nuclear factor-κB (NF-κB) pathway in response to C. t. infection, including phosphorylation of the inhibitor of κB (IκB), the nuclear translocation of NF-κB subunit (NF-κB-P65 ) and the recruitment of NF-κB-P65 and RNA polymerase to the NF-κB response elements at the promoter region of the inflammatory genes. Lastly, the data confirm that C. t.-induced tumor necrosis factor mRNA expression in HEKs is toll-like receptor 2 (TLR2 ) dependent. CONCLUSION: Our results offer a mechanistic model for C. t.-induced inflammation in human keratinocytes via TLR2 and activation of IκB kinase and downstream signaling through the canonical NF-κB pathway. Relevance to chronic inflammatory diseases of the skin and cutaneous oncology is discussed.

Cytokine Levels in the In Vitro Response of T Cells to Planktonic and Biofilm Corynebacterium amycolatum.

Unravelling of the interplay between the immune system and non-diphtheria corynebacteria would contribute to understanding their increasing role as medically important microorganisms. We aimed at the analysis of pro- (TNF, IL-1ß, IL-6, IL-8, and IL-12p70) and anti-inflammatory (IL-10) cytokines produced by Jurkat T cells in response to planktonic and biofilm Corynebacterium amycolatum. Two reference strains: C. amycolatum ATCC 700207 (R-CA), Staphylococcus aureus ATCC 25923 (R-SA), and ten clinical strains of C. amycolatum (C-CA) were used in the study. Jurkat T cells were stimulated in vitro by the planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM) derived from the relevant cultures of the strains tested. The cytokine concentrations were determined in the cell culture supernatants using the flow cytometry. The levels of the cytokines analyzed were lower after stimulation with the BCM when compared to the PCM derived from the cultures of C-CA; statistical significance (p < 0.05) was observed for IL-1ß, IL-12 p70, and IL-10. Similarly, planktonic R-CA and R-SA stimulated a higher cytokine production than their biofilm counterparts. The highest levels of pro-inflammatory IL-8, IL-1ß, and IL-12p70 were observed after stimulation with planktonic R-SA whereas the strongest stimulation of anti-inflammatory IL-10 was noted for the BCM derived from the mixed culture of both reference species. Our results are indicative of weaker immunostimulatory properties of the biofilm C. amycolatum compared to its planktonic form. It may play a role in the persistence of biofilm-related infections. The extent of the cytokine response can be dependent on the inherent virulence of the infecting microorganism.Unravelling of the interplay between the immune system and non-diphtheria corynebacteria would contribute to understanding their increasing role as medically important microorganisms. We aimed at the analysis of pro- (TNF, IL-1ß, IL-6, IL-8, and IL-12p70) and anti-inflammatory (IL-10) cytokines produced by Jurkat T cells in response to planktonic and biofilm Corynebacterium amycolatum. Two reference strains: C. amycolatum ATCC 700207 (R-CA), Staphylococcus aureus ATCC 25923 (R-SA), and ten clinical strains of C. amycolatum (C-CA) were used in the study. Jurkat T cells were stimulated in vitro by the planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM) derived from the relevant cultures of the strains tested. The cytokine concentrations were determined in the cell culture supernatants using the flow cytometry. The levels of the cytokines analyzed were lower after stimulation with the BCM when compared to the PCM derived from the cultures of C-CA; statistical significance (p < 0.05) was observed for IL-1ß, IL-12 p70, and IL-10. Similarly, planktonic R-CA and R-SA stimulated a higher cytokine production than their biofilm counterparts. The highest levels of pro-inflammatory IL-8, IL-1ß, and IL-12p70 were observed after stimulation with planktonic R-SA whereas the strongest stimulation of anti-inflammatory IL-10 was noted for the BCM derived from the mixed culture of both reference species. Our results are indicative of weaker immunostimulatory properties of the biofilm C. amycolatum compared to its planktonic form. It may play a role in the persistence of biofilm-related infections. The extent of the cytokine response can be dependent on the inherent virulence of the infecting microorganism.

Toll-like receptor variants and cervical Atopobium vaginae infection in women with pelvic inflammatory disease.

PROBLEM: Toll-like (TLR) receptor genetic variants have been implicated in bacterial vaginosis (BV). We determined whether TLR variants are associated with fastidious BV-associated microbes that are linked with infertility following pelvic inflammatory disease (PID). METHOD OF STUDY: Sneathia spp., Atopobium vaginae, BVAB1, and Ureaplasma urealyticum were measured in 250 women from the PID Evaluation and Clinical Health (PEACH) study. Relative risk (RR) and 95% confidence intervals (CI) were calculated adjusting for chlamydia and gonorrhea. Principal component analysis was used to adjust for population stratification. A false discovery rate q-value of 0.05 was significant. RESULTS: TLR2-1733C>A (P = .003) and TLR2-616A>G (P = .004) were associated with cervical A. vaginae. TLR2-1733C>A and TLR6-438C>T were associated with A. vaginae detection in the endometrium, but this was not significant after adjustment for multiple comparisons (FDR q-value = 0.06). CONCLUSION: Host gene variants in TLR2 signaling pathways were modestly associated with cervical A. vaginae in women with clinical PID.

C. pseudotuberculosis Phop confers virulence and may be targeted by natural compounds.

The bacterial two-component system (TCS) regulates genes that are crucial for virulence in several pathogens. One of such TCS, the PhoPR system, consisting of a transmembrane sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP), has been reported to have a major role in mycobacterial pathogenesis. We knocked out the phoP in C. pseudotuberculosis, the causal organism of caseous lymphadenitis (CLA), and using a combination of in vitro and in vivo mouse system, we showed for the first time, that the PhoP of C. pseudotuberculosis plays an important role in the virulence and pathogenicity of this bacterium. Furthermore, we modeled the PhoP of C. pseudotuberculosis and our docking results showed that several natural compounds including Rhein, an anthraquinone from Rheum undulatum, and some drug-like molecules may target PhoP to inhibit the TCS of C. pseudotuberculosis, and therefore may facilitate a remarkable attenuation of bacterial pathogenicity being the CLA. Experiments are currently underway to validate these in silico docking results.

Modulation of immune response by interleukin-10 in systemic Corynebacterium kutscheri infection in mice.

Interleukin (IL)-10 is an anti-inflammatory cytokine that modulates sepsis by decreasing pro-inflammatory cytokine production and chemokine expression. In this study, IL-10-deficient and wild-type (WT) mice were infected with Corynebacterium kutscheri to determine if the absence of IL-10 altered the protective immunity and pathogenesis. After infection, IL-10 knockout (KO) mice had a higher survival rate than WT mice. The decrease of body weight and the increased weight of organs such as liver and spleen were greater in WT mice. Bacterial counts were significantly increased after inoculation in WT mice over those in IL-10 KO mice. WT mice had more granulomatous inflammation and coagulative necrosis in the liver and spleen, lymphocyte depletion in lymphoid follicles, and apoptosis of immune cells in the spleen. WT mice had significantly higher plasma concentrations of aspartate aminotransferase and alanine aminotransferase. Furthermore, more upregulation of tumor necrosis factor-α and IL-4 in the plasma, macrophage inflammatory protein-2, keratinocyte-derived chemokine, inducible nitric oxide synthase, and interferon-inducible protein 10 mRNA in the spleen were observed in WT mice after inoculation. These results suggest that the lack of IL-10 contributes to an increase in the systemic clearance of C. kutscheri, and that IL-10 plays a detrimental role in controlling systemic C. kutscheri infection.