Pal Rusha, Seleem Mohamed N
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
Center for Emerging Zoonotic and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
Microbiol Spectr. 2023 Sep 29;11(5):e0175523. doi: 10.1128/spectrum.01755-23.
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 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 α subunit () in . The designed anti- construct inhibited clinical isolates of (minimum inhibitory concentration values ranged between 4 and 8 µM) and exhibited bactericidal activity. Furthermore, silencing of the gene suppressed the expression of genes that encode virulence factors [toxin A (), toxin B ()] in , and the gene that encodes the transcription factor stage 0 sporulation protein (). 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 -TAT conjugate was very specific against and did not inhibit members of the beneficial gut microflora. Taken altogether, our study confirms that the gene can be a promising narrow-spectrum therapeutic target to curb infection. IMPORTANCE The widespread use of antibiotics can destroy beneficial intestinal microflora, opening the door for spores of 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 . Inhibition of the RNA polymerase α subunit gene (A) by PNA was found to be lethal for and could also disarm its virulence factors. Additionally, antisense inhibition of the A gene did not impact healthy microflora. We also propose a novel approach to manipulate gene expression in without the need for established genetic tools.
近年来，抗生素相关性腹泻和假膜性结肠炎的病原体已成为一种主要的肠道病原体。抗生素治疗会扰乱肠道微生物群稳态，这有利于该病原体在宿主肠道内的定植和增殖。矛盾的是，针对该感染的临床治疗药物包括万古霉素和/或非达霉素。目前的治疗方法并未解决受扰乱的肠道微生物群问题，这导致抗生素治疗停止后感染复发。肽核酸（PNA）是传统抗菌治疗的新型替代物，能够与RNA和DNA形成牢固且稳定的复合物，从而实现对特定基因的靶向抑制。在此，我们报告一种新型PNA，它可以靶向艰难梭菌中的RNA聚合酶α亚基（rpoA）。设计的抗rpoA构建体抑制了艰难梭菌临床分离株（最低抑菌浓度值在4至8 μM之间）并表现出杀菌活性。此外，rpoA基因的沉默抑制了艰难梭菌中编码毒力因子[毒素A（tcdA）、毒素B（tcdB）]的基因以及编码转录因子0期芽孢形成蛋白（spo0A）的基因的表达。有趣的是，即使在不同pH水平下针对高接种量的病原体进行测试，设计的PNA偶联物的功效仍不受影响。rpoA - TAT偶联物对艰难梭菌具有高度特异性，且不抑制有益肠道微生物群的成员。综上所述，我们的研究证实rpoA基因可能是抑制艰难梭菌感染的一个有前景的窄谱治疗靶点。重要性 抗生素的广泛使用会破坏有益的肠道微生物群，为艰难梭菌孢子在消化系统中肆虐打开大门，导致危及生命的腹泻。迫切需要针对这种致命病原体的替代方法。我们利用称为肽核酸（PNA）的靶向疗法来抑制艰难梭菌中的基因表达。发现PNA对RNA聚合酶α亚基基因（rpoA）的抑制对艰难梭菌具有致死性，并且还可以消除其毒力因子。此外，对rpoA基因的反义抑制不会影响健康的微生物群。我们还提出了一种无需成熟遗传工具即可操纵艰难梭菌基因表达的新方法。
