Centralin vivomechanisms by whichC. difficile’sproline reductase drives efficient metabolism, growth, and toxin production

Abstract

Clostridioides difficile (CD) is a sporulating and toxin-producing nosocomial pathogen that opportunistically infects the gut, particularly in patients with depleted microbiota after antibiotic exposure. Metabolically, CD rapidly generates energy and substrates for growth from Stickland fermentations of amino acids, with proline being a preferred reductive substrate. To investigate the in vivo effects of reductive proline metabolism on C. difficile virulence in an enriched gut nutrient environment, we evaluated wild-type and isogenic delta-prdB strains of ATCC43255 on pathogen behaviors and host outcomes in highly susceptible gnotobiotic mice. Mice infected with the delta-prdB mutant demonstrated extended survival via delayed colonization, growth and toxin production but ultimately succumbed to disease. In vivo transcriptomic analyses demonstrated how the absence of proline reductase activity more broadly disrupted pathogen metabolism including failure to recruit oxidative Stickland pathways, ornithine transformations to alanine, and additional pathways generating growth-promoting substrates, contributing to delayed growth, sporulation, and toxin production. Our findings illustrate the central role for proline reductase metabolism to support early stages of C. difficile colonization and subsequent impact on the pathogen s ability to rapidly expand and cause disease.

Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH

“…8 ). In line with previous studies ( 5 , 29 , 30 ), C. difficile WT displayed proline-dependent growth, leading to higher cell density at higher proline levels. In contrast, the growth of the Δ prdH -mutant was found to not respond to proline.…”

Distilling Mechanistic Models From Multi-Omics Data

“…5-aminovalerate and proline were highly correlated with both tcdA and tcdB , in agreement with previous results based on correlations between time points [13]. This also corroborates work that has suggested that the Stickland reaction is necessary for robust C. difficile growth [42, 33, 43, 44]. Hydroxyphenyl propionate, guanosine, and sedoheptulose were also partially correlated with tcdA and tcdB .…”

“…These results highlight the need to further investigate biochemical mechanisms underpinning these relationships. Both proline and 5-aminovalerate are involved in the Stickland reaction which provides energy to C. difficile [42, 33, 43, 44]. Through this reaction, C. difficile proline reductase reduces proline to 5-aminovalerate by using an electron donated to NADH by an amino acid (typically branched chain amino acids) from the oxidative branch of the Stick-land reaction [52].…”

“…These results highlight the need to further investigate biochemical mechanisms underpinning these relationships. Both proline and 5-aminovalerate are involved in the Stickland reaction which provides energy to C. difficile [42,33,43,44]. Through this reaction, C. difficile land reaction [52].…”

“…growth[42,33,43,44]. Hydroxyphenyl propionate, guanosine, and sedoheptulose were also partially correlated with tcdA and tcdB.A graphical representation of the metabolites with high partial corrrelations to tcdA and tcdB expression listed in Table1are shown in Figure3with solid lines indicating positive correlations, dashed lines indicating negative correlations, and line thickness representing the strength of the correlation.…”