Antisense inhibition of RNA polymerase subunit of Clostridioides difficile

Clostridioides difficile, the causative agent of antibiotic-associated diarrhea and pseudomembranous colitis, has emerged as a major enteric pathogen in recent years. Antibiotic treatment perturbs the gut microbiome homeostasis, which facilitates the colonization and proliferation of the pathogen in the host intestine. Paradoxically, the clinical repertoire for C. difficile infection includes the antibiotics vancomycin and/or fidaxomicin. The current therapies do not address the perturbed gut microbiome, which supports the recurrence of infection after cessation of antibiotic therapy. Peptide nucleic acids (PNAs) are novel alternatives to traditional antimicrobial therapy capable of forming strong and stable complexes with RNA and DNA, thus permitting targeted inhibition of specific genes. Here, we report a novel PNA that can target the RNA polymerase alpha subunit (rpoA) in C. difficile. The designed anti-rpoA construct inhibited clinical isolates of C. difficile (minimum inhibitory concentration values ranged between 4 and 8 mu M) and exhibited bactericidal activity. Furthermore, silencing of the rpoA gene suppressed the expression of genes that encode virulence factors [toxin A (tcdA), toxin B (tcdB)] in C. difficile, and the gene that encodes the transcription factor stage 0 sporulation protein (spoOA). Interestingly, the efficacy of the designed PNA conjugate remained unaffected even when tested at different pH levels and against a high inoculum of the pathogen. The rpoA-TAT conjugate was very specific against C. difficile and did not inhibit members of the beneficial gut microflora. Taken altogether, our study confirms that the rpoA gene can be a promising narrow-spectrum therapeutic target to curb C. difficile infection. IMPORTANCE The widespread use of antibiotics can destroy beneficial intestinal microflora, opening the door for spores of Clostridioides difficile to run rampant in the digestive system, causing life-threatening diarrhea. Alternative approaches to target this deadly pathogen are urgently needed. We utilized targeted therapeutics called peptide nucleic acids (PNAs) to inhibit gene expression in C. difficile. Inhibition of the RNA polymerase alpha subunit gene (rpoA) by PNA was found to be lethal for C. difficile and could also disarm its virulence factors. Additionally, antisense inhibition of the C. difficile rpoA gene did not impact healthy microflora. We also propose a novel approach to manipulate gene expression in C. difficile without the need for established genetic tools.