Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

Phage infection and sub-lethal antibiotic exposure mediate Enterococcus faecalis type VII secretion system dependent inhibition of bystander bacteria.

Bacteriophages (phages) are being considered as alternative therapeutics for the treatment of multidrug resistant bacterial infections. Considering phages have narrow host-ranges, it is generally accepted that therapeutic phages will have a marginal impact on non-target bacteria. We have discovered that lytic phage infection induces transcription of type VIIb secretion system (T7SS) genes in the pathobiont Enterococcus faecalis. Membrane damage during phage infection induces T7SS gene expression resulting in cell contact dependent antagonism of different Gram positive bystander bacteria. Deletion of essB, a T7SS structural component, abrogates phage-mediated killing of bystanders. A predicted immunity gene confers protection against T7SS mediated inhibition, and disruption of its upstream LXG toxin gene rescues growth of E. faecalis and Staphylococcus aureus bystanders. Phage induction of T7SS gene expression and bystander inhibition requires IreK, a serine/threonine kinase, and OG1RF_11099, a predicted GntR-family transcription factor. Additionally, sub-lethal doses of membrane targeting and DNA damaging antibiotics activated T7SS expression independent of phage infection, triggering T7SS antibacterial activity against bystander bacteria. Our findings highlight how phage infection and antibiotic exposure of a target bacterium can affect non-target bystander bacteria and implies that therapies beyond antibiotics, such as phage therapy, could impose collateral damage to polymicrobial communities.

Structural Differences in Complexes between the Master Regulator PrgX, Peptide Pheromones, and Operator Binding Sites Determine the Induction State for Conjugative Transfer of pCF10.

Pheromone-inducible conjugation in the Enterococcus faecalis pCF10 system is regulated by the PrgX transcription factor through binding interactions at two operator binding sites (XBS1 and XBS2) upstream of the transcription start site of the prgQ operon encoding the conjugation machinery. Repression of transcription requires the interaction of a PrgX tetramer with both XBSs via formation of a DNA loop. The ability of PrgX to regulate prgQ transcription is modulated by its interaction with two antagonistic regulatory peptides, ICF10 (I) and cCF10 (C); the former peptide inhibits prgQ transcription, while the latter peptide enhances prgQ transcription. In this report, we used electrophoretic mobility shift assays (EMSAs) and DNase footprinting to examine binding interactions between the XBS operator sites and various forms of PrgX (Apo-X, PrgX/I, and PrgX/C). Whereas a previous model based on high-resolution structures of PrgX proposed that the functional differences between PrgX/C and PrgX/I resulted from differences in PrgX oligomerization state, the current results show that specific differences in XBS2 occupancy by bound tetramers account for the differential regulatory properties of the two peptide/PrgX complexes and for the effects of XBS mutations on regulation. The results also confirmed a DNA looping model of PrgX function. IMPORTANCE Peptide pheromones regulate antibiotic resistance transfer in Enterococcus faecalis. Here, we present new data showing that pheromone-dependent regulation of transfer genes is mediated via effects on the structures of complexes between peptides, the intracellular peptide receptor, and operator sites on the target DNA.

The Phosphatase Bph and Peptidyl-Prolyl Isomerase PrsA Are Required for Gelatinase Expression and Activity in Enterococcus faecalis.

Enterococcus faecalis is a common commensal bacterium in the gastrointestinal tract as well as a frequent nosocomial pathogen. The secreted metalloprotease gelatinase (GelE) is an important E. faecalis virulence factor that contributes to numerous cellular activities, such as autolysis, biofilm formation, and biofilm-associated antibiotic resistance. Expression of gelE has been extensively studied and is regulated by the Fsr quorum sensing system. Here, we identify two additional factors regulating gelatinase expression and activity in E. faecalis OG1RF. The Bph phosphatase is required for expression of gelE in an Fsr-dependent manner. Additionally, the membrane-anchored protein foldase PrsA is required for GelE activity, but not fsr or gelE gene expression. Disrupting prsA also leads to increased antibiotic sensitivity in biofilms independent of the loss of GelE activity. Together, our results expand the model for gelatinase production in E. faecalis, which has important implications for fundamental studies of GelE function in Enterococcus and also E. faecalis pathogenesis. IMPORTANCE In Enterococcus faecalis, gelatinase (GelE) is a virulence factor that is also important for biofilm formation and interactions with other microbes as well as the host immune system. The long-standing model for GelE production is that the Fsr quorum sensing system positively regulates expression of gelE. Here, we update that model by identifying two additional factors that contribute to gelatinase production. The biofilm-associated Bph phosphatase regulates the expression of gelE through Fsr, and the peptidyl-prolyl isomerase PrsA is required for production of active GelE through an Fsr-independent mechanism. This provides important insight into how regulatory networks outside of the fsr locus coordinate expression of gelatinase.

