Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

Abstract

Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n ‫؍‬ 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA. V irulent phages have been shown to present a tremendous selective pressure on their bacterial host populations. Not only is phage predation a major driver of bacterial diversification (1, 2), but it may also select for hypermutators, which could increase the frequency of mutations in bacterial populations (3, 4). Whereas bacteria are limited to one cell division per generation, a single phage-infected cell can produce a burst ranging from less than 5 to over 1,000 progeny phages in a similar period of time (5-7). Phages consequently have the capability to rapidly outgrow their bacterial hosts and can significantly reduce or eliminate susceptible bacteria in the local environment (8, 9). Therefore, the potential for bacterial strains to persist in an environment containing lytic phages may be contingent upon that strain accumulating spontaneous mutations that grant resistance to phage infection (10). These phage-resistant mutant strains most typically resist phage infection through mechanisms of adsorption inhibition, i.e., alterations of the cell surface that affect phage attachment (11). However, one study reported that nearly all phage-resistant mutant strains of Streptococcus thermophilus had acquired CRISPR spacers that matched invading phage genomes (12); these S. thermophilus mutant strains would be expected to resist phage infection after the adsorption step. Phage-resistant mutant strains that resist infection through mechanisms of adsorption inh...

A Viral Immunity Chromosome in the Marine Picoeukaryote, Ostreococcus tauri

“…Surface glycans are known to be important for host–virus interactions, where they mediate initial binding and recognition events of both host cells and pathogens at the cell surfaces [53]. In particular, rhamnan is a polymer important for bacterial host–virus interactions [54,55], and sulphated derivatives of rhamnans are known to have anti-viral activity in mammals [56]. The metazoan-derived CMP N-acetylneuraminic acid hydroxylase (ostta19g00610), in humans makes the influenza A virus receptor, N-acetylneuraminic acid, whose decorations affect receptor specificity [57].…”

High-throughput mapping of the phage resistance landscape inE. coli

“…Despite nearly a century of pioneering molecular work, the mechanistic insights into phage specificity for a given host, infection pathways, and the breadth of bacterial responses to different phages have largely focused on a handful of individual bacterium-phage systems [9][10][11][12][13]. Bacterial sensitivity/resistance to phages is typically characterized using phenotypic methods such as cross-infection patterns against a panel of phages [14][15][16][17][18][19][20][21][22][23][24][25][26][27] or by whole-genome sequencing of phage-resistant mutants [28][29][30][31][32]. As such, our understanding of bacterial resistance mechanisms against phages remains limited, and the field is therefore in need of improved methods to characterize phage-host interactions, determine the generality and diversity of phage resistance mechanisms in nature, and identify the degree of specificity for each bacterial resistance mechanism across diverse phage types [13,25,26,[33][34][35][36][37][38][39][40][41][42][43][44][45][46][47].…”

Using Phage PSM‐1 to Control Shewanella marisflavi Infection in Juvenile Sea Cucumber, Apostichopus japonicus

“…However, there is an increase in the OD 600 value in the later period of incubation for the cultures infected with phage PSM‐1, which might be caused by the generation of phage‐resistant strains. Commonly, phage resistance tends to occur when a single phage is used individually to control bacterial growth in a laboratory‐controlled environment (Labrie et al ; Denes et al ).…”

“…Commonly, phage resistance tends to occur when a single phage is used individually to control bacterial growth in a laboratory-controlled environment (Labrie et al 2010;Denes et al 2015). Phage storage is considered to be an important component in the process of the application.…”