Publications by authors named "Gary Dunny"

112 Publications

Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

PLoS Genet 2017 Jul 3;13(7):e1006878. Epub 2017 Jul 3.

Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, United States of America.

In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.
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http://dx.doi.org/10.1371/journal.pgen.1006878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515443PMC
July 2017

Mechanisms of peptide sex pheromone regulation of conjugation in Enterococcus faecalis.

Microbiologyopen 2017 08 19;6(4). Epub 2017 May 19.

Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN, USA.

In many gram positive bacteria, horizontal transfer and virulence are regulated by peptide-mediated cell-cell signaling. The heptapeptide cCF10 (C) activates conjugative transfer of the Enterococcus faecalis plasmid pCF10, whereas the iCF10 (I) peptide inhibits transfer. Both peptides bind to the same domain of the master transcription regulator PrgX, a repressor of transcription of the prgQ operon encoding conjugation genes. We show that repression of prgQ by PrgX tetramers requires formation of a pCF10 DNA loop where each of two PrgX DNA-binding sites is occupied by a dimer. I binding to PrgX enhances prgQ repression, while C binding has the opposite effect. Previous models suggested that differential effects of these two peptides on the PrgX oligomerization state accounted for their distinct functions. Our new results demonstrate that both peptides have similar, high-binding affinity for PrgX, and that both peptides actually promote formation of PrgX tetramers with higher DNA-binding affinity than Apo-PrgX. We propose that differences in repression ability of PrgX/peptide complexes result from subtle differences in the structures of DNA-bound PrgX/peptide complexes. Changes in the induction state of a donor cell likely results from replacement of one type of DNA-bound peptide/PrgX tetramer with the other.
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http://dx.doi.org/10.1002/mbo3.492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552905PMC
August 2017

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range.

J Bacteriol 2017 06 25;199(12). Epub 2017 May 25.

Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA.

Tools for regulated gene expression in are extremely limited. In this report, we describe the construction of an expression vector for , designated pCIE, utilizing the P pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two family toxin-antitoxin (TA) loci present in , of the pAD1 plasmid and located on the chromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of TA system function as well as the regulated expression of other genes in is an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the P pheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function in and to further elucidate its potential roles in cell physiology.
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http://dx.doi.org/10.1128/JB.00065-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446624PMC
June 2017

PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis.

Mol Microbiol 2017 02 16;103(3):398-412. Epub 2016 Dec 16.

Department of Microbiology and Molecular Genetics, McGovern Medical School, 6431 Fannin St, Houston, Texas, 77030, USA.

Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. Here, we report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10-encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10-encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA-binding fold, and prgB-prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA-binding regulators that act to mitigate toxicity accompanying overproduction of PrgB-like adhesins in E. faecalis and other clinically-important Gram-positive species.
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http://dx.doi.org/10.1111/mmi.13563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5263107PMC
February 2017

Enterococcal Metabolite Cues Facilitate Interspecies Niche Modulation and Polymicrobial Infection.

Cell Host Microbe 2016 Oct;20(4):493-503

Singapore Centre for Environmental Life Science Engineering, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. Electronic address:

Enterococcus faecalis is frequently associated with polymicrobial infections of the urinary tract, indwelling catheters, and surgical wound sites. E. faecalis co-exists with Escherichia coli and other pathogens in wound infections, but mechanisms that govern polymicrobial colonization and pathogenesis are poorly defined. During infection, bacteria must overcome multiple host defenses, including nutrient iron limitation, to persist and cause disease. In this study, we investigated the contribution of E. faecalis to mixed-species infection when iron availability is restricted. We show that E. faecalis significantly augments E. coli biofilm growth and survival in vitro and in vivo by exporting L-ornithine. This metabolic cue facilitates E. coli biosynthesis of the enterobactin siderophore, allowing E. coli growth and biofilm formation in iron-limiting conditions that would otherwise restrict its growth. Thus, E. faecalis modulates its local environment by contributing growth-promoting cues that allow co-infecting organisms to overcome iron limitation and promotes polymicrobial infections.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076562PMC
http://dx.doi.org/10.1016/j.chom.2016.09.004DOI Listing
October 2016

Enterococcus faecalis readily colonizes the entire gastrointestinal tract and forms biofilms in a germ-free mouse model.

Virulence 2017 04 25;8(3):282-296. Epub 2016 Aug 25.

a Departments of Microbiology & Immunology , University of Minnesota Medical School , Minneapolis , MN , USA.