Two ABC transport systems carry out peptide uptake in Enterococcus faecalis: Their roles in growth and in uptake of sex pheromones.

Enterococcal pheromone-inducible plasmids encode a predicted OppA-family secreted lipoprotein. In the case of plasmid pCF10, the protein is PrgZ, which enhances the mating response to cCF10 pheromone. OppA proteins generally function with associated OppBCDF ABC transporters to import peptides. In this study, we analyzed the potential interactions of PrgZ with two host-encoded Opp transporters using two pheromone-inducible fluorescent reporter constructs. Based on our results, we propose renaming these loci opp1 (OG1RF_10634-10639) and opp2 (OG1RF_12366-12370). We also examined the ability of the Opp1 and Opp2 systems to mediate import in the absence of PrgZ. Cells expressing PrgZ were able to import pheromone if either opp1 or opp2 was functional, but not if both opp loci were disrupted. In the absence of PrgZ, pheromone import was dependent on a functional opp2 system, including opp2A. Comparative structural analysis of the peptide-binding pockets of PrgZ, Opp1A, Opp2A, and the related Lactococcus lactis OppA protein, suggested that the robust pheromone-binding ability of PrgZ relates to a nearly optimal fit of the hydrophobic peptide, whereas binding ability of Opp2A likely results from a more open, promiscuous peptide-binding pocket similar to L. lactis OppA.

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

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.

Probiotic Bacillus Affects Enterococcus faecalis Antibiotic Resistance Transfer by Interfering with Pheromone Signaling Cascades.

Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis subsp. natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from chromosomally encoded CF10 (cCF10) without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30- to 50-kDa extracellular protease present in B. subtilis subsp. natto. Nattokinase of B. subtilis subsp. natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into LVTL plus VFV fragments. Moreover, the cleavage product LVTL (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis subsp. natto can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis subsp. natto and L peptide to mitigate E. faecalis antibiotic resistance transfer. IMPORTANCE Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, antibiotic resistance genes can undergo efficient intra- and interspecies transfer via E. faecalis peptide pheromone-mediated plasmid transfer systems. Therefore, this study provided the first experimental demonstration that probiotics are a feasible approach for interfering with conjugative plasmid transfer between E. faecalis strains to stop the transfer of antibiotic resistance. We found that the extracellular protease(s) of Bacillus subtilis subsp. natto cleaved peptide pheromones without affecting the growth of E. faecalis, thereby reducing the frequency of conjugative plasmid transfer. In addition, a specific cleaved pheromone fragment interfered with conjugative plasmid transfer. These findings provide a potential probiotic-based method for interfering with the transfer of antibiotic resistance between E. faecalis strains.

Single-Cell Analysis Reveals that the Enterococcal Sex Pheromone Response Results in Expression of Full-Length Conjugation Operon Transcripts in All Induced Cells.

