Parallel genomics uncover novel enterococcal-bacteriophage interactions

Chatterjee, Anushila; Willett, Julia L. E.; Nguyen, Uyen Thy; Monogue, Brendan; Palmer, Kelli L.; Dunny, Gary M.; Duerkop, Breck A.

doi:10.1101/858506

Cited by 14 publications

(28 citation statements)

References 98 publications

(71 reference statements)

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“…There have been few attempts to use genetic approaches for studying genome-wide host factors essential in phage infection. These loss-of-function (LOF) genetic screens broadly use bacterial saturation mutagenesis [48,53,[57][58][59][60] or an arrayed library of single-gene deletion strains for studying phage-host interactions [49,50,52,61,62]. Consequently, these studies have generally involved laborious experiments on relatively few phages and their hosts, and scaling the approach to characterize hundreds of phages is challenging.…”

Section: Introductionmentioning

confidence: 99%

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

Mutalik¹,

Adler²,

Rishi³

et al. 2020

Preprint

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Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and highthroughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phagemediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.3 Introduction:

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Section: Introductionmentioning

confidence: 99%

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

Mutalik¹,

Adler²,

Rishi³

et al. 2020

Preprint

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“…The T7SS has been shown to play an important role in virulence in multiple bacterial species such as Staphylococcus, Listeria and Bacillus (45). In E. faecalis, genes in the T7SS locus have been shown to be induced during phage infection (46). We observed that transposon mutants for esaB (OG1RF_11103), a putative cytoplasmic accessory protein; OG1RF_11109…”

Section: Ethanolamine Utilization and T7ss Genes Contribute To E Faementioning

confidence: 89%

“…For example, OG1RF_12241, a homolog of the oxidative stress regulator hypR, was underrepresented at all time points (Table S4A). We have recently shown that this gene is involved in phage VPE25 infection of E. faecalis OG1RF (46). Furthermore, enterococcal mutants in the CRISPR/cas9 locus (OG1RF_10404 and OG1RF_10407) were underrepresented at all three time points (Table S4A).…”

Section: Ethanolamine Utilization and T7ss Genes Contribute To E Faementioning

confidence: 94%

“…Transposon-junction DNA library preparation and Illumina NovaSeq 6000 DNA sequencing (150 base paired end mode) was performed by the Microarray and Genomics Core at the University of Colorado Anschutz Medical Campus as previously described (46). For downstream analysis of transposonjunctions, we used only the R1 reads generated by paired end sequencing.…”

Section: Transposon Library Sequencing and Data Analysismentioning

confidence: 99%

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Genome-wide mutagenesis identifies factors involved inEnterococcus faecalisvaginal adherence and persistence

Alhajjar

Chatterjee

Spencer

et al. 2020

Preprint

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21Enterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an 22 opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive 23 tract and reports suggest vaginal colonization increases following antibiotic treatment or in patients with 24 aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal 25 colonization and subsequent infection. We developed a mouse model of E. faecalis vaginal carriage and 26 demonstrate that both vancomycin resistant and sensitive strains colonize the murine vaginal tract. 27Following vaginal colonization, we observed E. faecalis in vaginal, cervical and uterine tissue. A mutant 28 lacking endocarditis-and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with 29 human vaginal and cervical cells in vitro as well as ascend to the cervix and uterus in vivo. To further 30 identify novel factors that promote vaginal colonization, we screened a low-complexity transposon (Tn) 31 mutant library to identify genes important for E. faecalis persistence in the vaginal tract. This screen 32 revealed 383 mutants that were underrepresented during vaginal colonization at all time points compared 33 to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism and the 34 type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal 35 the complex nature of vaginal colonization and suggest that multiple factors contribute to E. 36 faecalis persistence in the reproductive tract. 37 38 IMPORTANCE 39Despite increasing prevalence and association of E. faecalis with aerobic vaginitis, essentially nothing is 40 known about the bacterial factors that influence E. faecalis vaginal colonization. We have established an 41 animal model of vaginal colonization that supports colonization of multiple E. faecalis strains. 42Additionally, we determined that pili, ethanolamine utilization, and type VII secretion system genes 43 3 contribute to vaginal persistence and ascension to reproductive tract tissues. Identification of factors 44 important for vaginal colonization and persistence provides potential targets for the development of 45 therapeutics. This study is the first to identify key determinants that promote vaginal colonization by E. 46 faecalis, which may represent an important reservoir for antibiotic resistant enterococci. 47 48 INTRODUCTION 49Enterococcus faecalis is an opportunistic pathogen that resides in the human gastrointestinal and 50 urogenital tracts [1, 2]. While E. faecalis colonization is normally asymptomatic, certain populations are 51 at risk for severe disease including urinary tract infections (UTIs), wound infections, pelvic inflammatory 52 disease (PID), infective endocarditis, and adverse birth effects during pregnancy [3, 4]. Enterococcal 53 infections are often associated with the production of biofilms, assemblages of microbes enclosed in an 54 extracellula...

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“…Mutation of epaR and epaX results in E. faecalis phage resistance (9,10,14) and recently it was determined that the epaR and epaX genes of E. faecalis V583 participate in wall teichoic acid biosynthesis (25). Considering that mutation of the epaX homologs epaOX and epaOX2 from E. faecalis OG1RF conferred phage VPE25-resistance by limiting phage adsorption (10,11), we suspect that teichoic acids also mediate adsorption of phage 9183 to E. faecium 1,141,733. We were surprised that we did not find any phage 9183 resistant strains with mutations in PIP EF, given the high protein homology and similar genome organization observed between phages 9183, VPE25 and VFW, the latter two which use PIPEF as a receptor (11) (Fig.…”

Section: Faeciummentioning

confidence: 96%

Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium

Canfield

Chatterjee

Espinosa³

et al. 2020

Preprint

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Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-arginine-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium. Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could potentially serve as antimicrobial adjuvants in treating E. faecium infections.

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Parallel genomics uncover novel enterococcal-bacteriophage interactions

Cited by 14 publications

References 98 publications

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

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

Genome-wide mutagenesis identifies factors involved inEnterococcus faecalisvaginal adherence and persistence

Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium

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