Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains

The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment

Increased sporulation underpins adaptation of Clostridium difficile strain 630 to a biologically–relevant faecal environment, with implications for pathogenicity

Abstract Clostridium difficile virulence is driven primarily by the processes of toxinogenesis and sporulation, however many in vitro experimental systems for studying C. difficile physiology have arguably limited relevance to the human colonic environment. We therefore created a more physiologically–relevant model of the colonic milieu to study gut pathogen biology, incorporating human faecal water (FW) into growth media and assessing the physiological effects of this on C. difficile strain 630. We identified a novel set of C. difficile–derived metabolites in culture supernatants, including hexanoyl– and pentanoyl–amino acid derivatives by LC-MSn. Growth of C. difficile strain 630 in FW media resulted in increased cell length without altering growth rate and RNA sequencing identified 889 transcripts as differentially expressed (p < 0.001). Significantly, up to 300–fold increases in the expression of sporulation–associated genes were observed in FW media–grown cells, along with reductions in motility and toxin genes’ expression. Moreover, the expression of classical stress–response genes did not change, showing that C. difficile is well–adapted to this faecal milieu. Using our novel approach we have shown that interaction with FW causes fundamental changes in C. difficile biology that will lead to increased disease transmissibility

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability

Abstract Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37–41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis

Semiquantitative Analysis of Clinical Heat Stress in Clostridium difficile Strain 630 Using a GeLC/MS Workflow with emPAI Quantitation.

<div><p><i>Clostridium difficile</i> is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 <i>C. difficile</i> proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.</p></div

Pleiotropic Role of the RNA Chaperone Protein Hfq in the Human Pathogen Clostridium difficile

International audienceClostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria

Structure and assembly of a Clostridioides difficile spore polar appendage

Clostridioides difficile, a strict anaerobic spore-former, is the main cause of nosocomial disease associated to antibiotic therapy in adults and a growing concern in the community. Spores are the main infectious, persistence and transmission vehicle. Spore germination occurs in the intestine and the resulting vegetative cells will produce the toxins responsible for the disease symptoms, and spores. During sporulation, a wild type population bifurcates into two main spore morphotypes, with or without a thick exosporium. We show that this bifurcation extends to the formation of spores with a robust polar appendage or spores with a short appendage or that lack this structure. The cysteine-rich CdeM protein localizes to the appendage and around the entire surface of the spore, and is a major structural component of the exosporium, which we show is continuous with the appendage. In a CdeM mutant, when present, the polar appendage is short and disorganized. We show that wild type and cdeM spores with a short or no appendage germinate poorly in response to taurocholate, compared to those with an appendage. cdeM spores of the two types, however, germinate faster than their wild type counterparts. Thus, while the absence of CdeM may increase the permeability of spores to taurocholate, proper assembly of the appendage is also important for germination. Consistent with an overall enhancement of germination, a cdeM mutant shows increased virulence in a hamster model of disease. For a wild type population, spores with a short or no appendage germinate slower than the appendage-bearing spores. Differences in transmission, persistence and disease severity may result, in part, from their proportion in a spore population.<br/

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile▿†

Toxin synthesis in Clostridium difficile increases as cells enter into stationary phase. We first compared the expression profiles of strain 630E during exponential growth and at the onset of stationary phase and showed that genes involved in sporulation, cellular division, and motility, as well as carbon and amino acid metabolism, were differentially expressed under these conditions. We inactivated the sigH gene, which encodes an alternative sigma factor involved in the transition to post-exponential phase in Bacillus subtilis. Then, we compared the expression profiles of strain 630E and the sigH mutant after 10 h of growth. About 60% of the genes that were differentially expressed between exponential and stationary phases, including genes involved in motility, sporulation, and metabolism, were regulated by SigH, which thus appears to be a key regulator of the transition phase in C. difficile. SigH positively controls several genes required for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The spo0A and CD2492 genes, encoding the master regulator of sporulation and one of its associated kinases, and the spoIIA operon were transcribed from a SigH-dependent promoter. The expression of tcdA and tcdB, encoding the toxins, and of tcdR, encoding the sigma factor required for toxin production, increased in a sigH mutant. Finally, SigH regulates the expression of genes encoding surface-associated proteins, such as the Cwp66 adhesin, the S-layer precursor, and the flagellum components. Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting a SigH consensus in their promoter regions are good candidates for direct SigH targets