Loss-of-Function Mutations in
            <i>epaR</i>
            Confer Resistance to ϕNPV1 Infection in Enterococcus faecalis OG1RF

Ho, Khang; Huo, Wenwen; Pas, Savannah; Dao, Ryan; Palmer, Kelli L.

doi:10.1128/aac.00758-18

Cited by 55 publications

(70 citation statements)

References 42 publications

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“…3 and Table S1). This is consistent with a recent study demonstrating that mutation of epaR in E. faecalis OG1RF results in resistance to infection by the phage NPV1 (16). The remaining mutations in the phage-resistant isolates mapped to variable-region epa genes, including epaS, epaW, epaX, and epaAC ( Fig.…”

Section: Resultssupporting

confidence: 92%

“…Previous studies have demonstrated that core epa genes are important for phage infection of E. faecalis (16)(17)(18). To confirm the role of epa variable genes in facilitating phage infection of the NMRC phages, we generated in-frame deletion mutants of epaS and epaAC in E. faecalis V583 using allelic replacement.…”

Section: Resultsmentioning

confidence: 99%

“…To date, only a single membrane protein, PIP EF (phage infection protein of E. faecalis), has been definitively identified as a phage receptor for E. faecalis (15). The enterococcal polysaccharide antigen (Epa) is involved in phage adsorption to E. faecalis cells and may act as a phage receptor (16)(17)(18). Considering that at least a dozen well-characterized lytic enterococcal phages have the potential to be used for phage therapy (11), identification of receptors used by phages could allow for the generation of more-efficient phage cocktails to be used for the treatment of enterococcal infections.…”

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confidence: 99%

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Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci

et al. 2019

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Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalis epa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci

et al. 2019

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“…Tn-Seq identified numerous E. faecalis genes that govern VPE25 susceptibility. Mutations in epa variable genes conferred VPE25 resistance by preventing phage adsorption, similar to other E. faecalis phages (23,(35)(36)(37). We discovered that phage infection of mismatch repair gene mutants results in the emergence of phage adsorption deficiencies; thus, the mismatch repair system likely fails to correct DNA damage of epa genes during phage infection.…”

Section: Discussionmentioning

confidence: 78%

“…The epa genes encode proteins involved in the formation of a cell surface-associated rhamnose-containing polysaccharide (34). We and others have previously demonstrated that enterococcal phages unrelated to VPE25 utilize Epa to adsorb and infect E. faecalis (23,(35)(36)(37). Initial work from our group showed that mutation of the VPE25 receptor Pip EF prevented VPE25 DNA entry into E. faecalis V583; yet, phages could still adsorb to receptor mutants (32), suggesting that the factors that promote phage infection via surface adsorption remained to be identified.…”

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confidence: 99%

Parallel Genomics Uncover Novel Enterococcal-Bacteriophage Interactions

Chatterjee

Willett

Nguyen

et al. 2020

mBio

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Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intraand interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy. IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

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Genetically distant bacteriophages select for unique genomic changes in Enterococcus faecalis

et al. 2022

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The human microbiota harbors diverse bacterial and bacteriophage (phage) communities. Bacteria evolve to overcome phage infection, thereby driving phage evolution to counter bacterial resistance. Understanding how phages select for genetic alterations in medically relevant bacteria is important as phages become established biologics for the treatment of multidrug‐resistant (MDR) bacterial infections. Before phages can be widely used as standalone or combination antibacterial therapies, we must obtain a deep understanding of the molecular mechanisms of phage infection and how host bacteria alter their genomes to become resistant. We performed coevolution experiments using a single Enterococcus faecalis strain and two distantly related phages to determine how phage pressure impacts the evolution of the E. faecalis genome. Whole‐genome sequencing of E. faecalis following continuous exposure to these two phages revealed mutations previously demonstrated to be essential for phage infection. We also identified mutations in genes previously unreported to be associated with phage infection in E. faecalis . Intriguingly, there was only one shared mutation in the E. faecalis genome that was selected by both phages tested, demonstrating that infection by two genetically distinct phages selects for diverse variants. This knowledge serves as the basis for the continued study of E. faecalis genome evolution during phage infection and can be used to inform the design of future therapeutics, such as phage cocktails, intended to target MDR E. faecalis .

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Loss-of-Function Mutations in epaR Confer Resistance to ϕNPV1 Infection in Enterococcus faecalis OG1RF

Cited by 55 publications

References 42 publications

Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci

Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci

Parallel Genomics Uncover Novel Enterococcal-Bacteriophage Interactions

Genetically distant bacteriophages select for unique genomic changes in Enterococcus faecalis

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