The mammalian gastrointestinal (GI) tract is a complex organ system with a twist-a significant portion of its composition is a community of microbial symbionts. The microbiota plays an increasingly appreciated role in many clinically-relevant conditions. It is important to understand the details of biofilm development in the GI tract since bacteria in this state not only use biofilms to improve colonization, biofilm bacteria often exhibit high levels of resistance to common, clinically relevant antibacterial drugs. Here we examine the initial colonization of the germ-free murine GI tract by Enterococcus faecalis-one of the first bacterial colonizers of the naïve mammalian gut. We demonstrate strong morphological similarities to our previous in vitro E. faecalis biofilm microcolony architecture using 3 complementary imaging techniques: conventional tissue Gram stain, immunofluorescent imaging (IFM) of constitutive fluorescent protein reporter expression, and low-voltage scanning electron microscopy (LV-SEM). E. faecalis biofilm microcolonies were readily identifiable throughout the entire lower GI tract, from the duodenum to the colon. Notably, biofilm development appeared to occur as discrete microcolonies directly attached to the epithelial surface rather than confluent sheets of cells throughout the GI tract even in the presence of high (>10) fecal bacterial loads. An in vivo competition experiment using a pool of 11 select E. faecalis mutant strains containing sequence-defined transposon insertions showed the potential of this model to identify genetic factors involved in E. faecalis colonization of the murine GI tract.
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http://dx.doi.org/10.1080/21505594.2016.1208890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411234PMC
April 2017

Antagonistic Donor Density Effect Conserved in Multiple Enterococcal Conjugative Plasmids.

Appl Environ Microbiol 2016 08 15;82(15):4537-45. Epub 2016 Jul 15.

Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, USA

Unlabelled: Enterococcus faecalis, a common causative agent of hospital-acquired infections, is resistant to many known antibiotics. Its ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious concern for public health. In some cases, induction of transfer of E. faecalis plasmids results from peptide pheromones produced by plasmid-free recipient cells, which are sensed by the plasmid-bearing donor cells. These plasmids generally encode an inhibitory peptide that competes with the pheromone and suppresses self-induction of donors. We recently demonstrated that the inhibitor peptide encoded on plasmid pCF10 is part of a unique quorum-sensing system in which it functions as a "self-sensing signal," reducing the response to the pheromone in a density-dependent fashion. Based on the similarities between regulatory features controlling conjugation in pAD1 and pAM373 and those controlling conjugation in pCF10, we hypothesized that these plasmids are likely to exhibit similar quorum-sensing behaviors. Experimental findings indicate that for both pAD1 and pAM373, high donor densities indeed resulted in decreased induction of the conjugation operon and reduced conjugation frequencies. This effect was restored by the addition of exogenous inhibitor, confirming that the inhibitor serves as an indicator for donor density. Donor density also affects cross-species conjugative plasmid transfer. Based on our experimental results, we propose models for induction and shutdown of the conjugation operon in pAD1 and pAM373.

Importance: Enterococcus faecalis is a leading cause of hospital-acquired infections. Its ability to transfer antibiotic resistance and virulence determinants by sharing its genetic material with other bacteria through direct cell-cell contact via conjugation poses a serious threat. Two antagonistic signaling peptides control the transfer of plasmids pAD1 and pAM373: a peptide pheromone produced by plasmid-free recipients triggers the conjugative transfer in plasmid-containing donors, and an inhibitor peptide encoded on the plasmid and produced by donor cells serves to modulate the donor response in accordance with the relative abundance of donors and recipients. We demonstrate that high donor density reduces the conjugation frequency of both of these plasmids, which is a consequence of increased inhibitor concentration in high-donor-density cultures. While most antibiotic strategies end up selecting resistant strains and disrupting the community balance, manipulating bacterial signaling mechanisms can serve as an alternate strategy to prevent the spread of antibiotic resistance.
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http://dx.doi.org/10.1128/AEM.00363-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4984291PMC
August 2016

Enterococcal Sex Pheromones: Evolutionary Pathways to Complex, Two-Signal Systems.

J Bacteriol 2016 06 13;198(11):1556-1562. Epub 2016 May 13.

