Pleiotropic Clostridioides difficile Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses.

The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.

PrsA2 (CD630_35000) of Clostridioides difficile Is an Active Parvulin-Type PPIase and a Virulence Modulator.

Clostridioides difficile is the main cause for nosocomial antibiotic associated diarrhea and has become a major burden for the health care systems of industrial countries. Its main virulence factors, the small GTPase glycosylating toxins TcdA and TcdB, are extensively studied. In contrast, the contribution of other factors to development and progression of C. difficile infection (CDI) are only insufficiently understood. Many bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) have been described in the context of virulence. Among them are the parvulin-type PrsA-like PPIases of Gram-positive bacteria. On this basis, we identified CD630_35000 as the PrsA2 homolog in C. difficile and conducted its enzymatic and phenotypic characterization in order to assess its involvement during C. difficile infection. For this purpose, wild type CdPrsA2 and mutant variants carrying amino acid exchanges mainly in the PPIase domain were recombinantly produced. Recombinant CdPrsA2 showed PPIase activity toward the substrate peptide Ala-Xaa-Pro-Phe with a preference for positively charged amino acids preceding the proline residue. Mutation of conserved residues in its active site pocket impaired the enzymatic activity. A PrsA2 deficient mutant was generated in the C. difficile 630Δerm background using the ClosTron technology. Inactivation of prsA2 resulted in a reduced germination rate in response to taurocholic acid, and in a slight increase in resistance to the secondary bile acids LCA and DCA. Interestingly, in the absence of PrsA2 colonization of mice by C. difficile 630 was significantly reduced. We concluded that CdPrsA2 is an active PPIase that acts as a virulence modulator by influencing crucial processes like sporulation, germination and bile acid resistance resulting in attenuated mice colonization.

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.

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.