scholarly journals LpxC Inhibitors as New Antibacterial Agents and Tools for Studying Regulation of Lipid A Biosynthesis in Gram-Negative Pathogens

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Andrew P. Tomaras ◽  
Craig J. McPherson ◽  
Michael Kuhn ◽  
Arlene Carifa ◽  
Lisa Mullins ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Lipid A ◽  
Resistance Mechanisms ◽  
Gram Negative ◽  
Content Type ◽  
Cellular Targets ◽  
Depth Analysis ◽  
Lipid A Biosynthesis

ABSTRACT The problem of multidrug resistance in serious Gram-negative bacterial pathogens has escalated so severely that new cellular targets and pathways need to be exploited to avoid many of the preexisting antibiotic resistance mechanisms that are rapidly disseminating to new strains. The discovery of small-molecule inhibitors of LpxC, the enzyme responsible for the first committed step in the biosynthesis of lipid A, represents a clinically unprecedented strategy to specifically act against Gram-negative organisms such as Pseudomonas aeruginosa and members of the Enterobacteriaceae. In this report, we describe the microbiological characterization of LpxC-4, a recently disclosed inhibitor of this bacterial target, and demonstrate that its spectrum of activity extends to several of the pathogenic species that are most threatening to human health today. We also show that spontaneous generation of LpxC-4 resistance occurs at frequencies comparable to those seen with marketed antibiotics, and we provide an in-depth analysis of the mechanisms of resistance utilized by target pathogens. Interestingly, these isolates also served as tools to further our understanding of the regulation of lipid A biosynthesis and enabled the discovery that this process occurs very distinctly between P. aeruginosa and members of the Enterobacteriaceae. Finally, we demonstrate that LpxC-4 is efficacious in vivo against multiple strains in different models of bacterial infection and that the major first-step resistance mechanisms employed by the intended target organisms can still be effectively treated with this new inhibitor. IMPORTANCE New antibiotics are needed for the effective treatment of serious infections caused by Gram-negative pathogens, and the responsibility of identifying new drug candidates rests squarely on the shoulders of the infectious disease community. The limited number of validated cellular targets and approaches, along with the increasing amount of antibiotic resistance that is spreading throughout the clinical environment, has prompted us to explore the utility of inhibitors of novel targets and pathways in these resistant organisms, since preexisting target-based resistance should be negligible. Lipid A biosynthesis is an essential process for the formation of lipopolysaccharide, which is a critical component of the Gram-negative outer membrane. In this report, we describe the in vitro and in vivo characterization of novel inhibitors of LpxC, an enzyme whose activity is required for proper lipid A biosynthesis, and demonstrate that our lead compound has the requisite attributes to warrant further consideration as a novel antibiotic.

scholarly journals Clinically Relevant Gram-Negative Resistance Mechanisms Have No Effect on the Efficacy of MC-1, a Novel Siderophore-Conjugated Monocarbam

2012 ◽  
Vol 56 (12) ◽  
pp. 6334-6342 ◽  
Author(s):  
Craig J. McPherson ◽  
Lisa M. Aschenbrenner ◽  
Brian M. Lacey ◽  
Kelly C. Fahnoe ◽  
Margaret M. Lemmon ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Negative Resistance ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Gram Negative ◽  
Content Type ◽  
Pharmacologically Active

ABSTRACTThe incidence of hospital-acquired infections with multidrug-resistant (MDR) Gram-negative pathogens is increasing at an alarming rate. Equally alarming is the overall lack of efficacious therapeutic options for clinicians, which is due primarily to the acquisition and development of various antibiotic resistance mechanisms that render these drugs ineffective. Among these mechanisms is the reduced permeability of the outer membrane, which prevents many marketed antibiotics from traversing this barrier. To circumvent this, recent drug discovery efforts have focused on conjugating a siderophore moiety to a pharmacologically active compound that has been designed to hijack the bacterial siderophore transport system and trick cells into importing the active drug by recognizing it as a nutritionally beneficial compound. MC-1, a novel siderophore-conjugated β-lactam that promotes its own uptake into bacteria, has exquisite activity against many Gram-negative pathogens. While the inclusion of the siderophore was originally designed to facilitate outer membrane penetration into Gram-negative cells, here we show that this structural moiety also renders other clinically relevant antibiotic resistance mechanisms unable to affect MC-1 efficacy. Resistance frequency determinations and subsequent characterization of first-step resistant mutants identified PiuA, a TonB-dependent outer membrane siderophore receptor, as the primary means of MC-1 entry intoPseudomonas aeruginosa. While the MICs of these mutants were increased 32-fold relative to the parental strainin vitro, we show that this resistance phenotype is not relevantin vivo, as alternative siderophore-mediated uptake mechanisms compensated for the loss of PiuA under iron-limiting conditions.

