Tracking gene expression and oxidative damage of O2-stressed Clostridioides difficile by a multi-omics approach.

Clostridioides difficile is the major pathogen causing diarrhea following antibiotic treatment. It is considered to be a strictly anaerobic bacterium, however, previous studies have shown a certain and strain-dependent oxygen tolerance. In this study, the model strain C. difficile 630Δerm was shifted to micro-aerobiosis and was found to stay growing to the same extent as anaerobically growing cells with only few changes in the metabolite pattern. However, an extensive change in gene expression was determined by RNA-Seq. The most striking adaptation strategies involve a change in the reductive fermentation pathways of the amino acids proline, glycine and leucine. But also a far-reaching restructuring in the carbohydrate metabolism was detected with changes in the phosphotransferase system (PTS) facilitated uptake of sugars and a repression of enzymes of glycolysis and butyrate fermentation. Furthermore, a temporary induction in the synthesis of cofactor riboflavin was detected possibly due to an increased demand for flavin mononucleotid (FMN) and flavin adenine dinucleotide (FAD) in redox reactions. However, biosynthesis of the cofactors thiamin pyrophosphate and cobalamin were repressed deducing oxidation-prone enzymes and intermediates in these pathways. Micro-aerobically shocked cells were characterized by an increased demand for cysteine and a thiol redox proteomics approach revealed a dramatic increase in the oxidative state of cysteine in more than 800 peptides after 15 min of micro-aerobic shock. This provides not only a catalogue of oxidation-prone cysteine residues in the C. difficile proteome but also puts the amino acid cysteine into a key position in the oxidative stress response. Our study suggests that tolerance of C. difficile towards O2 is based on a complex and far-reaching adjustment of global gene expression which leads to only a slight change in phenotype.

High metabolic versatility of different toxigenic and non-toxigenic Clostridioides difficile isolates.

Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630Δerm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major differences in the fermentation products obtained after growth in a medium containing casamino acids and glucose as carbon and energy source. While the metabolism of branched chain amino acids remained comparable in all isolates, the aromatic amino acid uptake and metabolism and the central carbon metabolism-associated fermentation pathways varied strongly between the isolates. The patterns obtained followed neither the classification of the clades nor the ribotyping patterns nor the toxin distribution. As the toxin formation is strongly connected to the metabolism, our data allow an improved differentiation of C. difficile strains. The observed metabolic flexibility provides the optimal basis for the adaption in the course of infection and to changing conditions in different environments including the human gut.

Time-resolved amino acid uptake of Clostridium difficile 630Δerm and concomitant fermentation product and toxin formation.

BACKGROUND: Clostridium difficile is one of the major nosocomial threats causing severe gastrointestinal infections. Compared to the well documented clinical symptoms, little is known about the processes in the bacterial cell like the regulation and activity of metabolic pathways. In this study, we present time-resolved and global data of extracellular substrates and products. In a second part, we focus on the correlation of fermentation products and substrate uptake with toxin production. RESULTS: Formation of different fermentation products during growth in a comparison between the two different media in a global approach was studied using non-targeted gas chromatography-mass spectrometry (GC-MS) based analysis. During cultivation in a casamino acids medium and minimal medium, the clinical isolate C. difficile 630Δerm showed major differences in amino acid utilization: In casamino acids medium, C. difficile preferred proline, leucine and cysteine as carbon and energy sources while glutamate and lysine were not or hardly used. In contrast, proline and leucine were consumed at a significantly later stage in minimal medium. Due to the more complex substrate mixture more fermentation products were detectable in the casamino acids medium, accompanied by major changes in the ratios between oxidative and reductive Stickland products. Different glucose consumption dynamics were observed in presence of either casamino acids or the minimal set of amino acids, accompanied by major changes in butanoate formation. This was associated with a variation in both the toxin yield and a change in the ratio of toxin A to toxin B. CONCLUSIONS: Since in all media compositions, more than one substrate was available as a suitable carbon source, availability of different carbon sources and their metabolic fate appears to be the key factor for toxin formation.

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.

Clostridioides difficile Toxin CDT Induces Cytotoxic Responses in Human Mucosal-Associated Invariant T (MAIT) Cells.

Clostridioides difficile is the major cause of antibiotic-associated colitis (CDAC) with increasing prevalence in morbidity and mortality. Severity of CDAC has been attributed to hypervirulent C. difficile strains, which in addition to toxin A and B (TcdA, TcdB) produce the binary toxin C. difficile transferase (CDT). However, the link between these toxins and host immune responses as potential drivers of immunopathology are still incompletely understood. Here, we provide first experimental evidence that C. difficile toxins efficiently activate human mucosal-associated invariant T (MAIT) cells. Among the tested toxins, CDT and more specifically, the substrate binding and pore-forming subunit CDTb provoked significant MAIT cell activation resulting in selective MAIT cell degranulation of the lytic granule components perforin and granzyme B. CDT-induced MAIT cell responses required accessory immune cells, and we suggest monocytes as a potential CDT target cell population. Within the peripheral blood mononuclear cell fraction, we found increased IL-18 levels following CDT stimulation and MAIT cell response was indeed partly dependent on this cytokine. Surprisingly, CDT-induced MAIT cell activation was found to be partially MR1-dependent, although bacterial-derived metabolite antigens were absent. However, the role of antigen presentation in this process was not analyzed here and needs to be validated in future studies. Thus, MR1-dependent induction of MAIT cell cytotoxicity might be instrumental for hypervirulent C. difficile to overcome cellular barriers and may contribute to pathophysiology of CDAC.

Characterization of Clostridioides difficile DSM 101085 with A-B-CDT+ Phenotype from a Late Recurrent Colonization.

During the last decades, hypervirulent strains of Clostridioides difficile with frequent disease recurrence and increased mortality appeared. Clostridioides difficile DSM 101085 was isolated from a patient who suffered from several recurrent infections and colonizations, likely contributing to a fatal outcome. Analysis of the toxin repertoire revealed the presence of a complete binary toxin locus and an atypical pathogenicity locus consisting of only a tcdA pseudogene and a disrupted tcdC gene sequence. The pathogenicity locus shows upstream a transposon and has been subject to homologous recombination or lateral gene transfer events. Matching the results of the genome analysis, neither TcdA nor TcdB production but the expression of cdtA and cdtB was detected. This highlights a potential role of the binary toxin C. difficile toxin in this recurrent colonization and possibly further in a host-dependent virulence. Compared with the C. difficile metabolic model strains DSM 28645 (630Δerm) and DSM 27147 (R20291), strain DSM 101085 showed a specific metabolic profile, featuring changes in the threonine degradation pathways and alterations in the central carbon metabolism. Moreover, products originating from Stickland pathways processing leucine, aromatic amino acids, and methionine were more abundant in strain DSM 101085, indicating a more efficient use of these substrates. The particular characteristics of strain C. difficile DSM 101085 may represent an adaptation to a low-protein diet in a patient with recurrent infections.

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.