Dataset Information

The C. difficile clnRAB operon initiates adaptations to the host environment in response to LL-37.

ABSTRACT: To cause disease, Clostridioides (Clostridium) difficile must resist killing by innate immune effectors in the intestine, including the host antimicrobial peptide, cathelicidin (LL-37). The mechanisms that enable C. difficile to adapt to the intestine in the presence of antimicrobial peptides are unknown. Expression analyses revealed an operon, CD630_16170-CD630_16190 (clnRAB), which is highly induced by LL-37 and is not expressed in response to other cell-surface active antimicrobials. This operon encodes a predicted transcriptional regulator (ClnR) and an ABC transporter system (ClnAB), all of which are required for function. Analyses of a clnR mutant indicate that ClnR is a pleiotropic regulator that directly binds to LL-37 and controls expression of numerous genes, including many involved in metabolism, cellular transport, signaling, gene regulation, and pathogenesis. The data suggest that ClnRAB is a novel regulatory mechanism that senses LL-37 as a host signal and regulates gene expression to adapt to the host intestinal environment during infection.

SUBMITTER: Woods EC

PROVIDER: S-EPMC6117091 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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The C. difficile clnRAB operon initiates adaptations to the host environment in response to LL-37.

Woods Emily C EC Edwards Adrianne N AN Childress Kevin O KO Jones Joshua B JB McBride Shonna M SM

PLoS pathogens 20180820 8

To cause disease, Clostridioides (Clostridium) difficile must resist killing by innate immune effectors in the intestine, including the host antimicrobial peptide, cathelicidin (LL-37). The mechanisms that enable C. difficile to adapt to the intestine in the presence of antimicrobial peptides are unknown. Expression analyses revealed an operon, CD630_16170-CD630_16190 (clnRAB), which is highly induced by LL-37 and is not expressed in response to other cell-surface active antimicrobials. This ope ...[more]

PMID: 30125334

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Project description:Candida albicans remains the predominant cause of fungal infections, where adhered microbial cells form biofilms - densely packed communities. The central feature of C. albicans biofilms is the production of an extracellular matrix (ECM) consisting of polymers and extracellular nucleic acids (eDNA, eRNA), which significantly impedes the infiltration of host cells. Neutrophils, as crucial players in the innate host defense, employ several mechanisms to eradicate the fungal infection, including NETosis, endocytosis, or the release of granules containing, among others, antimicrobial peptides (AMPs). The main representative of these is the positively charged peptide LL-37 formed from an inactive precursor (hCAP18). In addition to its antimicrobial functions, this peptide possesses a propensity to interact with negatively charged molecules, including nucleic acids. Our in vitro studies have demonstrated that LL-37 contacting with C. albicans nucleic acids, isolated from biofilm, are complexed by the peptide and its shorter derivatives, as confirmed by electrophoretic mobility shift assays. We indicated that the generation of the complexes induces discernible alterations in the neutrophil response to fungal nucleic acids compared to the effects of unconjugated molecules. Our analyses involving fluorescence microscopy, flow cytometry, and Western blotting revealed that stimulation of neutrophils with DNA:LL-37 or RNA:LL-37 complexes hamper the activation of pro-apoptotic caspases 3 and 7 and fosters increased activation of anti-apoptotic pathways mediated by the Mcl-1 protein. Furthermore, the formation of complexes elicits a dual effect on neutrophil immune response. Firstly, they facilitate increased nucleic acid uptake, as evidenced by microscopic observations, and enhance the pro-inflammatory response, promoting IL-8 production. Secondly, the complexes detection suppresses the production of reactive oxygen species and attenuates NETosis activation. In conclusion, these findings may imply that the neutrophil immune response shifts toward mobilizing the immune system as a whole, rather than inactivating the pathogen locally. Our findings shed new light on the intricate interplay between the constituents of the C. albicans biofilm and the host's immune response and indicate possible reasons for the elimination of NETosis from the arsenal of the neutrophil response during contact with the fungal biofilm.

Project description:Clostridioides difficile (formerly Clostridium difficile) colonizes the gastrointestinal tract following disruption of the microbiota and can initiate a spectrum of clinical manifestations ranging from asymptomatic to life-threatening colitis. Following antibiotic treatment, luminal oxygen concentrations increase, exposing gut microbes to potentially toxic reactive oxygen species (ROS). Though typically regarded as a strict anaerobe, C. difficile can grow at low oxygen concentrations. How the bacterium adapts to a microaerobic environment and whether those responses to oxygen are conserved amongst strains is not entirely understood. Here, two C. difficile strains (630 and CD196) were cultured in 1.5% oxygen and the transcriptional response was evaluated via RNA-sequencing. During growth in a microaerobic environment, several genes predicted to protect against oxidative stress were upregulated, including ruberythrins and rubredoxins. Genes involved in metal homeostasis were positively correlated with increasing oxygen levels and were also amongst the most differentially transcribed. These included ferrous iron transporters (feo), a zinc transporter (zupT), and predicted siderophore transporters. To directly compare the transcriptional landscape between C. difficile strains, a ‘consensus-genome’ was generated. On the basis of the identified conserved genes, basal transcriptional differences as well as variations in the response to oxygen were evaluated. While several responses were similar between the strains, there were significant differences in the abundance of transcripts for amino acid and carbohydrate metabolism. Furthermore, homologous metal homeostasis genes were similarly transcribed, but the intracellular metal concentrations significantly varied both in an oxygen-dependent and independent manner. Overall, these results indicate that C. difficile adapts to grow in a low oxygen environment through transcriptional changes, though the specific strategy employed varies between strains.

Dataset Information

The C. difficile clnRAB operon initiates adaptations to the host environment in response to LL-37.

Publications

The C. difficile clnRAB operon initiates adaptations to the host environment in response to LL-37.

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