For high-frequency transfer of pCF10 between E. faecalis cells, induced expression of the pCF10 genes encoding conjugative machinery from the prgQ operon is required. This process is initiated by the cCF10 (C) inducer peptide produced by potential recipient cells. The expression timing of prgB, an "early" gene just downstream of the inducible promoter, has been studied extensively in single cells. However, several previous studies suggest that only 1 to 10% of donors induced for early prgQ gene expression actually transfer plasmids to recipients, even at a very high recipient population density. One possible explanation for this is that only a minority of pheromone-induced donors actually transcribe the entire prgQ operon. Such cells would not be able to functionally conjugate but might play another role in the group behavior of donors. Here, we sought to (i) simultaneously assess the presence of RNAs produced from the proximal (early induced transcripts [early Q]) and distal (late Q) portions of the prgQ operon in individual cells, (ii) investigate the prevalence of heterogeneity in induced transcript length, and (iii) evaluate the temporality of induced transcript expression. Using fluorescent in situ hybridization chain reaction (HCR) transcript labeling and single-cell microscopic analysis, we observed that most cells expressing early transcripts (QL, prgB, and prgA) also expressed late transcripts (prgJ, pcfC, and pcfG). These data support the conclusion that, after induction is initiated, transcription likely extends through the end of the conjugation machinery operon for most, if not all, induced cells.IMPORTANCE In Enterococcus faecalis, conjugative plasmids like pCF10 often carry antibiotic resistance genes. With antibiotic treatment, bacteria benefit from plasmid carriage; however, without antibiotic treatment, plasmid gene expression may have a fitness cost. Transfer of pCF10 is mediated by cell-to-cell signaling, which activates the expression of conjugation genes and leads to efficient plasmid transfer. Yet, not all donor cells in induced populations transfer the plasmid. We examined whether induced cells might not be able to functionally conjugate due to premature induced transcript termination. Single-cell analysis showed that most induced cells do, in fact, express all of the genes required for conjugation, suggesting that premature transcription termination within the prgQ operon does not account for failure of induced donor cell gene transfer.

Genome-Wide Mutagenesis Identifies Factors Involved in Enterococcus faecalis Vaginal Adherence and Persistence.

Enterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive tract, and reports suggest vaginal colonization increases following antibiotic treatment or in patients with aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal colonization and subsequent infection. We modified an established mouse vaginal colonization model to explore E. faecalis vaginal carriage and demonstrate that both vancomycin-resistant and -sensitive strains colonize the murine vaginal tract. Following vaginal colonization, we observed E. faecalis in vaginal, cervical, and uterine tissue. A mutant lacking endocarditis- and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with human vaginal and cervical cells in vitro but did not contribute to colonization in vivo Thus, we screened a low-complexity transposon (Tn) mutant library to identify novel genes important for E. faecalis colonization and persistence in the vaginal tract. This screen revealed 383 mutants that were underrepresented during vaginal colonization at 1, 5, and 8 days postinoculation compared to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism or in the type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal the complex nature of vaginal colonization and suggest that multiple factors contribute to E. faecalis persistence in the reproductive tract.

A new flavor of entry exclusion in ICE elements provides a selective advantage for the element and its host.

Entry exclusion has been described in many bacterial conjugation systems, but their molecular mechanisms are not well understood. In the current issue, Avello et al. describe a new exclusion system in the conjugative element ICEBs1. They identify the yddJ gene as the functional exclusion gene and its target as the protein product of the conG gene. They provide evidence for a possible mechanism and for the contribution of the system to reduce fitness costs of ICE expression.

Effects of endogenous levels of master regulator PrgX and peptide pheromones on inducibility of conjugation in the enterococcal pCF10 system.

Enterococcal pheromone responsive conjugative plasmids like pCF10 promote horizontal spread of antibiotic resistance genes following induction of plasmid-containing cells by potential recipients. Transcription of conjugation genes from promoter PQ is inhibited by the master regulator PrgX, further repressed when PrgX is in complex with the inhibitory I peptide, and allowed when PrgX is in complex with the C inducing peptide. Single-cell analysis has shown that heterogeneity in the pheromone response is prevalent. Here, we systematically varied levels of regulatory molecules to better understand why some individual cells have increased propensity for induction. In this study, PrgX was confirmed to repress PQ in the absence of exogenous peptides in vivo, but cells with increased levels of PrgX were shown to be more prone to induction. Further, ablation of endogenous I reduced PrgX levels, resulting in reduced basal repression and loss of inducibility. Reduction of both endogenous peptides by washing increased the inducibility of cells. Together, these results show that endogenous PrgX, C, and I levels can impact the induction potential of a cell and establish the importance of basal I for regulation. These results also suggest that PrgX/C complexes may directly activate prgQ transcription, contrary to a long-standing working model.

