Deletion of Prophages in Clostridioides difficile

Abstract

Clostridioides difficile (C. difficile) formally known as Clostridium difficile, is a Gram-positive spore forming anaerobe responsible for almost all cases of pseudomembranous colitis and is implicated in 25-33% of antibiotic associated diarrhoea. Many C. difficile strains are lysogens; that is, they carry bacterial viruses known as phages, which have been shown to affect bacterial cellular processes. Phages can be virulent or temperate; a temperate phage infects a bacterial cell, then integrates its genome (prophage) into the bacterial chromosome. All phages currently identified within C. difficile are temperate. C. difficile 630 (CD630) was the first isolate to be sequenced and is a reference strain. CD630 harbours two temperate phages, ΦCD630-1, and ΦCD630-2 of 55,850 bp and 49,178 bp, respectively. Both phage genomes have a similar organisation and above 60% genome nucleotide homology. To study whether prophages contribute to host virulence, persistence, and transmission in CD630, prophage deletion is a first step. Due to the large size of the prophage genomes, precise deletion is challenging. This study aims to delete a prophage of CD630.

Two plasmids, pRK6301 and pRK6302, based on pMTL-SC7315 and codA-based counter selection, were generated to delete ΦCD630-1 and ΦCD630-2, respectively from the CD630 genome by allele coupled exchange. Homology arms (HA) of 1kb flanking each prophage genome were cloned, such that pRK6301 and pRK6302 were ~8.8kb. Of 200 transconjugants screened, three colonies lost pRK6301 and two colonies lost pRK6302, hence were double crossover mutants. However, all double crossover mutants were found to retain the prophage integrated junctions, hence no prophage deletion occurred.

Another recombinant plasmid, pHHCD630-2sgRNA_HA, based on pMTL83151 and CRISPR-Cas9 selection of viable mutants, was generated to delete ΦCD630-2 by targeting the phage integrase gene. The single guide RNA (sgRNA) was constitutively expressed, while Cas9 was inducible by anhydrotetracycline (aTC). The same homology arm from pRK6302 was cloned, such that pHHCD630-2sgRNA_HA was ~11.9 kb. Nine C. difficile transconjugant colonies containing either pHHCD630-2sgRNA_HA, control plasmid without HA (pHH_sgRNA), or control empty plasmid (pHH) were screened by PCR. All 9 colonies contained pHHCD630-2sgRNA_HA. Six colonies were confirmed for carrying pHH_sgRNA plasmid (without HA), and nine colonies contained pHH.

A transconjugant of pHHCD630-2sgRNA_HA was induced with 30 ng/mL of aTC and screened for viable mutants. Five colonies were negative for the phage integrase gene by colony PCR, suggesting the ΦCD630-2 prophage or integrase gene was lost. However, the colonies remained resistant to thiamphenicol, and all retained the plasmid, confirmed by PCR, after efforts to cure the plasmid by serial passage. Further work is needed to confirm their identity. In conclusion, there are genetic engineering tools currently available which allow precise manipulation of genomes including the deletion of prophages. This study generated plasmids and protocols for prophage deletion in CD630, however, did not confirm the deletion of a prophage.