Three-component Regulatory System Research Articles

C-di-AMP levels modulate Staphylococcus aureus cell wall thickness, response to oxidative stress, and antibiotic resistance and tolerance.

Beta-lactam antibiotics are widely used to treat infections caused by the important human pathogen Staphylococcus aureus. Resistance to beta-lactams, as found in methicillin-resistant S. aureus, renders effective treatment difficult. The second messenger cyclic di-3',5'-adenosine monophosphate (c-di-AMP) promotes beta-lactam resistance in clinical S. aureus isolates. C-di-AMP plays a crucial role in the regulation of cellular processes such as virulence, cell wall homeostasis, and resistance to beta-lactams in many bacterial species. In S. aureus, c-di-AMP synthesis is mediated by the diadenylate cyclase DacA, while its degradation is carried out by the phosphodiesterases GdpP and Pde2. In this work, we assessed the effect of altered c-di-AMP levels due to mutations in dacA, gdpP, or gdpP/pde2 on virulence determinants. We report that a previously described growth defect in bacteria producing high c-di-AMP levels is mainly attributable to smaller cell size. High c-di-AMP levels also led to decreased survival upon oxidative stress, reduced production of the antioxidant staphyloxanthin, increased oxacillin and fosfomycin resistance, and increased cell wall thickness. While resistance to ceftaroline was not affected, high c-di-AMP levels promoted tolerance to this antibiotic. In response to cell wall stress induced by antibiotics, the three-component regulatory system VraTSR mediates an increase in cell wall synthesis via the cell wall stress stimulon (CWSS). Increased c-di-AMP levels led to an activation of the CWSS. Upon deletion of vraR, resistance to oxacillin and fosfomycin as well as cell wall thickness diminished in the ΔgdpP mutant, indicating a contribution of the VraTSR system to the cell wall related phenotypes.IMPORTANCEAntibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3', 5'-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting.

HptA Mutation May Mediate Fosfomycin Resistance in Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

Fosfomycin can be used alone or in combination to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. However, fosfomycin resistance has been observed in MRSA. In S. aureus, fosfomycin resistance is mediated by the fosfomycin-modifying enzyme FosB, or mutations in the target enzyme MurA. Mutations in the chromosomal glpT and uhpT genes, which encode fosfomycin transporters, also result in fosfomycin resistance. The three-component regulatory system HptRSA mediates the expression of uhpT and glpT in S. aureus. This study aimed to investigate the role of hptRSA mutation in fosfomycin resistance in MRSA clinical isolates. We found that hptRSA mutations were common in MRSA strains isolated from our hospital. Most mutations were amino acid substitutions and widely distributed in fosfomycin-sensitive and fosfomycin-resistant strains. However, HptA-truncated mutations were only found in fosB-negative fosfomycin-resistant strains with wild-type uhpT and glpT genes. Quantitative real-time PCR results showed that the transcription level of uhpT decreased by 13.7-25.6-fold in the HptA-truncated strains. Concordantly, the fosfomycin minimum inhibitory concentration (MIC) of HptA-truncated strains was 64-128 μg/mL, while SA240 was 2 μg/mL. The low transcription level of uhpT and high increase in MIC suggest that hptA mutation may lead to fosfomycin resistance in MRSA. We complemented hptA in one of the HptA-truncated clinical strains (SA179), showing reversal of fosfomycin resistance (from 128 to 32 μg/mL). Then we knocked out hptA in S. aureus Newman; fosfomycin MIC increased from 4 to 64 μg/mL, suggesting that HptA mutation may play an important role in fosfomycin resistance.

