Biology subjects

Hamiot, A.

Publications and source records attributed to Hamiot, A..

3 recordsLinked to original sources

bioRxiv2021

c-di-AMP signaling is required for bile salts resistance and long-term colonization by Clostridioides difficile

To cause disease, the important human enteropathogen Clostridioides difficile must colonize the gastro-intestinal tract but little is known on how this organism senses and responds to the harsh host environment to adapt and multiply. Nucleotide second messengers are signaling molecules used by bacteria to respond to changing environmental conditions. In this study, we showed for the first time that c-di-AMP is produced by C. difficile and controls the uptake of potassium, making it essential for growth. We found that c-di-AMP is involved in biofilm formation, cell wall homeostasis, osmotolerance as well as detergent and bile salt resistance in C. difficile. In a colonization mouse model, a strain lacking GdpP, a c-di-AMP degrading enzyme, failed to persist in the gut in contrast to the parental strain. We identified OpuR as a new regulator that binds c-di-AMP and represses the expression of the compatible solute transporter OpuC. Interestingly, an opuR mutant is highly resistant to a hyperosmotic or bile salt stress compared to the parental strain while an opuCA mutant is more susceptible A short exposure of C. difficile cells to bile salts resulted in a decrease of the c-di-AMP concentrations reinforcing the hypothesis that changes in membrane characteristics due to variations of the cellular turgor or membrane damages constitute a signal for the adjustment of the intracellular c-di-AMP concentration. Thus, c-di-AMP is a signaling molecule with pleiotropic effects that controls osmolyte uptake to confer osmotolerance and bile salt resistance in C. difficile and that is important for colonization of the host. One Sentence Summaryc-di-AMP is an essential regulatory molecule conferring resistance to osmotic and bile salt stresses by controlling osmolyte uptake and contributing to gut persistence in the human enteropathogen Clostridioides difficile.

microbiology↗

bioRxiv2021

Metabolic adaption to extracellular pyruvate triggers biofilm formation in Clostridioides difficile

Clostridioides difficile infections are associated with gut microbiome dysbiosis and are the leading cause of hospital acquired diarrhoea. The infectious process is strongly influenced by the microbiota and successful infection relies on the absence of specific microbiota-produced metabolites. Deoxycholic acid (DOC) and short chain fatty acids are microbiota-produced metabolites that limit the growth of C. difficile and protect the host against this infection. In a previous study, we showed that DOC causes C. difficile to form strongly adherent biofilms after 48 h. Here, our objectives were to identify and characterize key molecules and events required for biofilm formation in the presence of DOC. We applied time-course transcriptomics and genetics to identify sigma factors, metabolic processes and type IV pili that drive biofilm formation. These analyses revealed that extracellular pyruvate induces biofilm formation in the presence of DOC. In the absence of DOC, pyruvate supplementation was sufficient to induce biofilm formation in a process that was dependent on pyruvate uptake by the membrane protein CstA. In the context of the human gut, microbiota-generated pyruvate is a metabolite that limits pathogen colonization. Taken together our results suggest that pyruvate-induced biofilm formation might act as a key process driving C. difficile persistence in the gut.

microbiology↗

bioRxiv2020

Type I toxin-antitoxin systems contribute to mobile genetic elements maintenance in Clostridioides difficile and can be used as a counter-selectable marker for chromosomal manipulation

Toxin-antitoxin (TA) systems are widespread on mobile genetic elements as well as in bacterial chromosomes. According to the nature of the antitoxin and its mode of action for toxin inhibition, TA systems are subdivided into different types. The first type I TA modules were recently identified in the human enteropathogen Clostridioides (formerly Clostridium) difficile. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA during normal growth. Here, we report the characterization of five additional type I TA systems present within phiCD630-1 and phiCD630-2 prophage regions of C. difficile 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest. Growth is restored when the antitoxin RNAs, transcribed from the opposite strand, are co-expressed together with the toxin genes. In addition, we show that type I TA modules located within the phiCD630-1 prophage contribute to its stability and mediate phiCD630-1 heritability. Type I TA systems were found to be widespread in genomes of C. difficile phages, further suggesting their functional importance. We have made use of a toxin gene from one of type I TA modules of C. difficile as a counter-selectable marker to generate an efficient mutagenesis tool for this bacterium. This tool enabled us to delete all identified toxin genes within the phiCD630-1 prophage, thus allowing investigation of the role of TA in prophage maintenance. Furthermore, we were able to delete the large 49 kb phiCD630-2 prophage region using this improved procedure.

microbiology↗