scholarly journals Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

2017 ◽  
Vol 199 (17) ◽  
Author(s):  
Benjamin I. Baarda ◽  
Sarah Emerson ◽  
Philip J. Proteau ◽  
Aleksandra E. Sikora
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Phenotype Microarray ◽  
Negative Effects ◽  
Content Type ◽  
Vaccine Candidates ◽  
Phenotype Microarrays

ABSTRACTThe function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such asNeisseria gonorrhoeae. A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates—NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344—in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamEmutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for anN. gonorrhoeaephenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such asN. gonorrhoeae.IMPORTANCEInnovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation intoNeisseria gonorrhoeaeto probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overallN. gonorrhoeaephysiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins ofN. gonorrhoeaediscovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.

scholarly journals Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

2014 ◽  
Vol 80 (14) ◽  
pp. 4226-4233 ◽  
Author(s):  
Ana M. Esteves ◽  
Sanjeev K. Chandrayan ◽  
Patrick M. McTernan ◽  
Nuno Borges ◽  
Michael W. W. Adams ◽  
...  
Keyword(s):  
Heat Stress ◽  
Pyrococcus Furiosus ◽  
Inositol Phosphate ◽  
Compatible Solutes ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Cell Protection ◽  
Content Type ◽  
Molecular Events ◽  
Supraoptimal Temperatures

ABSTRACTMarine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities.Pyrococcus furiosusis a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed forP. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.

scholarly journals Appropriate Regulation of the σE-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Hervé Nicoloff ◽  
Saumya Gopalkrishnan ◽  
Sarah E. Ades
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Stress Responses ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Point Mutations ◽  
Content Type ◽  
Envelope Stress ◽  
Outer Membrane Porins

ABSTRACT The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σE-directed cell lysis and the role of σE in stationary phase, we investigated the effects of elevated σE activity in cultures grown for 10 days. We demonstrate that high σE activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σE activity, due primarily to point mutations in the region of σE that binds the −35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σE activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the σE-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σE activity. Our results demonstrate that appropriate regulation of σE activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability. IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σE response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σE is tightly regulated to match the production of σE regulon members to the needs of the cell. In E. coli, loss of σE results in lethality. Here we demonstrate that excessive σE activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

scholarly journals Carboxydocella manganica sp. nov., a thermophilic, dissimilatory Mn(IV)- and Fe(III)-reducing bacterium from a Kamchatka hot spring

2012 ◽  
Vol 62 (Pt_4) ◽  
pp. 890-894 ◽  
Author(s):  
G. B. Slobodkina ◽  
A. N. Panteleeva ◽  
T. G. Sokolova ◽  
E. A. Bonch-Osmolovskaya ◽  
A. I. Slobodkin
Keyword(s):  
Hot Spring ◽  
Phylogenetic Analyses ◽  
Optimal Growth ◽  
Kamchatka Peninsula ◽  
Growth Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Physiological Properties ◽  
Bacterium Strain

A thermophilic, anaerobic, dissimilatory Mn(IV)- and Fe(III)-reducing bacterium (strain SLM 61T) was isolated from a terrestrial hot spring on the Kamchatka peninsula. The cells were straight rods, 0.5–0.6 µm in diameter and 1.0–6.0 µm long, and exhibited tumbling motility by means of peritrichous flagellation. The strain grew at 26–70 °C, with an optimum at 58–60 °C, and at pH 5.5–8.0, with an optimum at pH 6.5. Growth of SLM 61T was observed at 0–2.0 % (w/v) NaCl, with an optimum at 0.5 % (w/v). The generation time under optimal growth conditions was 40 min. Strain SLM 61T grew and reduced Mn(IV), Fe(III) or nitrate with a number of organic acids and complex proteinaceous compounds as electron donors. It was capable of chemolithoautotrophic growth using molecular hydrogen as an electron donor, Fe(III) but not Mn(IV) or nitrate as an electron acceptor and CO2 as a carbon source. It also was able to ferment pyruvate, yeast extract, glucose, fructose, sucrose and maltose. The G+C content of DNA of strain SLM 61T was 50.9 mol%. 16S rRNA gene sequence analysis revealed that the closest relative of the isolated organism was Carboxydocella thermautotrophica 41T (96.9 % similarity). On the basis of its physiological properties and phylogenetic analyses, the isolate is considered to represent a novel species, for which the name Carboxydocella manganica sp. nov. is proposed. The type strain is SLM 61T ( = DSM 23132T  = VKM B-2609T). C. manganica is the first described representative of the genus Carboxydocella that possesses the ability to reduce metals and does not utilize CO.