2350. LiaF is required for LiaX mediated protection against Daptomycin and LL-37 in Enterococcus faecalis OG1RF

Abstract Background Daptomycin (DAP) resistance in enterococci is regulated by the LiaFSR three-component regulatory system. Mutations in liaF have been linked to activation of this system, with increased expression of the protein LiaX, and resistance to DAP in clinical isolates. LiaX functions by recognizing DAP in the extracellular medium, and serves as a signal transduction molecule. However, the role of LiaF in signaling and its relationship with LiaX are unknown. Methods We generated a liaF null mutant in E. faecalis OG117 by adding four stop codons at amino acid positions 11–14 (OG117liaF*11-14) using a CRISPR-Cas9 system. The mutant was complemented by restoring wild-type liaF in its chromosomal location (OG117liaF*11-14::liaF). Mutants were confirmed with whole genome sequencing (WGS). Anionic phospholipid (AP) microdomain distribution was assessed on microscopy using 10-N-nonyl-acridine-orange (NAO). LiaFSR activation was assessed by surface expression of LiaX via ELISA. DAP MICs were performed by broth microdilution in the presence/absence of exogenous LiaX (eLiaX). LL37 (50µg/ml) and DAP (1.5µg/ml) killing assays with eLiaX were also performed in triplicate. Results Truncation of liaF did not have any effect on DAP MICs, AP microdomain distribution or LiaX surface expression compared to OG117. No additional mutations were observed by WGS in the mutants. In the presence eLiaX, DAP MIC increased 8-fold in wild-type OG117 but remained the same (1 µg/mL) in OG117liaF*11-14. Complementation of liaF restored the increase in DAP MIC in the presence of eLiaX. In the LL37 killing assay, survival of OG117 was significantly increased on the addition of eLiaX (2.9% vs 11.8%, respectively, P < .002). Addition of eLiaX was unable to rescue OG117liaF*11-14 in the presence of LL-37 (2.4% vs 2.5% respectively, P=0.51), but was able to increase survival in the complemented strain (2.8% vs 7.6%, respectively, P < .002). A similar trend was seen in the DAP killing assay, with increased survival for OG117 (8.7% vs 20%) and OG117liaF*11-14::liaF (11.6% vs 17%), but not OG117liaF*11-14 (0.38% vs 1%). Conclusion LiaF is required for full tolerance to the cathelicidin LL37 and DAP in E. faecalis OG1RF. These results suggest that LiaF may interact with LiaX as a part of the LiaFSR stress response. Disclosures William R. Miller, MD, Entasis: Grant/Research Support|UpToDate: Honoraria Cesar A. Arias, MD, PhD, Entasis: Grant/Research Support|MeMed Diagnostics Ltd: Grant/Research Support|Merck: Grant/Research Support.

Revisiting the Role of VraTSR in Staphylococcus aureus Response to Cell Wall-Targeting Antibiotics.

ABSTRACTExposure of Staphylococcus aureus to cell wall inhibitors leads to the activation of the VraTSR three-component sensory regulatory system. This system is composed of VraS, a membrane histidine kinase; VraR, its cognate response regulator, and VraT, a protein required for the full activity of VraTSR. The exact function of VraT remains mostly uncharacterized, although it has been proposed to detect the unknown stimulus sensed by the VraTSR system. Here, we elucidate the topology of VraT, showing that its C-terminal domain is extracellular. We also demonstrate that the signal sensed by VraTSR is not an intermediate in the peptidoglycan synthesis pathway, as previously suggested. Instead, the specific inhibition of the penicillin-binding protein (PBP)2 leads to strong activation of the system.IMPORTANCE The Gram-positive bacterial pathogen Staphylococcus aureus is currently the second most frequent cause of global deaths associated with antibiotic resistance. Its response to cell wall-targeting antibiotics requires the VraTSR three-component system, which senses cell wall damage. Here, we show that the signal sensed by VraTSR is not an intermediate in the peptidoglycan synthesis pathway, as previously suggested. Instead, the specific inhibition of the penicillin-binding protein (PBP)2, the major peptidoglycan synthase in S. aureus, leads to strong activation of the system. Identifying the exact cell wall damage signal is key to fully understanding the response of S. aureus to cell wall-targeting antibiotics.

Ubericin K, a New Pore-Forming Bacteriocin Targeting mannose-PTS.