Gut dysbiosis during antileukemia chemotherapy versus allogeneic hematopoietic cell transplantation.

BACKGROUND: In the field of malignant hematology, most microbiome studies have focused on recipients of allogeneic hematopoietic cell transplantation (allo-HCT). As a result, this population has remained the primary target for novel microbiota therapeutics. Because the types of insults to the microbiome are similar during hematopoietic cell transplantation and intensive antileukemia therapy, this study evaluated whether the dysbiosis states are similar in the 2 settings. METHODS: This study compared gut microbiota assemblages and community domination states in 2 cohorts of patients: patients with intensively treated acute leukemia (AL) and allo-HCT recipients. 16S ribosomal RNA gene profiling of thrice weekly stool samples was performed. Linear discriminant analysis effect size was used to determine differentially abundant taxa in groups of interest, and mixed modes were used to determine the predictors of microbiome states. RESULTS: Microbiome changes in both cohorts were characterized by a marked loss of diversity and domination of low-diversity communities by Enterococcus. In the AL cohort, the relative abundance of Lactobacillus was also inversely correlated with diversity. Communities dominated by these genera were compositionally different. CONCLUSIONS: Similarities in microbiota assemblages between the 2 cohorts support a broader scope for microbiota-directed therapeutics than previously considered, whereas specific differences suggest a personalized aspect to such therapeutics with the possibility of a differential response.

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

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.

Role of epaQ, a Previously Uncharacterized Enterococcus faecalis Gene, in Biofilm Development and Antimicrobial Resistance.

Enterococcus faecalis is a commensal of the human gastrointestinal tract; it is also an opportunistic pathogen and one of the leading causes of hospital-acquired infections. E. faecalis produces biofilms that are highly resistant to antibiotics, and it has been previously reported that certain genes of the epa operon contribute to biofilm-associated antibiotic resistance. Despite several studies examining the epa operon, many gene products of this operon remain annotated as hypothetical proteins. Here, we further explore the epa operon; we identified epaQ, currently annotated as encoding a hypothetical membrane protein, as being important for biofilm formation in the presence of the antibiotic daptomycin. Mutants with disruptions of epaQ were more susceptible to daptomycin relative to the wild type, suggesting its importance in biofilm-associated antibiotic resistance. Furthermore, the ΔepaQ mutant exhibited an altered biofilm architectural arrangement and formed small aggregates in liquid cultures. Our cumulative data show that epa mutations result in altered polysaccharide content, increased cell surface hydrophobicity, and decreased membrane potential. Surprisingly, several epa mutations significantly increased resistance to the antibiotic ceftriaxone, indicating that the way in which the epa operon impacts antibiotic resistance is antibiotic dependent. These results further define the key role of epa in antibiotic resistance in biofilms and in biofilm architecture.IMPORTANCEE. faecalis is a common cause of nosocomial infection, has a high level of antibiotic resistance, and forms robust biofilms. Biofilm formation is associated with increased antibiotic resistance. Therefore, a thorough understanding of biofilm-associated antibiotic resistance is important for combating resistance. Several genes from the epa operon have previously been implicated in biofilm-associated antibiotic resistance, pathogenesis, and competitive fitness in the GI tract, but most genes in this locus remain uncharacterized. Here, we examine epaQ, which has not been characterized functionally. We show that the ΔepaQ mutant exhibits reduced biofilm formation in the presence of daptomycin, altered biofilm architecture, and increased resistance to ceftriaxone, further expanding our understanding of the contribution of this operon to intrinsic enterococcal antibiotic resistance and biofilm growth.

Expression of Adhesive Pili and the Collagen-Binding Adhesin Ace Is Activated by ArgR Family Transcription Factors in Enterococcus faecalis.