scholarly journals Reinforcing Lipid A Acylation on the Cell Surface of Acinetobacter baumannii Promotes Cationic Antimicrobial Peptide Resistance and Desiccation Survival

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Joseph M. Boll ◽  
Ashley T. Tucker ◽  
Dustin R. Klein ◽  
Alexander M. Beltran ◽  
Jennifer S. Brodbelt ◽  
...  
Keyword(s):  
Health Care ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Lipid A ◽  
Treatment Options ◽  
Cationic Antimicrobial Peptide ◽  
Gram Negative ◽  
Content Type ◽  
Hospital Environments ◽  
Lipid A Biosynthesis

ABSTRACTAcinetobacter baumanniiis an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments,A. baumanniiwithstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, includingA. baumannii, fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas inEscherichia coliandSalmonella, increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier,A. baumanniidoes not carry a gene that encodes a PagP homolog. Instead,A. baumanniihas evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adaptedA. baumanniigenetic recombineering system, we characterized two putative acyltransferases inA. baumanniidesignated LpxLAb(A. baumanniiLpxL) and LpxMAb(A. baumanniiLpxM), which transfer one and two lauroyl (C12:0) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation ofA. baumanniilipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxMAb-dependent acylation of lipid A is essential forA. baumanniidesiccation survival, a key resistance mechanism for survival in hospital environments. Compounds that inhibit LpxMAb-dependent hepta-acylation of lipid A could act synergistically with CAMPs to provide innovative transmission prevention strategies and treat multidrug-resistant infections.IMPORTANCEAcinetobacter baumanniiinfections can be life threatening, and disease can progress in a variety of host tissues. Current antibiotic regimen and disinfectant strategies have failed to limit nosocomialA. baumanniiinfections. Instead, the rate ofA. baumanniiinfection among health care communities has skyrocketed due to the bacterium's adaptability. Its aptitude for survival over extended periods on inanimate objects, such as catheters, respirators, and surfaces in intensive care units, or on the hands of health care workers and its ability to rapidly develop antibiotic resistance makeA. baumanniia threat to health care communities. Emergence of multidrug- and extremely drug-resistantA. baumanniiillustrates the ineffectiveness of current prevention and treatment options. Our analysis to understand howA. baumanniiresists cationic antimicrobial peptide (CAMP)-mediated and desiccative killing revealed two lipid A acyltransferases that produce protective hepta-acylated lipid A. Our work suggests that inhibiting lipid A biosynthesis by targeting the acyltransferase LpxMAb(A. baumanniiLpxM) could provide a novel target to combat this pathogen.

scholarly journals Update on Antimicrobial Resistance in Clostridium difficile: Resistance Mechanisms and Antimicrobial Susceptibility Testing

2017 ◽  
Vol 55 (7) ◽  
pp. 1998-2008 ◽  
Author(s):  
Zhong Peng ◽  
Dazhi Jin ◽  
Hyeun Bum Kim ◽  
Charles W. Stratton ◽  
Bin Wu ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Clostridium Difficile ◽  
Gene Mutations ◽  
Antibiotic Resistance Genes ◽  
Resistance Mechanisms ◽  
Test Methods ◽  
Genotypic Resistance ◽  
Altered Expression ◽  
Content Type