ABSTRACTBovine mastitis infection in dairy cattle is a significant economic burden for the dairy industry globally. To reduce the use of antibiotics in treatment of clinical mastitis, new alternative treatment options are needed. Antimicrobial peptides from bacteria, also known as bacteriocins, are potential alternatives for combating mastitis pathogens. In search of novel bacteriocins against mastitis pathogens, we screened samples of Norwegian bovine raw milk and found a Streptococcus uberis strain with potent antimicrobial activity toward Enterococcus, Streptococcus, Listeria, and Lactococcus. Whole-genome sequencing of the strain revealed a multibacteriocin gene cluster encoding one class IIb bacteriocin, two class IId bacteriocins, in addition to a three-component regulatory system and a dedicated ABC transporter. Isolation and purification of the antimicrobial activity from culture supernatants resulted in the detection of a 6.3-kDa mass peak by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, a mass corresponding to the predicted size of one of the class IId bacteriocins. The identification of this bacteriocin, called ubericin K, was further confirmed by in vitro protein synthesis, which showed the same inhibitory spectrum as the purified antimicrobial compound. Ubericin K shows highest sequence similarity to the class IId bacteriocins bovicin 255, lactococcin A, and garvieacin Q. We found that ubericin K uses the sugar transporter mannose phosphotransferase (PTS) as a target receptor. Further, by using the pHlourin sensor system to detect intracellular pH changes due to leakage across the membrane, ubericin K was shown to be a pore former, killing target cells by membrane disruption.IMPORTANCE Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance. Compounds other than antibiotics also exist that kill bacteria causing infections in cows; these compounds, known as bacteriocins, are natural products produced by other bacteria in the environment. In this work, we discover a new bacteriocin that we call ubericin K, which kills several species of bacteria known to cause infections in dairy cows. We also use in vitro synthesis as a novel method for rapidly characterizing bacteriocins directly from genomic data, which could be useful for other researchers. We believe that ubericin K and the methods described in this work will aid in the transition away from antibiotics in the dairy industry.

Molecular characterization of the possible regulation of multiple bacteriocin production through a three-component regulatory system in Enterococcus faecium NKR-5-3

Enterococcus faecium NKR-5-3 produces multiple-bacteriocins, enterocins NKR-5-3A, B, C, D, and Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). However, the biosynthetic mechanisms on how their productions are regulated are yet to be fully understood. In silico analysis revealed putative promoters and terminators in the enterocin NKR-5-3ACDZ gene cluster, and the putative direct repeats (5'-ATTTTAGGATA-3') were conserved upstream of each promoter. Transcriptional analysis by quantitative real-time polymerase chain reaction (PCR) of the biosynthetic genes for the enterocins NKR-5-3 suggested that an inducing peptide (Ent53D) regulates the transcription of the structure genes and corresponding biosynthetic genes of enterocins NKR-5-3, except for Ent53B (a circular bacteriocin), thus consequently regulating their production. Moreover, transcriptional analysis of some knock-out mutants showed that the production of Ent53A, C, D and Z is controlled by a three-component regulatory system (TCS) consisting of Ent53D, EnkR (response regulator), and EnkK (histidine kinase). The production of the circular bacteriocin Ent53B appeared to be independent from this TCS. Nevertheless, disrupting the TCS by deletion of a single component (enkD, enkR and enkK) resulted in a slight increase of enkB transcription and consequently the production of Ent53B, presumably, as an indirect consequence of the increase of available energy to the strain NKR-5-3. Here, we demonstrate the regulatory control of the multiple bacteriocin production of strain NKR-5-3 likely through the TCS consisting of Ent53D, EnkR, and EnkK. The information of the sharing of the regulatory machinery between bacteriocins in strain NKR-5-3 can be useful in its future application such as designing strategies to effectively dispense its multiple bacteriocin arsenal.

