Biology subjects

Dunny, G. M.

Publications and source records attributed to Dunny, G. M..

10 recordsLinked to original sources

bioRxiv2024

Arginine impacts aggregation, biofilm formation, and antibiotic susceptibility in Enterococcus faecalis

Enterococcus faecalis is a commensal bacterium in the gastrointestinal tract (GIT) of humans and other organisms. E. faecalis also causes infections in root canals, wounds, the urinary tract, and on heart valves. E. faecalis metabolizes arginine through the arginine deiminase (ADI) pathway, which converts arginine to ornithine and releases ATP, ammonia, and CO2. E. faecalis arginine metabolism also affects virulence of other pathogens during co-culture. E. faecalis may encounter elevated levels of arginine in the GIT or the oral cavity, where arginine is used as a dental therapeutic. Little is known about how E. faecalis responds to growth in arginine in the absence of other bacteria. To address this, we used RNAseq and additional assays to measure growth, gene expression, and biofilm formation in E. faecalis OG1RF grown in arginine. We demonstrate that arginine decreases E. faecalis biofilm production and causes widespread differential expression of genes related to metabolism, quorum sensing, and polysaccharide synthesis. Growth in arginine also increases aggregation of E. faecalis and promotes decreased susceptibility to the antibiotics ampicillin and ceftriaxone. This work provides a platform for understanding of how the presence of arginine in biological niches affects E. faecalis physiology and virulence of surrounding microbes.

microbiology↗

bioRxiv2023

Optimized replication of arrayed bacterial mutant libraries increase access to biological resources

Biological collections, including arrayed libraries of single transposon or deletion mutants, greatly accelerate the pace of bacterial genetics research. Despite the importance of these resources, few protocols exist for the replication and distribution of these materials. Here, we describe a protocol for creating multiple replicates of an arrayed bacterial Tn library consisting of approximately 6,800 mutants in 73 x 96-well plates. Our protocol provides multiple checkpoints to guard against contamination and minimize genetic drift caused by freeze/thaw cycles. This approach can also be scaled for arrayed culture collections of various sizes. Overall, this protocol is a valuable resource for other researchers considering the construction and distribution of arrayed culture collection resources for the benefit of the greater scientific community. ImportanceArrayed mutant collections drive robust genetic screens, yet few protocols exist for replication of these resources and subsequent quality control. Increasing distribution of arrayed biological collections will increase accessibility to and use of these resources. Developing standardized techniques for replication of these resources is essential for ensuring their quality and usefulness to the scientific community.

microbiology↗

bioRxiv2022

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. ImportanceIn 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.

microbiology↗

bioRxiv2021

Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation

Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation. ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

microbiology↗

bioRxiv2020

Enterococcal PrgU mitigates PrgB overexpression toxicity by binding to intergenic RNA downstream of the PQ promoter

Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the sex pheromone cCF10, which induces the expression of the Type 4 Secretion System (T4SS) genes controlled by the PQ promoter. The pheromone responsive PQ promoter is strictly regulated to prevent overproduction of the prgQ operon, which contains the T4SS, and to limit the cell toxicity caused by overproduction of PrgB, a T4SS adhesin involved in cellular aggregation. PrgU plays an important role in regulating this toxicity by decreasing PrgB production. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. With a combination of lacZ reporter fusion, northern blot, and RNAseq analyses, we provide evidence that PrgU binds a specific RNA sequence within the intergenic region (IGR), ca 400 bp downstream of the PQ promoter. PrgU-IGR binding reduces levels of downstream transcripts, with the strongest decrease seen for prgB messages. Consistent with these findings, we determined that pCF10-carrying cells expressing prgU decreased transcript levels more rapidly than isogenic cells deleted of prgU. Finally, purified PrgU bound RNA in vitro, but without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors to bind its RNA target in vivo. Together, our results support a working model where PrgU binding to the IGR serves to recruit RNase(s) for targeted degradation of downstream transcripts.Importance Bacteria utilize Type 4 Secretion Systems (T4SS) to efficiently transfer DNA from donor to recipient cells, thereby spreading genes encoding for antibiotic resistance as well as various virulence factors. The conjugative plasmid pCF10 from Enterococcus faecalis, originally isolated from clinical isolates, serves as a model system for these processes in Gram-positive bacteria. It is very important to strictly regulate the expression of the T4SS proteins for the bacteria, as some of these proteins are highly toxic to the cell. Here, we identify the mechanism by which PrgU performs its delicate fine tuning of the expression levels. As prgU genes are present in various conjugative plasmids and transposons, this provides an important new insight into the bacterial repertoire of regulation mechanisms of these clinically important systems.View Full Text

molecular biology↗

bioRxiv2020

Enterococcal PrgA extends far outside the cell and provides surface exclusion to protect against unwanted conjugation

Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via Type 4 Secretion Systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a 2nd copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggest that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

biochemistry↗

bioRxiv2020

Phage infection mediates 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. Author SummaryRenewed interest in phages as alternative therapeutics to combat multi-drug resistant bacterial infections, highlights the importance of understanding the consequences of phage-bacteria interactions in the context of microbial communities. Although it is well established that phages are highly specific for their host bacterium, there is no clear consensus on whether or not phage infection (and thus phage therapy) would impose collateral damage to non-target bacteria in polymicrobial communities. Here we provide direct evidence of how phage infection of a clinically relevant pathogen triggers an intrinsic type VII secretion system (T7SS) antibacterial response that consequently restricts the growth of neighboring bacterial cells that are not susceptible to phage infection. Phage induction of T7SS activity is a stress response and in addition to phages, T7SS antagonism can be induced using sub-inhibitory concentrations of antibiotics that facilitate membrane or DNA damage. Together these data show that a bacterial pathogen responds to diverse stressors to induce T7SS activity which manifests through the antagonism of neighboring non-kin bystander bacterial cells.

microbiology↗

bioRxiv2020

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 post-inoculation 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. IMPORTANCEDespite increasing prevalence and association of E. faecalis with aerobic vaginitis, essentially nothing is known about the bacterial factors that influence E. faecalis vaginal colonization. We have adapted an animal model of vaginal colonization that supports colonization of multiple E. faecalis strains. Additionally, we determined that ethanolamine utilization and type VII secretion system genes contribute to vaginal colonization and persistence. Identification of factors important for vaginal colonization and persistence provides potential targets for the development of therapeutics. This study is the first to identify key determinants that promote vaginal colonization by E. faecalis, which may represent an important reservoir for antibiotic resistant enterococci.

microbiology↗

bioRxiv2019

Parallel genomics uncover novel enterococcal-bacteriophage interactions

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 employed two distinct genomic approaches, transposon (Tn) 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 intra and inter-bacterial 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. ImportanceWe 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 of their intended host targets.

microbiology↗

bioRxiv2017

Extracellular Electron Transfer Powers Enterococcus faecalis Biofilm Metabolism

Enterococci are important human commensals and significant opportunistic pathogens associated with endocarditis, urinary tract infections, wound and surgical site infections, and medical device associated infections. These infections often become chronic upon the formation of biofilm. The biofilm matrix establishes properties that distinguish this state from free-living bacterial cells and increase tolerance to antimicrobial interventions. The metabolic versatility of the Enterococci is reflected in the diversity and complexity of environments and communities in which they thrive. Understanding metabolic factors governing colonization and persistence in different host niches can reveal factors influencing the transition from commensal to opportunistic pathogen. Here, we report a new form of iron-dependent metabolism for Enterococcus faecalis where, in the absence of heme, respiration components can be utilised for extracellular electron transfer (EET). Iron augments E. faecalis biofilm growth and generates alterations in biofilm matrix, cell spatial distribution, and biofilm matrix properties. We identify the genes involved in iron-augmented biofilm growth and show that it occurs by promoting EET to iron within biofilm.\n\nSignificanceBacterial metabolic versatility is often key in dictating the outcome of host-pathogen interactions, yet determinants of metabolic shifts are difficult to resolve. The bacterial biofilm matrix provides the structural and functional support that distinguishes this state from free-living bacterial cells. Here, we show that the biofilm matrix provides access to resources necessary for metabolism and growth which are otherwise inaccessible in the planktonic state. Our data shows that in the absence of heme, components of Enterococcus faecalis respiration (l-lactate dehydrogenase and acetaldehyde dehydrogenase) may function as initiators of EET through the cytoplasmic membrane quinone pool and utilize matrix-associated iron to carry out EET. The presence of iron resources within the biofilm matrix leads to enhanced biofilm growth.

microbiology↗