ABSTRACT Oral antibiotics such as metronidazole, vancomycin and fidaxomicin are therapies of choice for Clostridium difficile infection. Several important mechanisms for C. difficile antibiotic resistance have been described, including the acquisition of antibiotic resistance genes via the transfer of mobile genetic elements, selective pressure in vivo resulting in gene mutations, altered expression of redox-active proteins, iron metabolism, and DNA repair, as well as via biofilm formation. This update summarizes new information published since 2010 on phenotypic and genotypic resistance mechanisms in C. difficile and addresses susceptibility test methods and other strategies to counter antibiotic resistance of C. difficile .

scholarly journals Molecular and Clinical Characterization of Multidrug-Resistant and Hypervirulent Klebsiella pneumoniae Strains from Liver Abscess in Taiwan

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Yi-Tsung Lin ◽  
Yi-Hsiang Cheng ◽  
Chien Chuang ◽  
Sheng-Hua Chou ◽  
Wan-Hsin Liu ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Liver Abscess ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Virulence Plasmid ◽  
Content Type ◽  
Sequence Types

ABSTRACT Hypervirulent Klebsiella pneumoniae strains are the major cause of liver abscesses throughout East Asia, and these strains are usually antibiotic susceptible. Recently, multidrug-resistant and hypervirulent (MDR-HV) K. pneumoniae strains have emerged due to hypervirulent strains acquiring antimicrobial resistance determinants or the transfer of a virulence plasmid into a classic MDR strain. In this study, we characterized the clinical and microbiological properties of K. pneumoniae liver abscess (KPLA) caused by MDR-HV strains in Taiwan. Patients with community onset KPLA were retrospectively identified at Taipei Veterans General Hospital during January 2013 to May 2018. Antimicrobial resistance mechanisms, capsular types, and sequence types were determined. MDR-HV strains and their parental antimicrobial-susceptible strains further underwent whole-genome sequencing (WGS) and in vivo mice lethality tests. Thirteen MDR-HV strains were identified from a total of 218 KPLA episodes. MDR-HV strains resulted in similar outcomes to antimicrobial-susceptible strains. All MDR-HV strains were traditional hypervirulent clones carrying virulence capsular types. The major resistance mechanisms were the overexpression of efflux pumps and/or the acquisition of ESBL or AmpC β-lactamase genes. WGS revealed that two hypervirulent strains had evolved to an MDR phenotype due to mutation in the ramR gene and the acquisition of an SHV-12-bearing plasmid, respectively. Both these MDR-HV strains retained high virulence compared to their parental strains. The spread of MDR-HV K. pneumoniae strains in the community raises significant public concerns, and measures should be taken to prevent the further acquisition of carbapenemase and other resistance genes among these strains in order to avoid the occurrence of untreatable KPLA.

scholarly journals LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Jun-Rong Wei ◽  
Daryl L. Richie ◽  
Mina Mostafavi ◽  
Louis E. Metzger ◽  
Christopher M. Rath ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cell Growth ◽  
Acinetobacter Baumannii ◽  
Biosynthetic Pathway ◽  
Lipid A ◽  
Fatty Acid Biosynthesis ◽  
Gram Negative ◽  
Content Type ◽  
Lipid A Biosynthesis ◽  
Chemical Inhibition