599. LiaF is an Activator of the LiaR-Mediated Response Against Daptomycin and Antimicrobial Peptides in Multidrug-Resistant Enterococcus faecalis (Efs)

BackgroundDaptomycin (DAP) is a key first-line agent for the treatment of vancomycin-resistant enterococcal infections. Resistance to DAP in enterococci is regulated by the liaFSR three-component regulatory system that consists of a histidine kinase sensor (LiaS), a response regulator (LiaR) and a transmembrane protein of unknown function (LiaF). Previous studies indicate that deletion of isoleucine in position 177 of LiaF results in DAP tolerance and is sufficient to change membrane architecture. Here, we dissect the role of LiaF in DAP resistanceMethodsWe generated three liaF mutants in OG1RF, a DAP-susceptible laboratory strain of Efs (DAP MIC = 2 µg/mL): (i) a non-polar, C-terminal truncation of liaF (OG1RFliaF∆152), (ii) a null liaF mutant with a premature stop-codon (OG1RFliaF*11), and (iii) an isoleucine deletion at position 177 (OG1RFliaF177). We determined DAP MIC by Etest and characterized the localization of anionic phospholipids microdomains using 10-nonyl-acridine-orange (NAO). The expression of the liaXYZ (the main target of LiaR) and liaFSR clusters were evaluated by qRT-PCR and relative expression ratios (Log2 fold change) were calculated by normalizing to gyrB expression. We assessed activation of LiaFSR by evaluating surface exposure of LiaX by ELISA. We used the bacterial adenylate cyclase two-hybrid system (BACTH) to evaluate the protein-protein interaction between LiaF and LiaS.ResultsFull deletion of liaF or the C-terminal truncation of LiaF did not have any effect on DAP MICs, membrane architecture or a significant increase in LiaX surface exposure compared with parental strain OG1RF. In contrast, deletion of the codon encoding isoleucine in position 177 of LiaF caused a major increase (8-fold) in LiaX exposure and redistribution of anionic phospholipid microdomains away from the septum without changes in the actual DAP MIC. Transcriptional analyses indicated upregulation (>2 log2-fold) in the liaXYZ gene cluster indicating activation of the stress response. We also observed a positive interaction between LiaF and LiaS.ConclusionLiaF is likely a key activator of the LiaFSR stress response and the critical regulatory domain appears to be located in a stretch of four isoleucines toward the C-terminal of the protein.Disclosures All authors: No reported disclosures.

Sodium Lactate Negatively Regulates Shewanella putrefaciens CN32 Biofilm Formation via a Three-Component Regulatory System (LrbS-LrbA-LrbR).

The capability of biofilm formation has a major impact on the industrial and biotechnological applications of Shewanella putrefaciens CN32. However, the detailed regulatory mechanisms underlying biofilm formation in this strain remain largely unknown. In the present report, we describe a three-component regulatory system which negatively regulates the biofilm formation of S. putrefaciens CN32. This system consists of a histidine kinase LrbS (Sputcn32_0303) and two cognate response regulators, including a transcription factor, LrbA (Sputcn32_0304), and a phosphodiesterase, LrbR (Sputcn32_0305). LrbS responds to the signal of the carbon source sodium lactate and subsequently activates LrbA. The activated LrbA then promotes the expression of lrbR, the gene for the other response regulator. The bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) phosphodiesterase LrbR, containing an EAL domain, decreases the concentration of intracellular c-di-GMP, thereby negatively regulating biofilm formation. In summary, the carbon source sodium lactate acts as a signal molecule that regulates biofilm formation via a three-component regulatory system (LrbS-LrbA-LrbR) in S. putrefaciens CN32.IMPORTANCE Biofilm formation is a significant capability used by some bacteria to survive in adverse environments. Numerous environmental factors can affect biofilm formation through different signal transduction pathways. Carbon sources are critical nutrients for bacterial growth, and their concentrations and types significantly influence the biomass and structure of biofilms. However, knowledge about the underlying mechanism of biofilm formation regulation by carbon source is still limited. This work elucidates a modulation pattern of biofilm formation negatively regulated by sodium lactate as a carbon source via a three-component regulatory system in S. putrefaciens CN32, which may serve as a good example for studying how the carbon sources impact biofilm development in other bacteria.

