Cellular adaptation of Clostridioides difficile to high salinity encompasses a compatible solute-responsive change in cell morphology.

Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrhoeas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt-stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of l-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress.

A Point Mutation in the Transcriptional Repressor PerR Results in a Constitutive Oxidative Stress Response in Clostridioides difficile 630Δerm.

The human pathogen Clostridioides difficile has evolved into the leading cause of nosocomial diarrhea. The bacterium is capable of spore formation, which even allows survival of antibiotic treatment. Although C. difficile features an anaerobic lifestyle, we determined a remarkably high oxygen tolerance of the laboratory reference strain 630Δerm A mutation of a single nucleotide (single nucleotide polymorphism [SNP]) in the DNA sequence (A to G) of the gene encoding the regulatory protein PerR results in an amino acid substitution (Thr to Ala) in one of the helices of the helix-turn-helix DNA binding domain of this transcriptional repressor in C. difficile 630Δerm PerR is a sensor protein for hydrogen peroxide and controls the expression of genes involved in the oxidative stress response. We show that PerR of C. difficile 630Δerm has lost its ability to bind the promoter region of PerR-controlled genes. This results in a constitutive derepression of genes encoding oxidative stress proteins such as a rubrerythrin (rbr1) whose mRNA abundance under anaerobic conditions was increased by a factor of about 7 compared to its parental strain C. difficile 630. Rubrerythrin repression in strain 630Δerm could be restored by the introduction of PerR from strain 630. The permanent oxidative stress response of C. difficile 630Δerm observed here should be considered in physiological and pathophysiological investigations based on this widely used model strain.IMPORTANCE The intestinal pathogen Clostridioides difficile is one of the major challenges in medical facilities nowadays. In order to better combat the bacterium, detailed knowledge of its physiology is mandatory. C. difficile strain 630Δerm was generated in a laboratory from the patient-isolated strain C. difficile 630 and represents a reference strain for many researchers in the field, serving as the basis for the construction of insertional gene knockout mutants. In our work, we demonstrate that this strain is characterized by an uncontrolled oxidative stress response as a result of a single-base-pair substitution in the sequence of a transcriptional regulator. C. difficile researchers working with model strain 630Δerm should be aware of this permanent stress response.

Influence of L-lactate and low glucose concentrations on the metabolism and the toxin formation of Clostridioides difficile.

The virulence of Clostridioides difficile (formerly Clostridium difficile) is mainly caused by its two toxins A and B. Their formation is significantly regulated by metabolic processes. Here we investigated the influence of various sugars (glucose, fructose, mannose, trehalose), sugar derivatives (mannitol and xylitol) and L-lactate on toxin synthesis. Fructose, mannose, trehalose, mannitol and xylitol in the growth medium resulted in an up to 2.2-fold increase of secreted toxin. Low glucose concentration of 2 g/L increased the toxin concentration 1.4-fold compared to growth without glucose, while high glucose concentrations in the growth medium (5 and 10 g/L) led to up to 6.6-fold decrease in toxin formation. Transcriptomic and metabolic investigation of the low glucose effect pointed towards an inactive CcpA and Rex regulatory system. L-lactate (500 mg/L) significantly reduced extracellular toxin formation. Transcriptome analyses of the later process revealed the induction of the lactose utilization operon encoding lactate racemase (larA), electron confurcating lactate dehydrogenase (CDIF630erm_01321) and the corresponding electron transfer flavoprotein (etfAB). Metabolome analyses revealed L-lactate consumption and the formation of pyruvate. The involved electron confurcation process might be responsible for the also observed reduction of the NAD+/NADH ratio which in turn is apparently linked to reduced toxin release from the cell.

Redefining the Clostridioides difficile σB Regulon: σB Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide.

In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation.IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.

Clostridioides difficile Activates Human Mucosal-Associated Invariant T Cells.