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

Gram-positive bacteria carry out intercellular communication using secreted peptides. Important examples of this type of communication are the enterococcal sex pheromone systems, in which the transfer of conjugative plasmids is controlled by intercellular signaling among populations of donors and recipients. This review focuses on the pheromone response system of the conjugative plasmid pCF10. The peptide pheromones regulating pCF10 transfer act by modulating the ability of the PrgX transcription factor to repress the transcription of an operon encoding conjugation functions. Many Gram-positive bacteria regulate important processes, including the production of virulence factors, biofilm formation, sporulation, and genetic exchange using peptide-mediated signaling systems. The key master regulators of these systems comprise the RRNPP (RggRap/NprR/PlcR/PrgX) family of intracellular peptide receptors; these regulators show conserved structures. While many RRNPP systems include a core module of two linked genes encoding the regulatory protein and its cognate signaling peptide, the enterococcal sex pheromone plasmids have evolved to a complex system that also recognizes a second host-encoded signaling peptide. Additional regulatory genes not found in most RRNPP systems also modulate signal production and signal import in the enterococcal pheromone plasmids. This review summarizes several structural studies that cumulatively demonstrate that the ability of three pCF10 regulatory proteins to recognize the same 7-amino-acid pheromone peptide arose by convergent evolution of unrelated proteins from different families. We also focus on the selective pressures and structure/function constraints that have driven the evolution of pCF10 from a simple, single-peptide system resembling current RRNPPs in other bacteria to the current complex inducible plasmid transfer system.
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http://dx.doi.org/10.1128/JB.00128-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959283PMC
June 2016

The Influence of Biofilms in the Biology of Plasmids.

Microbiol Spectr 2014 Oct;2(5)

The field of plasmid biology has historically focused on bacteria growing in liquid culture. Surface-attached communities of bacterial biofilms have recently been understood to be the normal environment of bacteria in the natural world. Thus, studies examining plasmid replication, maintenance, and transfer in biofilms are essential for a true understanding of bacterial plasmid biology. This article reviews the current knowledge of the interplay between bacterial biofilms and plasmids, focusing on the role of plasmids in biofilm development and the role of biofilms in plasmid maintenance, copy-number control, and transfer. The studies examined herein highlight the importance of biofilms as an important ecological niche in which bacterial plasmids play an essential role.
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http://dx.doi.org/10.1128/microbiolspec.PLAS-0012-2013DOI Listing
October 2014

Evaluation of the Enterococcus faecalis Biofilm-Associated Virulence Factors AhrC and Eep in Rat Foreign Body Osteomyelitis and In Vitro Biofilm-Associated Antimicrobial Resistance.

PLoS One 2015 15;10(6):e0130187. Epub 2015 Jun 15.

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, United States of America; Division of Infectious Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.

Enterococcus faecalis can cause healthcare-associated biofilm infections, including those of orthopedic devices. Treatment of enterococcal prosthetic joint infection is difficult, in part, due to biofilm-associated antimicrobial resistance. We previously showed that the E. faecalis OG1RF genes ahrC and eep are in vitro biofilm determinants and virulence factors in animal models of endocarditis and catheter-associated urinary tract infection. In this study, we evaluated the role of these genes in a rat acute foreign body osteomyelitis model and in in vitro biofilm-associated antimicrobial resistance. Osteomyelitis was established for one week following the implantation of stainless steel orthopedic wires inoculated with E. faecalis strains OG1RF, ΩahrC, and ∆eep into the proximal tibiae of rats. The median bacterial loads recovered from bones and wires did not differ significantly between the strains at multiple inoculum concentrations. We hypothesize that factors present at the infection site that affect biofilm formation, such as the presence or absence of shear force, may account for the differences in attenuation in the various animal models we have used to study the ΩahrC and ∆eep strains. No differences among the three strains were observed in the planktonic and biofilm antimicrobial susceptibilities to ampicillin, vancomycin, daptomycin, linezolid, and tetracycline. These findings suggest that neither ahrC nor eep directly contribute to E. faecalis biofilm-associated antimicrobial resistance. Notably, the experimental evidence that the biofilm attachment mutant ΩahrC displays biofilm-associated antimicrobial resistance suggests that surface colonization alone is sufficient for E. faecalis cells to acquire the biofilm antimicrobial resistance phenotype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130187PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467866PMC
April 2016

Multiple roles for Enterococcus faecalis glycosyltransferases in biofilm-associated antibiotic resistance, cell envelope integrity, and conjugative transfer.

Antimicrob Agents Chemother 2015 Jul 27;59(7):4094-105. Epub 2015 Apr 27.

