Campylobacter jejuni Developed the Resistance to Bacteriophage CP39 by Phase Variable Expression of 06875 Encoding the CGPTase.

Yuanyue Tang,Xin’An Jiao,Jinlin Huang,Jie Li,Hangning Ying,Zhaojun Song,Yuexuan Wang,Linghua Kong

doi:10.3390/v14030485

Abstract

Bacteriophage (phage) is regarded as an antimicrobial alternative for Campylobacter in food production. However, the development of phage resistance to the host is a main concern for the phage application. This study characterized the phage CP39 and investigated the phage resistance of CP39 in Campylobacter jejuni NCTC12662. We determined that phage CP39 belonged to the Myoviridae family by the WGS and phylogenetic analysis. Phage CP39 was confirmed as a capsular polysaccharide (CPS)-dependent phage by primary C. jejuni phage typing. It was further confirmed that the phage could not be adsorbed by the acapsular mutant ΔkpsM but showed the same lytic ability in both the wild-type strain NCTC 12662 and the ΔmotA mutant lacking motile flagella filaments. We further determined that the 06875 gene encoding CDP-glycerol:poly (glycerophosphate) glycerophosphotransferase (CGPTase) in the CPS loci was related to phage CP39 adsorption by SNP analysis and observed a rapid development of phage resistance in NCTC 12662 during the phage infection. Furthermore, we observed a high mutation frequency of 06875 (32%), which randomly occurred in nine different sites in the gene according to colony PCR sequencing. The mutation of the 06875 gene could cause the phase variable expression of non-functional protein and allow the bacteria against the phage infection by modifying the CPS. Our study confirmed the 06875 gene responsible for the CPS-phage adsorption for the first time and demonstrated the phase variable expression as a main mechanism for the bacteria to defend phage CP39. Our study provided knowledge for the evolutionary adaption of bacteria against the bacteriophage, which could add more information to understand the phage resistance mechanism before applying in the industry.

Highlights

Bacteriophages play an essential part in shaping the bacterial ecosystem by the lytic process, while bacteria usually react constantly to promote a defense action during phage infection [1]
Our results demonstrated that CP39 could form a clear plaque on wild type (Figure 2A) and ∆motA mutant (Figure 2B), while no plaque was observed on ∆kpsM (Figure 2C), indicating that the absence of capsular polysaccharides (CPS) in the recipient strain could influence the phage adsorption
Our study demonstrated that phage CP39 was a CPS-dependent phage belonging to the Myoviridae family

Summary

Introduction

Bacteriophages (or phages) play an essential part in shaping the bacterial ecosystem by the lytic process, while bacteria usually react constantly to promote a defense action during phage infection [1]. The phage resistance mechanisms can either be the external prevention of phage to inject DNA by blocking or modifying phage binding receptors on the cell membrane or the internal degradation of the foreign phage genomic DNA, including the restriction–modification systems, abortive infection systems, and the CRISPR-cas interference [2]. The initial defense strategy is to modify the phage receptor and prevent phage absorption, which is the most common bacterial resistance strategy during phage–host interaction [2]. Phage receptors in Gram-negative bacteria include the outer membrane proteins, lipopolysaccharides (LPS), capsular polysaccharides (CPS), pili, and flagella, which unique surface structures could ensure phage binding to the correct host [3]

Methods

Results

Conclusion