Clostridioides difficile is an important nosocomial pathogen that has been classified as an
“urgent threat” by CDC. Antibiotic use is the primary risk factor for the development of C.
difficile-associated disease because it disrupts healthy protective gut flora and enables C.
difficile to colonize the colon. C. difficile damage host tissue by secreting toxins and
disseminates by forming spores. The toxins encoding genes, tcdA, and tcdB are part of a
pathogenicity locus and are positively regulated by tcdR. Nutrient availability and other
environmental factors greatly influence toxin production in C. difficile. CodY, a protein found in
many Gram-positive bacterial pathogens, is an important factor that regulates major metabolic
pathways and virulence gene expression in response to nutrient availability. In C. difficile, CodY
senses the nutritional status of the cell by binding to ligands, GTP, and branched-chain amino
acids (Isoleucine, Leucine, and Valine-ILV), and this active CodY then represses toxin genes
transcription by binding to the promoter of tcdR and repressing its transcription. We have
identified that the codY gene in an epidemic C. difficile strain (R20291) carries a point mutation
that changes its Tyrosine residue at the 146th position to an Asparagine (codYY146N). Following
this observation, our search in the database leads us to the codYV58A allele in more than 170
different C. difficile strains of different origins. Similar to Y146 (at the dimer interface), V58 is
located (ILV binding domain) at a crucial position in CodY and can influence its activity.
Preliminary data indicated that CodYY146N and CodYV58A differ from wild-type CodY in their
function and activity. Any change in the activity of a master regulator can alter the gene
regulatory networks under its control. Proposed studies in this exploratory grant application will
identify the readjusted gene regulatory networks in C. difficile because of the production
CodYV58A or CodYY146N and will evaluate their effect in C. difficile physiology and pathogenesis.