scholarly journals The Stringent Stress Response Controls Proteases and Global Regulators under Optimal Growth Conditions in Pseudomonas aeruginosa

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Daniel Pletzer ◽  
Travis M. Blimkie ◽  
Heidi Wolfmeier ◽  
Yicong Li ◽  
Arjun Baghela ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stress Response ◽  
Stringent Response ◽  
Optimal Growth ◽  
Transcriptional Regulators ◽  
Growth Conditions ◽  
Rna Seq ◽  
Signaling Molecule ◽  
Content Type ◽  
Response Mutant

ABSTRACT The bacterial stringent stress response, mediated by the signaling molecule guanosine tetraphosphate, ppGpp, has recently gained attention as being important during normal cellular growth and as a potential new therapeutic target, which warrants detailed mechanistic understanding. Here, we used intracellular protein tracking in Pseudomonas aeruginosa PAO1, which indicated that RelA was bound to the ribosome, while SpoT localized at the cell poles. Transcriptome sequencing (RNA-Seq) was used to investigate the transcriptome of a ppGpp-deficient strain under nonstressful, nutrient-rich broth conditions where the mutant grew at the same rate as the parent strain. In the exponential growth phase, the lack of ppGpp led to >1,600 transcriptional changes (fold change cutoff of ±1.5), providing further novel insights into the normal physiological role of ppGpp. The stringent response was linked to gene expression of various proteases and secretion systems, including aprA, PA0277, impA, and clpP2. The previously observed reduction in cytotoxicity toward red blood cells in a stringent response mutant appeared to be due to aprA. Investigation of an aprA mutant in a murine skin infection model showed increased survival rates of mice infected with the aprA mutant, consistent with previous observations that stringent response mutants have reduced virulence. In addition, the overexpression of relA, but not induction of ppGpp with serine hydroxamate, dysregulated global transcriptional regulators as well as >30% of the regulatory networks controlled by AlgR, OxyR, LasR, and AmrZ. Together, these data expand our knowledge about ppGpp and its regulatory network and role in environmental adaptation. It also confirms its important role throughout the normal growth cycle of bacteria. IMPORTANCE Microorganisms need to adapt rapidly to survive harsh environmental changes. Here, we showed the broad influence of the highly studied bacterial stringent stress response under nonstressful conditions that indicate its general physiological importance and might reflect the readiness of bacteria to respond to and activate acute stress responses. Using RNA-Seq to investigate the transcriptional network of Pseudomonas aeruginosa cells revealed that >30% of all genes changed expression in a stringent response mutant under optimal growth conditions. This included genes regulated by global transcriptional regulators and novel downstream effectors. Our results help to understand the importance of this stress regulator in bacterial lifestyle under relatively unstressed conditions. As such, it draws attention to the consequences of targeting this ubiquitous bacterial signaling molecule.

scholarly journals Induction of Daptomycin Tolerance in Enterococcus faecalis by Fatty Acid Combinations

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
William Brewer ◽  
Johnathan Harrison ◽  
Holly E. Saito ◽  
Elizabeth M. Fozo
Keyword(s):  
Fatty Acids ◽  
Antibiotic Resistance ◽  
Fatty Acid ◽  
Stearic Acid ◽  
Enterococcus Faecalis ◽  
Cell Envelope ◽  
Negative Effects ◽  
Gram Positive ◽  
Bacterial Populations ◽  
Content Type

ABSTRACT Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid. IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

scholarly journals Salt-Induced Stress Stimulates a Lipoteichoic Acid-Specific Three-Component Glycosylation System inStaphylococcus aureus

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Kelvin Kho ◽  
Timothy C. Meredith
Keyword(s):  
Staphylococcus Aureus ◽  
Sigma Factor ◽  
Cell Envelope ◽  
Normal Growth ◽  
Lipoteichoic Acid ◽  
Growth Conditions ◽  
Growth Media ◽  
Alternative Sigma Factor ◽  
Content Type

