Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen.

Katherine R Hargreaves,Cesar O Flores,Martha R J Clokie,Trevor D Lawley,Farooq Azam,John Paul

doi:10.1128/mbio.01045-13

Abstract

ABSTRACTClostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity.

Highlights

The bacterium Clostridium difficile is a major nosocomial pathogen [1], which can be carried asymptomatically [2], is present in environmental and zoonotic reservoirs [3], and can transmit between livestock and humans [4]
The direct repeats (DRs) sequences from strain CD630 arrays are predicted to be folded, and a comparison of the DR sequences to those in Kunin et al [36] shows they cluster closest to groups 4 and 11
Phage CRISPR/Cas system dynamics have been explored in other systems, little analysis has been published examining the possible role of the CRISPR/Cas system in C. difficile

Summary

Introduction

The bacterium Clostridium difficile is a major nosocomial pathogen [1], which can be carried asymptomatically [2], is present in environmental and zoonotic reservoirs [3], and can transmit between livestock and humans [4]. Prophages are common within C. difficile genomes, and several temperate phages have been described which can infect specific strains by following a lytic cycle [7]. Prophage infection is prevalent in C. difficile [28], relatively few free or inducible prophages have been found that can propagate in a lytic manner on tested strains, despite large-scale screens for such phages [24, 27, 29] The mechanisms controlling these phage-host dynamics in C. difficile are unknown. An example of phages impacting the CRISPR dynamics can be seen in the ICR1 phage that infects Vibrio cholerae This phage has been shown to carry a functioning CRISPR/Cas system that targets a phage inhibitory chromosomal island, permitting infection of the host [31]. Our analyses suggest that the CRISPR/ Cas system is an important determinant of phage infection in C. difficile

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Conclusion