It was shown previously that the disruption of the ahrC gene encoding a predicted ArgR family transcription factor results in a severe defect in biofilm formation in vitro, as well as a significant attenuation of virulence of Enterococcus faecalis strain OG1RF in multiple experimental infection models. Using transcriptome sequencing (RNA-seq), we observed ahrC-dependent changes in the expression of more than 20 genes. AhrC-repressed genes included predicted determinants of arginine catabolism and several other metabolic genes and predicted transporters, while AhrC-activated genes included determinants involved in the production of surface protein adhesins. Most notably, the structural and regulatory genes of the ebp locus encoding adhesive pili were positively regulated, as well as the ace gene, encoding a collagen-binding adhesin. Using lacZ transcription reporter fusions, we determined that ahrC and a second argR transcription factor gene, argR2, both function to activate the expression of ebpR, which directly activates the transcription of the pilus structural genes. Our data suggest that in the wild-type E. faecalis, the low levels of EbpR limit the expression of pili and that biofilm biomass is also limited by the amount of pili expressed by the bacteria. The expression of ace is similarly enhanced by AhrC and ArgR2, but ace expression is not dependent on EbpR. Our results demonstrate the existence of novel regulatory cascades controlled by a pair of ArgR family transcription factors that might function as a heteromeric protein complex.IMPORTANCE Cell surface adhesins play critical roles in the formation of biofilms, host colonization, and the pathogenesis of opportunistic infections by Enterococcus faecalis Here, we present new results showing that the expression of two major enterococcal surface adhesins, ebp pili, and the collagen-binding protein Ace is positively regulated at the transcription level by two argR family transcription factors, AhrC and ArgR2. In the case of pili, the direct target of regulation is the ebpR gene, previously shown to activate the transcription of the pilus structural genes, while the activation of ace transcription appears to be directly impacted by the two ArgR proteins. These transcription factors may represent new targets for blocking enterococcal infections.

Mechanistic Features of the Enterococcal pCF10 Sex Pheromone Response and the Biology of Enterococcus faecalis in Its Natural Habitat.

Conjugative transfer of plasmids in enterococci is promoted by intercellular communication using peptide pheromones. The regulatory mechanisms that control transfer have been extensively studied in vitro However, the complicated systems that regulate the spread of these plasmids did not evolve in the laboratory test tube, and remarkably little is known about this form of signaling in the intestinal tract, the primary niche of these organisms. Because the evolution of Enterococcus faecalis strains and their coresident pheromone-inducible plasmids, such as pCF10, have occurred in the gastrointestinal (GI) tract, it is important to consider the functions controlled by pheromones in light of this ecology. This review summarizes our current understanding of the pCF10-encoded pheromone response. We consider how selective pressures in the natural environment may have selected for the complex and very tightly regulated systems controlling conjugation, and we pay special attention to the ecology of enterococci and the pCF10 plasmid as a gut commensal. We summarize the results of recent studies of the pheromone response at the single-cell level, as well as those of the first experiments demonstrating a role for pheromone signaling in plasmid transfer and in GI tract competitive fitness. These results will serve as a foundation for further in vivo studies that could lead to novel interventions to reduce opportunistic infections and the spread of antibiotic resistance.

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

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.

Pretransplant Gut Colonization with Intrinsically Vancomycin-Resistant Enterococci (E. gallinarum and E. casseliflavus) and Outcomes of Allogeneic Hematopoietic Cell Transplantation.

Pretransplant gut colonization with intrinsically vancomycin-resistant enterococci (iVRE) (Enterococcus gallinarum and Enterococcus casseliflavus) is uncommon and with unknown clinical impact. In a matched-pairs analysis of patients with versus without iVRE colonization (n = 18 in each group), we demonstrated significantly higher 2-year overall survival (86% [95% confidence interval, 52% to 96%] versus 35% [95% confidence interval, 8% to 65]; P <.01) and lower nonrelapse mortality (P <.01) among colonized patients. Putative metabolomes differentiated iVRE from E. faecalis/faecium and may contribute to a healthier gut microbiome in iVRE-colonized patients.

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

Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the PQ 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 par family toxin-antitoxin (TA) loci present in E. faecalis, parpAD1 of the pAD1 plasmid and parEF0409 located on the E. faecalis 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 par TA system function as well as the regulated expression of other genes in E. faecalisIMPORTANCEE. faecalis 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 PQ 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 E. faecalis and to further elucidate its potential roles in cell physiology.

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

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.