Clostridioides difficile nucleobase scavenging in the competitive gut environment

PROJECT SUMMARY Cells must synthesize nucleic acids to create genetic information that is used for protein synthesis, an essential process for all life. Nucleic acids are composed of nucleotides containing a nitrogenous base (nucleobase) that dictates base-pairing in the macromolecule and defines the genetic code. Nucleobases must be either

synthesized or salvaged from the environment for nucleic acid synthesis, and cellular energy demands often dictate which of these processes is used. Bacterial pathogens must synthesize or salvage nucleic acids for optimal growth and survival during infection, often through pathways that differ from the host, making nucleobase

metabolism an attractive target for therapeutic approaches. The vertebrate gastrointestinal tract is colonized by a cooperative group of microorganisms that prevent colonization by invading pathogens by depleting the gut environment of essential nutrients for colonization. The enteric pathogen Clostridioides difficile infects the host

gastrointestinal tract upon perturbation of the gut microbiota and is the leading cause of antibiotic-associated infections. Antibiotic perturbation of the gut microbiota alters the nutrient milieu in the gut environment, and C. difficile must compete with the host and microbiota to obtain critical nutrients to colonize and cause disease.

Amongst the nutrients altered in the gut following antibiotic treatment are nucleobases, and we hypothesize that C. difficile salvages nucleobases from the antibiotic perturbed gut. Our preliminary data indicate that C. difficile possesses a unique metabolic pathway to salvage a thio-modified uracil nucleobase, 4-thiouracil (4-TU), that is

present in the vertebrate gastrointestinal tract. C. difficile can metabolize 4-TU as a uracil source for growth instead of the energetically demanding pyrimidine biosynthetic pathway. Recently, an enzyme capable of metabolizing 4-TU has been described from an Aeromonas species, representing a member of a large family of

enzymes containing a DUF523 domain. However, the mechanism by which C. difficile metabolizes 4-TU has not been described. We have identified that two paralogous proteins (CD196_RS03875 and CD196_RS15345) contain a DUF523 domain in C. difficile. Furthermore, our work has uncovered that 4-TU is growth inhibitory to

Escherichia coli, which lacks a DUF523 homolog. We discovered that CD196_RS03875 which we named TudS (thiouracil desulfurase), is required for 4-TU metabolism and protects C. difficile from 4-TU mediated toxicity. We hypothesize that 4-TU metabolism enables C. difficile to thrive in the competitive gut environment, and

experiments in this proposal will test this hypothesis. In Specific Aim 1, we will define the molecular mechanism by which TudS converts 4-TU to uracil in C. difficile and identify other C. difficile gene products important for 4- TU metabolism through an innovative transposon screen. In Specific Aim 2, we will determine the contribution

of 4-TU metabolism to C. difficile pathogenesis using an animal model of infection with mutants defective in 4- TU metabolism. These studies have the potential to define a pathway for salvage and detoxification of an understudied, unconventional nucleobase that may contribute to the pathogenesis of an important gut pathogen.