The protein network of the Rnf-proline reductase complex required for respiratory energy generation in Clostridioides difficile

Abstract

Metabolic processes are central to toxin formation and corresponding infection success of the Gram-positive, anaerobic gut pathogen C. difficile. The bacterium utilizes a unique amino acid- based fermentation (Stickland reaction) in conjunction with a membrane-spanning ion-pumping Rnf complex for ATP generation. Here, it was demonstrated by interactomics that one of the major enzymes of the reductive Stickland fermentation D-proline reductase forms a stable supercomplex with the Rnf complex. Other enzymes of Stickland fermentation (subunits of the glycine reductase), of ATP generation (F-type and V-type ATPase), of electron bifurcation (Bcd2/Etf), butanoate metabolism (e.g. Buk2, Cat2, Crt2 and SucD), glycolysis (e.g. Fba, Gap and Pyk) and the TCA cycle (e.g. PycA) are also members of the supercomplex. Correspondingly, a still viable rnfC mutant contained an almost inactive D-proline reductase, showed changes in cell morphology, flagella formation and toxin production. Detailed holistic Omics-based analyses of compensatory changes in gene regulation, the protein inventory and metabolite composition revealed a significant reprogramming of the overall cell physiology. The NAD+/NADH ratio was imbalanced, a compensatory upregulation of the butanoate metabolism and TCA cycle as well as changes in the amino acid pathways were observed. Finally, the rnfC mutant showed clear cut defects in the colonization of the host as tested in a mouse model. In summary, obtained results underscores the importance of the Rnf-proline reductase supercomplex for the infection by C. difficile.