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA

The emergence of multidrug-resistant bacteria and the limited availability of new antibiotics are of increasing clinical concern. A compounding factor is the ability of microorganisms to form biofilms (communities of cells encased in a protective extracellular matrix) that are intrinsically resistant to antibiotics. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and also has the propensity to acquire resistance determinants via horizontal gene transfer. There is intense interest in the genetic basis for intrinsic and acquired antibiotic resistance in E. faecalis, since clinical isolates exhibiting resistance to multiple antibiotics are not uncommon. We performed a genetic screen using a library of transposon (Tn) mutants to identify E. faecalis biofilm-associated antibiotic resistance determinants. Five Tn mutants formed wild-type biofilms in the absence of antibiotics but produced decreased biofilm biomass in the presence of antibiotic concentrations that were subinhibitory to the parent strain. Genetic determinants responsible for biofilm-associated antibiotic resistance include components of the quorum-sensing system (fsrA, fsrC, and gelE) and two glycosyltransferase (GTF) genes (epaI and epaOX). We also found that the GTFs play additional roles in E. faecalis resistance to detergent and bile salts, maintenance of cell envelope integrity, determination of cell shape, polysaccharide composition, and conjugative transfer of the pheromone-inducible plasmid pCF10. The epaOX gene is located in a variable extended region of the enterococcal polysaccharide antigen (epa) locus. These data illustrate the importance of GTFs in E. faecalis adaptation to diverse growth conditions and suggest new targets for antimicrobial design.
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http://dx.doi.org/10.1128/AAC.00344-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468649PMC
July 2015

Transcriptome analysis of Enterococcus faecalis during mammalian infection shows cells undergo adaptation and exist in a stringent response state.

PLoS One 2014 29;9(12):e115839. Epub 2014 Dec 29.

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

As both a commensal and a major cause of healthcare-associated infections in humans, Enterococcus faecalis is a remarkably adaptable organism. We investigated how E. faecalis adapts in a mammalian host as a pathogen by characterizing changes in the transcriptome during infection in a rabbit model of subdermal abscess formation using transcriptional microarrays. The microarray experiments detected 222 and 291 differentially regulated genes in E. faecalis OG1RF at two and eight hours after subdermal chamber inoculation, respectively. The profile of significantly regulated genes at two hours post-inoculation included genes involved in stress response, metabolism, nutrient acquisition, and cell surface components, suggesting genome-wide adaptation to growth in an altered environment. At eight hours post-inoculation, 88% of the differentially expressed genes were down-regulated and matched a transcriptional profile consistent with a (p)ppGpp-mediated stringent response. Subsequent subdermal abscess infections with E. faecalis mutants lacking the (p)ppGpp synthetase/hydrolase RSH, the small synthetase RelQ, or both enzymes, suggest that intracellular (p)ppGpp levels, but not stringent response activation, influence persistence in the model. The ability of cells to synthesize (p)ppGpp was also found to be important for growth in human serum and whole blood. The data presented in this report provide the first genome-wide insights on E. faecalis in vivo gene expression and regulation measured by transcriptional profiling during infection in a mammalian host and show that (p)ppGpp levels affect viability of E. faecalis in multiple conditions relevant to mammalian infection. The subdermal abscess model can serve as a novel experimental system for studying the E. faecalis stringent response in the context of the mammalian immune system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115839PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278851PMC
October 2015

Enterococcus faecalis pCF10-encoded surface proteins PrgA, PrgB (aggregation substance) and PrgC contribute to plasmid transfer, biofilm formation and virulence.

Mol Microbiol 2015 Feb 30;95(4):660-77. Epub 2014 Dec 30.

Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, 6431 Fannin Street, Houston, TX, 77030, USA.

Enterococcus faecalis pCF10 transfers at high frequencies upon pheromone induction of the prgQ transfer operon. This operon codes for three cell wall-anchored proteins - PrgA, PrgB (aggregation substance) and PrgC - and a type IV secretion system through which the plasmid is delivered to recipient cells. Here, we defined the contributions of the Prg surface proteins to plasmid transfer, biofilm formation and virulence using the Caenorhabditis elegans infection model. We report that a combination of PrgB and extracellular DNA (eDNA), but not PrgA or PrgC, was required for extensive cellular aggregation and pCF10 transfer at wild-type frequencies. In addition to PrgB and eDNA, production of PrgA was necessary for extensive binding of enterococci to abiotic surfaces and development of robust biofilms. However, although PrgB is a known virulence factor in mammalian infection models, we determined that PrgA and PrgC, but not PrgB, were required for efficient killing in the worm infection model. We propose that the pheromone-responsive, conjugative plasmids of E. faecalis have retained Prg-like surface functions over evolutionary time for attachment, colonization and robust biofilm development. In natural settings, these biofilms are polymicrobial in composition and constitute optimal environments for signal exchange, mating pair formation and widespread lateral gene transfer.
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http://dx.doi.org/10.1111/mmi.12893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329047PMC
February 2015

The influence of biofilms in the biology of plasmids.