Genomic Pathways Associated with Daptomycin (DAP) Resistance in DAP-Susceptible Enterococcus faecium Harboring Substitutions in LiaFSR

Background DAP is used off-label for treatment of severe enterococcal infections. DAP resistance (R) in E. faecium has been associated with changes in LiaFSR, a three-component regulatory system that controls the cell envelope stress response to antibiotics. In particular, substitutions in LiaS (T120A) and LiaR (W73C) seem to predispose to development of DAP-R during therapy, without increasing the DAP MIC above the clinical breakpoint. Using a PK/PD model of simulated endocardial vegetations, we evaluated the genomic pathways for DAP-R under different DAP dose schemes.Methods A DAP-susceptible E. faecium (HOU503; MIC 3 mg/ml) harboring the above LiaSR substitutions, was subjected to simulated human doses of 6, 8, and 10 mg/kg/d in the model for 14 days using a starting inoculum of 109 CFU/ml. Sixteen DAP-R isolates were recovered from the SEV model: five isolates from 6 mg/kg (D6 isolates from days 2 to 14); five isolates from 8 mg/kg (D8 isolates from days 2 to 8); and six isolates from 10 mg/kg (D10 isolates from days 1 to 14) which were subjected to whole genome sequencing. Reads from each sequenced isolate were mapped against the HOU503 genome for SNP analyses. Variant calling was done with GATK, SamTools, and the low-frequency variant detector from CLC Genomics Workbench 8.5. Variants detected by the three callers were selected and annotated with SnpEff; then compared among the different groups of isolates accordingly to the DAP doses that were exposed.ResultsWe detected a total of 16 proteins exhibiting substitutions consistently in all the DAP-R sequenced isolates; including mobile genetic elements (9), hypothetical proteins (2), a bacteriocin, a cysteine desulfurase, a N-acetylglucosamine-specific PTS system, a N-acetylmannosamine-6-phosphate 2-epimerase and a MurR/RpiR, which is a transcriptional regulator that represses the operon MurPQ involved in the uptake and degradation of N-acetylmuramic acid. Notably, mutations in cardiolipin synthase were present only in isolates recovered under D8 dose. The LiaRS substitutions remained in all isolates.Conclusion Using a humanized SEV PK/PD model and SNP-based analyses, we were able to uncover possible novel genetic pathways associated with the development DAP-R via the LiaFSR system in enterococci.Disclosures M. J. Rybak, Allergen: Scientific Advisor, Consulting fee

Comparative Analysis of the Complete Genome of Lactobacillus plantarum GB-LP2 and Potential Candidate Genes for Host Immune System Enhancement

Acute respiratory virus infectious diseases are a growing health problem, particularly among children and the elderly. Much effort has been made to develop probiotics that prevent influenza virus infections by enhancing innate immunity in the respiratory tract until vaccines are available. Lactobacillus plantarum GB-LP2, isolated from a traditional Korean fermented vegetable, has exhibited preventive effects on influenza virus infection in mice. To identify the molecular basis of this strain, we conducted a whole-genome assembly study. The single circular DNA chromosome of 3,284,304 bp was completely assembled and 3,250 proteinencoding genes were predicted. Evolutionarily accelerated genes related to the phenotypic trait of anti-infective activities for influenza virus were identified. These genes encode three integral membrane proteins, a teichoic acid export ATP-binding protein and a glucosamine - fructose-6-phosphate aminotransferase involved in host innate immunity, the nonspecific DNA-binding protein Dps, which protects bacteria from oxidative damage, and the response regulator of the three-component quorum-sensing regulatory system, which is related to the capacity of adhesion to the surface of the respiratory tract and competition with pathogens. This is the first study to identify the genetic backgrounds of the antiviral activity in L. plantarum strains. These findings provide insight into the anti-infective activities of L. plantarum and the development of preventive probiotics.

Co-culture-inducible bacteriocin production in lactic acid bacteria.

It is common knowledge that microorganisms have capabilities, like the production of antimicrobial compounds, which do not normally appear in ideal laboratory conditions. Common antimicrobial discovery techniques require the isolation of monocultures and their individual screening against target microorganisms. One strategy to achieve expression of otherwise hidden antimicrobials is induction by co-cultures. In the area of bacteriocin-producing lactic acid bacteria, there has been some research focusing into the characteristics of co-culture-inducible bacteriocin production and particularly the molecular mechanism(s) of such interactions. No clear relationship has been seen between bacteriocin-inducing and bacteriocin-producing microorganisms. The three-component regulatory system seems to be playing a central role in the induction, but inducing compounds have not been identified or characterized. However, the presence of the universal messenger molecule autoinducer-2 has been associated in some cases with the co-culture-inducible bacteriocin phenotype and it may play the role in the additional regulation of the three-component regulatory system. Understanding the mechanisms of induction would facilitate the development of strategies for screening and development of co-culture bacteriocin-producing systems and novel products as well as the perseverance of such systems in food and down to the intestinal tract, possibly conferring a probiotic effect on the host.

