Impact of Phage CDHS-1 on the Transcription, Physiology and Pathogenicity of a Clostridioides difficile Ribotype 027 Strain, R20291

Janet Y Nale,Shaun Heaphy,Martha R J Clokie,Thekra Sideeq Al-Tayawi

doi:10.3390/v13112262

Abstract

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection.

Highlights

Clostridioides difficile is one of the most common causes of antibiotic related nosocomial infections with symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis and/or toxic megacolon
We have previously isolated, curated and studied a large bank of C. difficile phages which we have shown to have therapeutic potential for a wide range of human pathogenic strains in relevant infection models including biofilms, fermentation vessels spiked with combined multiple human gut microbiota, Galleria mellonella and hamster infection models [11,16,17,18]
To ascertain the impact of CDHS-1 infection on R20291, we identified the phases of the phage replication cycle and the genes expressed during these time points [22]

Summary

Introduction

Clostridioides difficile is one of the most common causes of antibiotic related nosocomial infections with symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis and/or toxic megacolon. The pathogenesis of C. difficile is commonly linked to enterotoxin and cytotoxin A, and a cytotoxin B, which are encoded by tcdA and tcdB genes respectively, and located on the pathogenicity locus, PaLoc [3]. The toxins belong to the Rho-glucosylating toxin or large Clostridial toxin family, which target and deactivate guanosine triphosphatases in the epithelial cells of infected guts [4,5] This can lead to disaggregation of the host cell cytoskeleton, loss of tight junctions between the epithelial cells and, apoptosis. In addition to the two toxins, a binary toxin is found in some strains such as those classified within the hypervirulent ribotype 027, where it contributes to increased pathogenicity [3]

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Results

Conclusion