Microbiol Spectr 2014 Oct;2(5):0012

Department of Microbiology, University of Minnesota, 1460 Mayo Bldg., MMC196, 420 Delaware St., SE, Minneapolis MN, 55455.

The field of plasmid biology has historically focused on bacteria growing in liquid culture. Surface attached communities of bacterial biofilms have recently been understood to be the normal environment of bacteria in the natural world. Thus, studies examining plasmid replication, maintenance, and transfer in biofilms are essential for a true understanding of bacterial plasmid biology. This chapter reviews the current knowledge of the interplay between bacterial biofilms and plasmids, focusing on the role of plasmids in biofilm development and the role of biofilms in plasmid maintenance, copy number control, and transfer. The studies examined herein highlight the importance of biofilms as an important ecological niche in which bacterial plasmids play an essential role.
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http://dx.doi.org/10.1128/microbiolspec.PLAS-0012-2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225719PMC
October 2014

Enterococcus faecalis 6-phosphogluconolactonase is required for both commensal and pathogenic interactions with Manduca sexta.

Infect Immun 2015 Jan 10;83(1):396-404. Epub 2014 Nov 10.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.

Enterococcus faecalis is a commensal and pathogen of humans and insects. In Manduca sexta, E. faecalis is an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigate E. faecalis factors required for commensalism, we identified E. faecalis genes that are upregulated in the gut of M. sexta using recombinase-based in vivo expression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designated pglA. A pglA deletion mutant was impaired in both pathogenesis and gut persistence in M. sexta and produced enhanced biofilms compared with the wild type in an in vitro polystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants for E. faecalis commensal and pathogenic interactions with M. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to prevent E. faecalis infections.
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http://dx.doi.org/10.1128/IAI.02442-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288895PMC
January 2015

A widely used in vitro biofilm assay has questionable clinical significance for enterococcal endocarditis.

PLoS One 2014 25;9(9):e107282. Epub 2014 Sep 25.

Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, United States of America.

Biofilm formation may play an important role in the pathogenesis of infections caused by Enterococcus faecalis, including endocarditis. Most biofilm studies use a polystyrene dish assay to quantify biofilm biomass. However, recent studies of E. faecalis strains in tissue and animal models suggest that polystyrene dish results need to be interpreted with caution. We evaluated 158 clinical E. faecalis isolates using a polystyrene dish assay and found variation in biofilm formation, with many isolates forming little biofilm even when different types of media were used. However, all tested clinical isolates were able to form biofilms on porcine heart valve explants. Dextrose-enhanced biofilm formation in the polystyrene dish assay was found in 6/12 (50%) of clinical isolates tested and may explain some, but not all of the differences between the polystyrene dish assay and the heart valve assay. These findings suggest that in studies assessing the clinical relevance of enterococcal biofilm-forming ability, ex vivo biofilm formation on a relevant tissue surface may be warranted to validate results of in vitro assays.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107282PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177788PMC
June 2015

Modified lactic acid bacteria detect and inhibit multiresistant enterococci.

ACS Synth Biol 2015 Mar 16;4(3):299-306. Epub 2014 Jun 16.

†Department of Chemical Engineering and Materials Science, ‡Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, United States.

We designed Lactococcus lactis to detect Enterococcus faecalis. Upon detection, L. lactis produce and secrete antienterococcal peptides. The peptides inhibit enterococcal growth and reduce viability of enterococci in the vicinity of L. lactis. The enterococcal sex pheromone cCF10 serves as the signal for detection. Expression vectors derived from pCF10, a cCF10-responsive E. faecalis sex-pheromone conjugative plasmid, were engineered in L. lactis for the detection system. Recombinant host strains were engineered to express genes for three bacteriocins, enterocin A, hiracin JM79 and enterocin P, each with potent antimicrobial activity against E. faecalis. Sensitive detection and specific inhibition occur both in agar and liquid media. The engineered L. lactis also inhibited growth of multidrug-resistant E. faecium strains, when induced by cCF10. The presented vectors and strains can be components of a toolbox for the development of alternative antibiotic technologies targeting enterococci at the site of infection.
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http://dx.doi.org/10.1021/sb500090bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384838PMC
March 2015

Environmental and animal-associated enterococci.