Purification and genetic characterization of gassericin E, a novel co-culture inducible bacteriocin from Lactobacillus gasseri EV1461 isolated from the vagina of a healthy woman.

BackgroundLactobacillus gasseri is one of the dominant Lactobacillus species in the vaginal ecosystem. Some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. In this work we have studied bacteriocin production of gassericin E (GasE), a novel bacteriocin produced by L. gasseri EV1461, a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. Biochemical and genetic characterization of this novel bacteriocin are addressed.ResultsWe found that the inhibitory spectrum of L. gasseri EV1461 was broad, being directed to species both related and non-related to the producing strain. Interestingly, L. gasseri EV1461 inhibited the grown of pathogens usually associated with bacterial vaginosis (BV). The antimicrobial activity was due to the production of a novel bacteriocin, gassericin E (GasE). Production of this bacteriocin in broth medium only was achieved at high cell densities. At low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing EV1461 culture (autoinduction), or through co-cultivation with several other Gram-positive strains (inducing bacteria). DNA sequence of the GasE locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin (gaeAXI), and in regulation, transport and processing (gaePKRTC). The gaePKR encodes a putative three-component regulatory system, involving an autoinducer peptide (GaeP), a histidine protein kinase (GaeK) and a response regulator (GaeR), while the gaeTC encodes for an ABC transporter (GaeT) and their accessory protein (GaeC), involved in transport and processing of the bacteriocin. The gaeAXI, encodes for the bacteriocin gassericin E (GasE), a putative peptide bacteriocin (GaeX), and their immunity protein (GaeI).ConclusionsThe origin of the strain (vagina of healthy woman) and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using L. gasseri EV1461 as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis (BV), since it could be better adapted to live and compete into the vaginal environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0663-1) contains supplementary material, which is available to authorized users.

Deletion of liaR Reverses Daptomycin Resistance in Enterococcus faecium Independent of the Genetic Background.

We have shown previously that changes in LiaFSR, a three-component regulatory system predicted to orchestrate the cell membrane stress response, are important mediators of daptomycin (DAP) resistance in enterococci. Indeed, deletion of the gene encoding the response regulator LiaR in a clinical strain of Enterococcus faecalis reversed DAP resistance (DAP-R) and produced a strain hypersusceptible to antimicrobial peptides. Since LiaFSR is conserved in Enterococcus faecium, we investigated the role of LiaR in a variety of clinical E. faecium strains representing the most common DAP-R genetic backgrounds. Deletion of liaR in DAP-R E. faecium R446F (DAP MIC of 16 μg/ml) and R497F (MIC of 24 μg/ml; harboring changes in LiaRS) strains fully reversed resistance (DAP MICs decreasing to 0.25 and 0.094 μg/ml, respectively). Moreover, DAP at concentrations of 13 μg/ml (achieved with human doses of 12 mg/kg body weight) retained bactericidal activity against the mutants. Furthermore, the liaR deletion derivatives of these two DAP-R strains exhibited increased binding of boron-dipyrromethene difluoride (BODIPY)-daptomycin, suggesting that high-level DAP-R mediated by LiaR in E. faecium involves repulsion of the calcium-DAP complex from the cell surface. In DAP-tolerant strains HOU503F and HOU515F (DAP MICs within the susceptible range but bacteria not killed by DAP concentrations of 5× the MIC), deletion of liaR not only markedly decreased the DAP MICs (0.064 and 0.047 μg/ml, respectively) but also restored the bactericidal activity of DAP at concentrations as low as 4 μg/ml (achieved with human doses of 4 mg/kg). Our results suggest that LiaR plays a relevant role in the enterococcal cell membrane adaptive response to antimicrobial peptides independent of the genetic background and emerges as an attractive target to restore the activity of DAP against multidrug-resistant strains.

