Multiple phase-variable mechanisms, including capsular polysaccharides, modify bacteriophage susceptibility in Bacteroides thetaiotaomicron

Abstract

21A variety of cell surface structures, including capsular polysaccharides (CPS), dictate 22interactions between bacteria and their environment including their viruses (bacteriophages). 23Members of the prominent human gut Bacteroidetes characteristically produce several phase-24 variable CPS, but their contributions to bacteriophage interactions are unknown. We used 25 engineered strains of the human symbiont Bacteroides thetaiotaomicron, which differ only in the 26 CPS they express, to isolate bacteriophages from two locations in the United States. Testing each 27 of 71 bacteriophages against a panel of strains that express wild-type phase-variable CPS, one of 28 eight different single CPS, or no CPS at all, revealed that each phage infects only a subset of 29 otherwise isogenic strains. Deletion of infection-permissive CPS from B. thetaiotaomicron was 30 sufficient to abolish infection for several individual bacteriophages, while infection of wild-type 31 B. thetaiotaomicron with either of two different bacteriophages rapidly selected for expression of 32 non-permissive CPS. Surprisingly, acapsular B. thetaiotaomicron also escapes complete killing 33 by these bacteriophages, but surviving bacteria exhibit increased expression of 8 distinct phase-34 variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes 35 resistance to multiple bacteriophages. Finally, both wild-type and acapsular B. thetaiotaomicron 36 were able to separately co-exist with one bacteriophage for over two months in the mouse gut, 37suggesting that phase-variation promotes resistance but also generates sufficient numbers of 38 susceptible revertants to allow bacteriophage persistence. Our results reveal important roles for 39Bacteroides CPS and other cell surface structures that allow these bacteria to persist despite 40 bacteriophage predation and hold important implications for using bacteriophages therapeutically 41 to target gut symbionts. 42 43Bacteroides thetaiotaomicron and Bacteroides fragilis each encode 8 different CPS 14,15 and there 61 is broad genetic diversity of cps loci among different strains within these species (e.g., 47 62 different cps biosynthetic loci were identified in just 14 strains of B. thetaiotaomicron) 8 . In 63Bacteroides, CPS structures appear to surround the entire bacterial cell 16,17 and the cps 64 biosynthetic loci that encode these surface coatings are often under the control of phase variable 65 promoters 8,15,18 . In conjunction with other regulatory mechanisms, phase variable CPS 66 4 expression generates phenotypic heterogeneity within an otherwise isogenic population that may 67 facilitate survival in the face of diverse disturbances 8,15,19,20 . 68 Bacterial viruses or bacteriophages (herein, phages), like the bacteria on which they prey, 69 vary greatly across individual gut microbiomes and are even responsive to host dietary changes 70 and disease states [21][22][23][24][25] . Compared to gut bacteria, far less is understood about the phages of the 71 gut microbi...

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].…”

Infection with novelBacteroides phage BV01alters host transcriptome and bile acid metabolism in a common human gut microbe

“…We demonstrate that BV01 is transducible in a gnotobiotic mouse model, suggesting that an unknown mammalian host factor is required for novel BV01 infection. Enigmatic infection dynamics may be the result of the phase variable polysaccharide capsule, as recent work suggests heterogeneity in capsular composition hinders phage infection on population-scales (17). Indeed, it has long been observed that finding phages in the Bacteroides using traditional techniques is difficult or impossible for most host strains (65, 66), making the host-first approach to phage discovery used here especially appealing.…”

“…The genus is known to degrade a diversity of complex carbohydrates (9, 10), and interact with host immune cells (11, 12). Within a single human host, many Bacteroides species and strains coexist, competing for nutrients under changing environmental conditions caused by host diet (13), host metabolites (14), host immune system activities (15, 16), and phage predation (17). Moreover, horizontal gene transfer plays an important role in shaping the evolution and function of Bacteroides genomes (18, 19).…”

“…CrAssphages demonstrate how traditional phage techniques ( e.g., agar overlay plaque assays) are not reliable for Bacteroides hosts (22), likely due to heterogeneity in capsular polysaccharide composition within isogenic cultures (23, 24). In fact, deletion of all capsular polysaccharide synthesis loci allows for the isolation of many phages on the host Bacteroides thetaiotaomicron VPI-5482 (17). Most phages isolated against Bacteroides hosts thus far exhibit an obligately lytic lifestyle (17, 22, 25, 26), despite the potentially large role of lysogeny in phage-host interactions in the gut, where at least 17% of the gut phageome is predicted to be temperate (27, 28).…”

“…In fact, deletion of all capsular polysaccharide synthesis loci allows for the isolation of many phages on the host Bacteroides thetaiotaomicron VPI-5482 (17). Most phages isolated against Bacteroides hosts thus far exhibit an obligately lytic lifestyle (17, 22, 25, 26), despite the potentially large role of lysogeny in phage-host interactions in the gut, where at least 17% of the gut phageome is predicted to be temperate (27, 28).…”