Adv Appl Microbiol 2014 ;87:147-86

BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, USA; Department of Soil, Water and Climate, University of Minnesota, St. Paul, Minnesota, USA. Electronic address:

Enterococci are generally commensal bacteria inhabiting the gastrointestinal tract of humans and animals. They have, however, been implicated as the etiological agent of a variety of illnesses and nosocomial infections. In addition to pathogenic potential, there is growing concern regarding the incidence of antibiotic resistance and genetic exchange among Enterococcus spp. within and among a variety of animal hosts. While primarily considered an enteric group, extra-enteric habitats in which enterococci persist and potentially grow have been studied for decades. Although many biotic (e.g., predation) and abiotic (e.g., sunlight, nutrients, and salinity) stressors have been thought to limit the success of enterococci in these secondary habitats, a growing body of evidence suggests that certain strains may become naturalized to environmental habitats. Enterococci have also been used for decades as indicators of fecal contamination in recreational waters where increased concentrations of this group have been linked to the incidence of illness in humans following recreational use of these waters. Persistence of enterococci in secondary habitats, however, suggests that their presence in ambient waters may prove to be a poor indicator of actual risks to public health. In this chapter, we provide a review of the existing body of literature concerning animal host associations, genetic exchange is reviewed, and emphasis is placed on the growing body of evidence for the persistence and growth of enterococci in secondary habitats.
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http://dx.doi.org/10.1016/B978-0-12-800261-2.00004-9DOI Listing
July 2014

Identification of a conserved branched RNA structure that functions as a factor-independent terminator.

Proc Natl Acad Sci U S A 2014 Mar 18;111(9):3573-8. Epub 2014 Feb 18.

Department of Microbiology, University of Minnesota, Minneapolis, MN 55455.

Anti-Q is a small RNA encoded on pCF10, an antibiotic resistance plasmid of Enterococcus faecalis, which negatively regulates conjugation of the plasmid. In this study we sought to understand how Anti-Q is generated relative to larger transcripts of the same operon. We found that Anti-Q folds into a branched structure that functions as a factor-independent terminator. In vitro and in vivo, termination is dependent on the integrity of this structure as well as the presence of a 3' polyuridine tract, but is not dependent on other downstream sequences. In vitro, terminated transcripts are released from RNA polymerase after synthesis. In vivo, a mutant with reduced termination efficiency demonstrated loss of tight control of conjugation function. A search of bacterial genomes revealed the presence of sequences that encode Anti-Q-like RNA structures. In vitro and in vivo experiments demonstrated that one of these functions as a terminator. This work reveals a previously unappreciated flexibility in the structure of factor-independent terminators and identifies a mechanism for generation of functional small RNAs; it should also inform annotation of bacterial sequence features, such as terminators, functional sRNAs, and operons.
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http://dx.doi.org/10.1073/pnas.1315374111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948284PMC
March 2014

Enterococcal sex pheromones: signaling, social behavior, and evolution.

Authors:
Gary M Dunny

Annu Rev Genet 2013 11;47:457-82. Epub 2013 Sep 11.

Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455; email:

In Enterococcus faecalis, lateral transfer of conjugative plasmids that encode antibiotic resistance and virulence determinants can be induced by peptide sex pheromones. The tetracycline-resistance plasmid pCF10 represents a paradigm for illustrating important conserved features of a large family of pheromone-responsive enterococcal plasmids. The pheromone is released into the growth medium by plasmid-free recipient cells and sensed by plasmid-containing donors. The activity of the pheromone is antagonized by a plasmid-encoded inhibitor peptide that prevents conjugation in the absence of an inducing signal and is also required to return the system to the ground state following an induction cycle. The pheromone response involves multiple transcriptional and posttranscriptional mechanisms as well as bi-stable biological switch behavior. Multiple layers of regulation are essential for proper function, and evolution of this tight control system may have been favored by reduction of the fitness cost of plasmid maintenance to the host cell.
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http://dx.doi.org/10.1146/annurev-genet-111212-133449DOI Listing
April 2014

Antagonistic self-sensing and mate-sensing signaling controls antibiotic-resistance transfer.

Proc Natl Acad Sci U S A 2013 Apr 8;110(17):7086-90. Epub 2013 Apr 8.

Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.

Conjugation is one of the most common ways bacteria acquire antibiotic resistance, contributing to the emergence of multidrug-resistant "superbugs." Bacteria of the genus Enterococcus faecalis are highly antibiotic-resistant nosocomial pathogens that use the mechanism of conjugation to spread antibiotic resistance between resistance-bearing donor cells and resistance-deficient recipient cells. Here, we report a unique quorum sensing-based communication system that uses two antagonistic signaling molecules to regulate conjugative transfer of tetracycline-resistance plasmid pCF10 in E. faecalis. A "mate-sensing" peptide sex pheromone produced by recipient cells is detected by donor cells to induce conjugative genetic transfer. Using mathematical modeling and experimentation, we show that a second antagonistic "self-sensing" signaling peptide, previously known to suppress self-induction of donor cells, also serves as a classic quorum-sensing signal for donors that functions to reduce antibiotic-resistance transfer at high donor density. This unique form of quorum sensing may provide a means of limiting the spread of the plasmid and present opportunities to control antibiotic-resistance transfer through manipulation of intercellular signaling, with implications in the clinical setting.
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http://dx.doi.org/10.1073/pnas.1212256110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3637703PMC
April 2013

AhrC and Eep are biofilm infection-associated virulence factors in Enterococcus faecalis.