ABSTRACTLipoteichoic acid (LTA) inStaphylococcus aureusis a poly-glycerophosphate polymer anchored to the outer surface of the cell membrane. LTA has numerous roles in cell envelope physiology, including regulating cell autolysis, coordinating cell division, and adapting to environmental growth conditions. LTA is often further modified with substituents, includingd-alanine and glycosyl groups, to alter cellular function. While the genetic determinants ofd-alanylation have been largely defined, the route of LTA glycosylation and its role in cell envelope physiology have remained unknown, in part due to the low levels of basal LTA glycosylation inS. aureus. We demonstrate here thatS. aureusutilizes a membrane-associated three-component glycosylation system composed of an undecaprenol (Und)N-acetylglucosamine (GlcNAc) charging enzyme (CsbB; SAOUHSC_00713), a putative flippase to transport loaded substrate to the outside surface of the cell (GtcA; SAOUHSC_02722), and finally an LTA-specific glycosyltransferase that adds α-GlcNAc moieties to LTA (YfhO; SAOUHSC_01213). We demonstrate that this system is specific for LTA with no cross recognition of the structurally similar polyribitol phosphate containing wall teichoic acids. We show that while wild-typeS. aureusLTA has only a trace of GlcNAcylated LTA under normal growth conditions, amounts are raised upon either overexpressing CsbB, reducing endogenousd-alanylation activity, expressing the cell envelope stress responsive alternative sigma factor SigB, or by exposure to environmental stress-inducing culture conditions, including growth media containing high levels of sodium chloride.IMPORTANCEThe role of glycosylation in the structure and function ofStaphylococcus aureuslipoteichoic acid (LTA) is largely unknown. By defining key components of the LTA three-component glycosylation pathway and uncovering stress-induced regulation by the alternative sigma factor SigB, the role ofN-acetylglucosamine tailoring during adaptation to environmental stresses can now be elucidated. As thedltand glycosylation pathways compete for the same sites on LTA and induction of glycosylation results in decreasedd-alanylation, the interplay between the two modification systems holds implications for resistance to antibiotics and antimicrobial peptides.

scholarly journals Alternative Oxidase Activity Reduces Stress in Vibrio fischeri Cells Exposed to Nitric Oxide

2018 ◽  
Vol 200 (15) ◽  
Author(s):  
Anne K. Dunn
Keyword(s):  
Nitric Oxide ◽  
Alternative Oxidase ◽  
Vibrio Fischeri ◽  
Membrane Integrity ◽  
Model Organism ◽  
Growth Conditions ◽  
Respiratory Pathway ◽  
Content Type ◽  
Atp Production ◽  
In Cells

ABSTRACT Alternative oxidase (Aox) is a non-energy-conserving respiratory oxidase found in certain eukaryotes and bacteria, whose role in physiology is not entirely clear. Using the genetically tractable bacterium Vibrio fischeri as a model organism, I have identified a role for Aox to reduce levels of stress in cells exposed to oxygen and nitric oxide (NO). In V. fischeri lacking the NO-detoxifying enzyme flavohemoglobin (Hmp), deletion of aox in cells grown in the presence of oxygen and NO results in alterations to the transcriptome that include increases in transcripts mapping to stress-related genes. Using fluorescence-based reporters, I identified corresponding increases in intracellular reactive oxygen species and decreases in membrane integrity in cells lacking aox. Under these growth conditions, activity of Aox is linked to a decrease in NADH levels, indicating coupling of Aox activity with NADH dehydrogenase activity. Taken together, these results suggest that Aox functions to indirectly limit production of ferrous iron and damaging hydroxyl radicals, effectively reducing cellular stress during NO exposure. IMPORTANCE Unlike typical respiratory oxidases, alternative oxidase (Aox) does not directly contribute to energy conservation, and its activity would presumably reduce the efficiency of respiration and associated ATP production. Aox has been identified in certain bacteria, a majority of which are marine associated. The presence of Aox in these bacteria poses the interesting question of how Aox function benefits bacterial growth and survival in the ocean. Using the genetically tractable marine bacterium Vibrio fischeri, I have identified a role for Aox in reduction of stress under conditions where electron flux through the aerobic respiratory pathway is inhibited. These results suggest that Aox activity could positively impact longer-term bacterial fitness and survival under stressful environmental conditions.

scholarly journals Optimization of Methanotrophic Growth and Production of Poly(3-Hydroxybutyrate) in a High-Throughput Microbioreactor System

2015 ◽  
Vol 81 (14) ◽  
pp. 4767-4773 ◽  
Author(s):  
Eric R. Sundstrom ◽  
Craig S. Criddle
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Medium Optimization ◽  
Nile Red ◽  
Bacterial Biomass ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Culture Conditions ◽  
High Mass ◽  
Content Type