Regulation of arsenite oxidation by the phosphate two-component system PhoBR in Halomonas sp. HAL1.

Previously, the expression of arsenite [As(III)] oxidase genes aioBA was reported to be regulated by a three-component regulatory system, AioXSR, in a number of As(III)-oxidizing bacterial strains. However, the regulation mechanism is still unknown when aioXSR genes are absent in some As(III)-oxidizing bacterial genomes, such as in Halomonas sp. HAL1. In this study, transposon mutagenesis and gene knock-out mutation were performed, and two mutants, HAL1-phoR931 and HAL1-▵phoB, were obtained in strain HAL1. The phoR and phoB constitute a two-component system which is responsible for phosphate (Pi) acquisition and assimilation. Both of the mutants showed negative As(III)-oxidation phenotypes in low Pi condition (0.1 mM) but not under normal Pi condition (1 mM). The phoBR complementation strain HAL1-▵phoB-C reversed the mutants' null phenotypes back to wild type status. Meanwhile, lacZ reporter fusions using pCM-lacZ showed that the expression of phoBR and aioBA were both induced by As(III) but were not induced in HAL1-phoR931 and HAL1-▵phoB. Using 15 consensus Pho box sequences, a putative Pho box was found in the aioBA regulation region. PhoB was able to bind to the putative Pho box in vivo (bacterial one-hybrid detection) and in vitro (electrophoretic mobility gel shift assay), and an 18-bp binding sequence containing nine conserved bases were determined. This study provided the evidence that PhoBR regulates the expression of aioBA in Halomonas sp. HAL1 under low Pi condition. The new regulation model further implies the close metabolic connection between As and Pi.

Competition between VanU(G) repressor and VanR(G) activator leads to rheostatic control of vanG vancomycin resistance operon expression.

Enterococcus faecalis BM4518 is resistant to vancomycin by synthesis of peptidoglycan precursors ending in D-alanyl-D-serine. In the chromosomal vanG locus, transcription of the resistance genes from the PYG resistance promoter is inducible and, upstream from these genes, there is an unusual three-component regulatory system encoded by the vanURSG operon from the PUG regulatory promoter. In contrast to the other van operons in enterococci, the vanG operon possesses the additional vanUG gene which encodes a transcriptional regulator whose role remains unknown. We show by DNase I footprinting, RT-qPCR, and reporter proteins activities that VanUG, but not VanRG, binds to PUG and negatively autoregulates the vanURSG operon and that it also represses PYG where it overlaps with VanRG for binding. In clinical isolate BM4518, the transcription level of the resistance genes was dependent on vancomycin concentration whereas, in a ΔvanUG mutant, resistance was expressed at a maximum level even at low concentrations of the inducer. The binding competition between VanUG and VanRG on the PYG resistance promoter allowed rheostatic activation of the resistance operon depending likely on the level of VanRG phosphorylation by the VanSG sensor. In addition, there was cross-talk between VanSG and VanR'G, a VanRG homolog, encoded elsewhere in the chromosome indicating a sophisticated and subtle regulation of vancomycin resistance expression by a complex two-component system.

RNA-seq analysis identifies new genes regulated by the histone-like nucleoid structuring protein (H-NS) affecting Vibrio cholerae virulence, stress response and chemotaxis.