Infect Immun 2013 May 4;81(5):1696-708. Epub 2013 Mar 4.

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.

Enterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species.
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http://dx.doi.org/10.1128/IAI.01210-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648002PMC
May 2013

Occurrence, genetic diversity, and persistence of enterococci in a Lake Superior watershed.

Appl Environ Microbiol 2013 May 1;79(9):3067-75. Epub 2013 Mar 1.

Biotechnology Institute, University of Minnesota, St. Paul, Minnesota, USA.

In 2012, the U.S. EPA suggested that coastal and Great Lakes states adopt enterococci as an alternative indicator for the monitoring of recreational water quality. Limited information, however, is available about the presence and persistence of enterococci in Lake Superior. In this study, the density, species composition, and persistence of enterococci in sand, sediment, water, and soil samples were examined at two sites in a Lake Superior watershed from May to September over a 2-year period. The genetic diversity of Enterococcus faecalis isolates collected from environmental samples was also studied by using the horizontal, fluorophore-enhanced repetitive PCR DNA fingerprinting technique. Results obtained by most-probable-number analyses indicated that enterococci were present in 149 (94%) of 159 samples and their densities were generally higher in the summer than in the other months examined. The Enterococcus species composition displayed spatial and temporal changes, with the dominant species being E. hirae, E. faecalis, E. faecium, E. mundtii, and E. casseliflavus. DNA fingerprint analyses indicated that the E. faecalis population in the watershed was genetically diverse and changed spatially and temporally. Moreover, some DNA fingerprints reoccurred over multiple sampling events. Taken together, these results suggest that some enterococci are able to persist and grow in the Lake Superior watershed, especially in soil, for a prolonged time after being introduced.
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http://dx.doi.org/10.1128/AEM.03908-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623127PMC
May 2013

Structure and mode of peptide binding of pheromone receptor PrgZ.

J Biol Chem 2012 Oct 4;287(44):37165-70. Epub 2012 Sep 4.

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, University of Groningen, 9747 AG Groningen, The Netherlands.

We present the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and OppA) explains the high specificity of PrgZ for hydrophobic heptapeptides versus the promiscuity of peptide binding in the homologous proteins.
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http://dx.doi.org/10.1074/jbc.M112.386334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3481316PMC
October 2012

Enterococcus faecalis produces abundant extracellular structures containing DNA in the absence of cell lysis during early biofilm formation.

mBio 2012 24;3(4):e00193-12. Epub 2012 Jul 24.

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.

Unlabelled: Enterococcus faecalis is a common Gram-positive commensal bacterium of the metazoan gastrointestinal tract capable of biofilm formation and an opportunistic pathogen of increasing clinical concern. Dogma has held that biofilms are slow-growing structures, often taking days to form mature microcolonies. Here we report that extracellular DNA (eDNA) is an integral structural component of early E. faecalis biofilms (≤4 h postinoculation). Combining cationic dye-based biofilm matrix stabilization techniques with correlative immuno-scanning electron microscopy (SEM) and fluorescent techniques, we demonstrate that--in early E. faecalis biofilms--eDNA localizes to previously undescribed intercellular filamentous structures, as well as to thick mats of extruded extracellular matrix material. Both of these results are consistent with previous reports that early biofilms are exquisitely sensitive to exogenous DNase treatment. High-resolution SEM demonstrates a punctate labeling pattern in both structures, suggesting the presence of an additional, non-DNA constituent. Notably, the previously described fratricidal or lytic mechanism reported as the source of eDNA in older (≥24 h) E. faecalis biofilms does not appear to be at work under these conditions; extensive visual examination by SEM revealed a striking lack of lysed cells, and bulk biochemical assays also support an absence of significant lysis at these early time points. In addition, some cells demonstrated eDNA labeling localized at the septum, suggesting the possibility of DNA secretion from metabolically active cells. Overall, these data are consistent with a model in which a subpopulation of viable E. faecalis cells secrete or extrude DNA into the extracellular matrix.