ABSTRACTProduction of poly(3-hydroxybutyrate) (P3HB) from methane has economic and environmental advantages over production by agricultural feedstock. Identification of high-productivity strains and optimal growth conditions is critical to efficient conversion of methane to polymer. Current culture conditions, including serum bottles, shake flasks, and agar plates, are labor-intensive and therefore insufficient for systematic screening and isolation. Gas chromatography, the standard method for analysis of P3HB content in bacterial biomass, is also incompatible with high-throughput screening. Growth in aerated microtiter plates coupled with a 96-well Nile red flow-cytometric assay creates an integrated microbioreactor system for high-throughput growth and analysis of P3HB-producing methanotrophic cultures, eliminating the need for individual manipulation of experimental replicates. This system was tested in practice to conduct medium optimization for P3HB production in pure cultures ofMethylocystis parvusOBBP. Optimization gave insight into unexpected interactions: for example, low calcium concentrations significantly enhanced P3HB production under nitrogen-limited conditions. Optimization of calcium and copper concentrations in the growth medium increased final P3HB content from 18.1% to 49.4% and P3HB concentration from 0.69 g/liter to 3.43 g/liter while reducing doubling time from 10.6 h to 8.6 h. The ability to culture and analyze thousands of replicates with high mass transfer in completely mixed culture promises to streamline medium optimization and allow the detection and isolation of highly productive strains. Applications for this system are numerous, encompassing analysis of biofuels and other lipid inclusions, as well as analysis of heterotrophic and photosynthetic systems.

scholarly journals Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection in a Murine Model of Lower Genital Tract Infection

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Dharanesh Gangaiah ◽  
Erica L. Raterman ◽  
Hong Wu ◽  
Kate R. Fortney ◽  
Hongyu Gao ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Murine Model ◽  
Response Regulator ◽  
Membrane Integrity ◽  
Gonococcal Infection ◽  
Genital Tract Infection ◽  
Content Type ◽  
Constitutive Activation ◽  
Lower Genital Tract Infection

ABSTRACT During infection, Neisseria gonorrhoeae senses and responds to stress; such responses may be modulated by MisRS (NGO0177 and NGO0176), a two-component system that is a homolog of CpxRA. In Escherichia coli, CpxRA senses and responds to envelope stress; CpxA is a sensor kinase/phosphatase for CpxR, a response regulator. When a cpxA mutant is grown in medium containing glucose, CpxR is phosphorylated by acetyl phosphate but cannot be dephosphorylated, resulting in constitutive activation. Kandler and coworkers (J. L. Kandler, C. L. Holley, J. L. Reimche, V. Dhulipala, J. T. Balthazar, A. Muszyński, R. W. Carlson, and W. M. Shafer, Antimicrob Agents Chemother 60:4690–4700, 2016, https://doi.org/10.1128/AAC.00823-16 ) showed that MisR (CpxR) is required for the maintenance of membrane integrity and resistance to antimicrobial peptides, suggesting a role in gonococcal survival in vivo. Here, we evaluated the contributions of MisR and MisS (CpxA) to gonococcal infection in a murine model of cervicovaginal colonization and identified MisR-regulated genes using RNA sequencing (RNA-Seq). The deletion of misR or misS severely reduced the capacity of N. gonorrhoeae to colonize mice or maintain infection over a 7-day period and reduced microbial fitness after exposure to heat shock. Compared to the wild type (WT), the inactivation of misR identified 157 differentially regulated genes, most of which encoded putative envelope proteins. The inactivation of misS identified 17 differentially regulated genes compared to the WT and 139 differentially regulated genes compared to the misR mutant, 111 of which overlapped those differentially expressed in the comparison of the WT versus the misR mutant. These data indicate that an intact MisRS system is required for gonococcal infection of mice. Provided the MisR is constitutively phosphorylated in the misS mutant, the data suggest that controlled but not constitutive activation is required for gonococcal infection in mice.

scholarly journals Multiple Roles for Enterococcus faecalis Glycosyltransferases in Biofilm-Associated Antibiotic Resistance, Cell Envelope Integrity, and Conjugative Transfer

2015 ◽  
Vol 59 (7) ◽  
pp. 4094-4105 ◽  
Author(s):  
Jennifer L. Dale ◽  
Julian Cagnazzo ◽  
Chi Q. Phan ◽  
Aaron M. T. Barnes ◽  
Gary M. Dunny
Keyword(s):  
Antibiotic Resistance ◽  
Enterococcus Faecalis ◽  
Cell Envelope ◽  
Opportunistic Pathogen ◽  
Growth Conditions ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Conjugative Transfer ◽  
Content Type ◽  
Resistance Determinants

ABSTRACTThe 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 faecalisis 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 inE. 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 identifyE. faecalisbiofilm-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, andgelE) and two glycosyltransferase (GTF) genes (epaIandepaOX). We also found that the GTFs play additional roles inE. faecalisresistance 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. TheepaOXgene is located in a variable extended region of the enterococcal polysaccharide antigen (epa) locus. These data illustrate the importance of GTFs inE. faecalisadaptation to diverse growth conditions and suggest new targets for antimicrobial design.

Sign in / Sign up

Export Citation Format

Share Document