The histone-like nucleoid structuring protein (H-NS) functions as a transcriptional silencer by binding to AT-rich sequences at bacterial promoters. However, H-NS repression can be counteracted by other transcription factors in response to environmental changes. The identification of potential toxic factors, the expression of which is prevented by H-NS could facilitate the discovery of new regulatory proteins that may contribute to the emergence of new pathogenic variants by anti-silencing. Vibrio cholerae hns mutants of the El Tor biotype exhibit altered virulence, motility and environmental stress response phenotypes compared to wild type. We used an RNA-seq analysis approach to determine the basis of the above hns phenotypes and identify new targets of H-NS transcriptional silencing. H-NS affected the expression of 18% of all predicted genes in a growth phase-dependent manner. Loss of H-NS resulted in diminished expression of numerous genes encoding methyl-accepting chemotaxis proteins as well as chemotaxis toward the attractants glycine and serine. Deletion of hns also induced an endogenous envelope stress response resulting in elevated expression of rpoE encoding the extracytoplamic sigma factor E (σE). The RNA-seq analysis identified new genes directly repressed by H-NS that can affect virulence and biofilm development in the El Tor biotype cholera bacterium. We show that H-NS and the quorum sensing regulator HapR silence the transcription of the vieSAB three-component regulatory system in El Tor biotype V. cholerae. We also demonstrate that H-NS directly represses the transcription of hlyA (hemolysin), rtxCA (the repeat in toxin or RTX), rtxBDE (RTX transport) and the biosynthesis of indole. Of these genes, H-NS occupancy at the hlyA promoter was diminished by overexpression of the transcription activator HlyU. We discuss the role of H-NS transcriptional silencing in phenotypic differences exhibited by V. cholerae biotypes.

Gene cluster responsible for secretion of and immunity to multiple bacteriocins, the NKR-5-3 enterocins.

Enterococcus faecium NKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA [ent53A], enkC [ent53C], enkD [ent53D], and enkZ [ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIaz and enkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies of enkT and ΔenkT mutant strains showed that enkT is responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRK mutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter.

The Role of the Staphylococcal VraTSR Regulatory System on Vancomycin Resistance and vanA Operon Expression in Vancomycin-Resistant Staphylococcus aureus

Vancomycin is often the preferred treatment for invasive methicillin-resistant Staphylococcus aureus (MRSA) infection. With the increase in incidence of MRSA infections, the use of vancomycin has increased and, as feared, isolates of vancomycin-resistant Staphylococcus aureus (VRSA) have emerged. VRSA isolates have acquired the entercoccal vanA operon contained on transposon (Tn) 1546 residing on a conjugal plasmid. VraTSR is a vancomycin and β-lactam-inducible three-component regulatory system encoded on the S. aureus chromosome that modulates the cell-wall stress response to cell-wall acting antibiotics. Mutation in vraTSR has shown to increase susceptibility to β-lactams and vancomycin in clinical VISA strains and in recombinant strain COLVA-200 which expresses a plasmid borne vanA operon. To date, the role of VraTSR in vanA operon expression in VRSA has not been demonstrated. In this study, the vraTSR operon was deleted from the first clinical VRSA strain (VRS1) by transduction with phage harvested from a USA300 vraTSR operon deletion strain. The absence of the vraTSR operon and presence of the vanA operon were confirmed in the transductant (VRS1Δvra) by PCR. Broth MIC determinations, demonstrated that the vancomycin MIC of VRS1Δvra (64 µg/ml) decreased by 16-fold compared with VRS1 (1024 µg/ml). The effect of the vraTSR operon deletion on expression of the van gene cluster (vanA, vanX and vanR) was examined by quantitative RT-PCR using relative quantification. A 2–5-fold decreased expression of the vanA operon genes occured in strain VRS1Δvra at stationary growth phase compared with the parent strain, VRS1. Both vancomycin resistance and vancomycin-induced expression of vanA and vanR were restored by complementation with a plasmid harboring the vraTSR operon. These findings demonstrate that expression in S. aureus of the horizontally acquired enterococcal vanA gene cluster is enhanced by the staphylococcal three-component cell wall stress regulatory system VraTSR, that is present in all S. aureus strains.

Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids

ABSTRACTTreatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.IMPORTANCE The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.

A liaF Codon Deletion Abolishes Daptomycin Bactericidal Activity against Vancomycin-Resistant Enterococcus faecalis

The genetic bases for antibiotic tolerance are obscure. Daptomycin (DAP) is a lipopeptide antibiotic with bactericidal activity against enterococci. Using time-kill assays, we provide evidence for the first time that a deletion of isoleucine in position 177 of LiaF, a member of the three-component regulatory system LiaFSR involved in the cell envelope response to antimicrobials, is directly responsible for a DAP-tolerant phenotype and is likely to negatively affect response to DAP therapy.