Clostridioides difficile infection (CDI) causes severe inflammatory responses at the intestinal mucosa but the immunological mechanisms underlying CDI-related immunopathology are still incompletely characterized. Here we identified for the first time that both, non-toxigenic strains as well as the hypervirulent ribotypes RT027 and RT023 of Clostridioides difficile (formerly Clostridium difficile), induced an effector phenotype in mucosal-associated invariant T (MAIT) cells. MAIT cells can directly respond to bacterial infections by recognizing MR1-presented metabolites derived from the riboflavin synthesis pathway constituting a novel class of antigens. We confirmed functional riboflavin synthesis of C. difficile and found fixed bacteria capable of activating primary human MAIT cells in a dose-dependent manner. C. difficile-activated MAIT cells showed an increased and MR1-dependent expression of CD69, proinflammatory IFNγ, and the lytic granule components granzyme B and perforin. Effector protein expression was accompanied by the release of lytic granules, which, in contrast to other effector functions, was mainly induced by IL-12 and IL-18. Notably, this study revealed hypervirulent C. difficile strains to be most competent in provoking MAIT cell responses suggesting MAIT cell activation to be instrumental for the immunopathology observed in C. difficile-associated colitis. In conclusion, we provide first evidence for a link between C. difficile metabolism and innate T cell-mediated immunity in humans.

Metabolic Reprogramming of Clostridioides difficile During the Stationary Phase With the Induction of Toxin Production.

The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism and the nutritional status of the growth environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A (CoA) biosynthesis. In contrast, different chaperones, several fermentation pathways, and cysteine, serine, and pantothenate biosynthesis were found upregulated. Focusing on the Stickland amino acid fermentation and the central carbon metabolism, we discovered the ability of C. difficile to replenish its favored amino acid cysteine by a pathway starting from the glycolytic 3-phosphoglycerate via L-serine as intermediate. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first to butanoate fermentation and then further to lactate fermentation. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular homeostasis and ensure survival under starvation conditions.

Iron Regulation in Clostridioides difficile.

The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (15 µM) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron and siderophore transporter genes were induced by Fur during low iron (0.2 µM) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some intermediates (phenylpyruvate, 2-oxo-isocaproate, 3-hydroxy-butyryl-CoA, crotonyl-CoA) accumulated, while end products like isocaproate and butyrate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB) of the F0F1-type was induced while the formation of a ATP-consuming, proton-pumping V-type ATPase (atpDBAFCEKI) was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. The fur mutant showed an increased sensitivity to vancomycin and polymyxin B. An intensive remodeling of the cell wall was observed, Polyamine biosynthesis (spe) was induced leading to an accumulation of spermine, spermidine, and putrescine. The fur mutant lost most of its flagella and motility. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.

Quality Control by Isoleucyl-tRNA Synthetase of Bacillus subtilis Is Required for Efficient Sporulation.

Isoleucyl-tRNA synthetase (IleRS) is an aminoacyl-tRNA synthetase whose essential function is to aminoacylate tRNAIle with isoleucine. Like some other aminoacyl-tRNA synthetases, IleRS can mischarge tRNAIle and correct this misacylation through a separate post-transfer editing function. To explore the biological significance of this editing function, we created a ileS(T233P) mutant of Bacillus subtilis that allows tRNAIle mischarging while retaining wild-type Ile-tRNAIle synthesis activity. As seen in other species defective for aminoacylation quality control, the growth rate of the ileS(T233P) strain was not significantly different from wild-type. When the ileS(T233P) strain was assessed for its ability to promote distinct phenotypes in response to starvation, the ileS(T233P) strain was observed to exhibit a significant defect in formation of environmentally resistant spores. The sporulation defect ranged from 3-fold to 30-fold and was due to a delay in activation of early sporulation genes. The loss of aminoacylation quality control in the ileS(T233P) strain resulted in the inability to compete with a wild-type strain under selective conditions that required sporulation. These data show that the quality control function of IleRS is required in B. subtilis for efficient sporulation and suggests that editing by aminoacyl-tRNA synthetases may be important for survival under starvation/nutrient limitation conditions.