Importance: This paper reports the production of extracellular DNA during early biofilm formation in Enterococcus faecalis. The work is significant because the mechanism of eDNA (extracellular DNA) production is independent of cell lysis and the DNA is confined to well-defined structures, suggesting a novel form of DNA secretion by viable cells. Previous models of biofilm formation in enterococci and related species propose cell lysis as the mechanism of DNA release.
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http://dx.doi.org/10.1128/mBio.00193-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413405PMC
October 2012

Ultrastructure of a novel bacterial form located in Staphylococcus aureus in vitro and in vivo catheter-associated biofilms.

J Histochem Cytochem 2012 Oct 21;60(10):770-6. Epub 2012 Jul 21.

Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA.

Bacterial biofilms are ubiquitous in nature, industry, and medicine, and understanding their development and cellular structure is critical in controlling the unwanted consequences of biofilm growth. Here, we report the ultrastructure of a novel bacterial form observed by scanning electron microscopy in the luminal vegetations of catheters from patients with active Staphylococcus aureus bacteremia. This novel structure had the general appearance of a normal staphylococcal cell but up to 10 to 15 times as large. Transmission electron microscopy indicated that these structures appeared as sacs enclosing multiple normal-sized (~0.6 µm) staphylococcal forms. Using in vitro cultivated biofilms, cytochemical studies using fluorescent reagents revealed that these structures were rich in lipids and appeared within 15 min after S. aureus inoculation onto clinically relevant abiotic surfaces. Because they appeared early in biofilm development, these novel bacterial forms may represent an unappreciated mechanism for biofilm surface adherence, and their prominent lipid expression levels could explain the perplexing increased antimicrobial resistance of biofilm-associated bacteria.
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http://dx.doi.org/10.1369/0022155412457573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3524562PMC
October 2012

In vivo and in vitro analyses of regulation of the pheromone-responsive prgQ promoter by the PrgX pheromone receptor protein.

J Bacteriol 2012 Jul 27;194(13):3386-94. Epub 2012 Apr 27.

Department of Microbiology, The Ohio State University, Columbus, Ohio, USA.

Expression of conjugative transfer and virulence functions of the Enterococcus faecalis antibiotic resistance plasmid pCF10 is regulated by the interaction of the pheromone receptor protein PrgX with two DNA binding operator sites (XBS1 and XBS2) upstream from the transcription start site of the prgQ operon (encoding the pCF10 transfer machinery) and by posttranscriptional mechanisms. Occupancy of both binding sites by PrgX dimers results in repression of the prgQ promoter. Structural and genetic studies suggest that the peptide pheromone cCF10 functions by binding to PrgX and altering its oligomerization state, resulting in reduced occupancy of XBSs and increased prgQ transcription. The DNA binding activity of PrgX has additional indirect regulatory effects on prgQ transcript levels related to the position of the convergently transcribed prgX operon. This has complicated interpretation of previous analyses of the control of prgQ expression by PrgX. We report here the results of in vivo and in vitro experiments examining the direct effects of PrgX on transcription from the prgQ promoter, as well as quantitative correlation between the concentrations of XBSs, PrgX protein, and prgQ promoter activity in vivo. The results of electrophoretic mobility shift assays and quantitative analysis of prgQ transcription in vitro and in vivo support the predicted roles of the PrgX DNA binding sites in prgQ transcription regulation. The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10 in vivo.
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http://dx.doi.org/10.1128/JB.00364-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434730PMC
July 2012

Use of recombinase-based in vivo expression technology to characterize Enterococcus faecalis gene expression during infection identifies in vivo-expressed antisense RNAs and implicates the protease Eep in pathogenesis.

Infect Immun 2012 Feb 5;80(2):539-49. Epub 2011 Dec 5.

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.

Enterococcus faecalis is a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades. E. faecalis must be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-based in vivo expression technology (RIVET) to identify promoters on the E. faecalis OG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putative in vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected in in vitro- and in vivo-grown cells, providing the first evidence of in vivo-expressed antisense RNAs in E. faecalis. Deletions in the in vivo-activated genes that encode glutamate 5-kinase (proB [EF0038]), the transcriptional regulator EbrA (ebrA [EF1809]), and the membrane metalloprotease Eep (eep [EF2380]) did not hinder biofilm formation in in vitro assays. In a rabbit model of endocarditis, the ΔebrA strain was fully virulent, the ΔproB strain was slightly attenuated, and the Δeep strain was severely attenuated. The Δeep virulence defect could be complemented by the expression of the wild-type gene in trans. Microscopic analysis of early Δeep biofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.
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http://dx.doi.org/10.1128/IAI.05964-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264308PMC
February 2012
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