ABSTRACT Acinetobacter baumannii is a Gram-negative pathogen for which new therapies are needed. The lipid A biosynthetic pathway has several potential enzyme targets for the development of anti-Gram-negative agents (e.g., LpxC). However, A. baumannii ATCC 19606 can grow in the absence of LpxC and, correspondingly, of lipid A. In contrast, we show that cellular depletion of LpxK, a kinase occurring later in the pathway, inhibits growth. Growth inhibition results from toxic accumulation of lipid A pathway intermediates, since chemical inhibition of LpxC or fatty acid biosynthesis rescues cell growth upon loss of LpxK. Overall, this suggests that targets such as LpxK can be essential for growth even in those Gram-negative bacteria that do not require lipid A biosynthesis per se. This strain provides an elegant tool to derive a better understanding of the steps in a pathway that is the focus of intense interest for the development of novel antibacterials. Acinetobacter baumannii ATCC 19606 can grow without lipid A, the major component of lipooligosaccharide. However, we previously reported that depletion of LpxH (the fourth enzyme in the lipid A biosynthetic pathway) prevented growth of this strain due to toxic accumulation of lipid A pathway intermediates. Here, we explored whether a similar phenomenon occurred with depletion of LpxK, a kinase that phosphorylates disaccharide 1-monophosphate (DSMP) at the 4′ position to yield lipid IVA. An A. baumannii ATCC 19606 derivative with LpxK expression under the control of an isopropyl β-d-1-thiogalactopyranoside (IPTG)-regulated expression system failed to grow without induction, indicating that LpxK is essential for growth. Light and electron microscopy of LpxK-depleted cells revealed morphological changes relating to the cell envelope, consistent with toxic accumulation of lipid A pathway intermediates disrupting cell membranes. Using liquid chromatography-mass spectrometry (LCMS), cellular accumulation of the detergent-like pathway intermediates DSMP and lipid X was shown. Toxic accumulation was further supported by restoration of growth upon chemical inhibition of LpxC (upstream of LpxK and the first committed step of lipid A biosynthesis) using CHIR-090. Inhibitors of fatty acid synthesis also abrogated the requirement for LpxK expression. Growth rescue with these inhibitors was possible on Mueller-Hinton agar but not on MacConkey agar. The latter contains outer membrane-impermeable bile salts, suggesting that despite growth restoration, the cell membrane permeability barrier was not restored. Therefore, LpxK is essential for growth of A. baumannii, since loss of LpxK causes accumulation of detergent-like pathway intermediates that inhibit cell growth. IMPORTANCE Acinetobacter baumannii is a Gram-negative pathogen for which new therapies are needed. The lipid A biosynthetic pathway has several potential enzyme targets for the development of anti-Gram-negative agents (e.g., LpxC). However, A. baumannii ATCC 19606 can grow in the absence of LpxC and, correspondingly, of lipid A. In contrast, we show that cellular depletion of LpxK, a kinase occurring later in the pathway, inhibits growth. Growth inhibition results from toxic accumulation of lipid A pathway intermediates, since chemical inhibition of LpxC or fatty acid biosynthesis rescues cell growth upon loss of LpxK. Overall, this suggests that targets such as LpxK can be essential for growth even in those Gram-negative bacteria that do not require lipid A biosynthesis per se. This strain provides an elegant tool to derive a better understanding of the steps in a pathway that is the focus of intense interest for the development of novel antibacterials.

scholarly journals Genetic Determinants Enabling Medium-Dependent Adaptation to Nafcillin in Methicillin-Resistant Staphylococcus aureus

mSystems ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Michael J. Salazar ◽  
Henrique Machado ◽  
Nicholas A. Dillon ◽  
Hannah Tsunemoto ◽  
Richard Szubin ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Resistance Mechanisms ◽  
Evolutionary Strategy ◽  
Continuous Exposure ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACT Antimicrobial susceptibility testing standards driving clinical decision-making have centered around the use of cation-adjusted Mueller-Hinton broth (CA-MHB) as the medium with the notion of supporting bacterial growth, without consideration of recapitulating the in vivo environment. However, it is increasingly recognized that various medium conditions have tremendous influence on antimicrobial activity, which in turn may have major implications on the ability of in vitro susceptibility assays to predict antibiotic activity in vivo. To elucidate differential growth optimization and antibiotic resistance mechanisms, adaptive laboratory evolution was performed in the presence or absence of the antibiotic nafcillin with methicillin-resistant Staphylococcus aureus (MRSA) TCH1516 in either (i) CA-MHB, a traditional bacteriological nutritionally rich medium, or (ii) Roswell Park Memorial Institute (RPMI), a medium more reflective of the in vivo host environment. Medium adaptation analysis showed an increase in growth rate in RPMI, but not CA-MHB, with mutations in apt, adenine phosphoribosyltransferase, and the manganese transporter subunit, mntA, occurring reproducibly in parallel replicate evolutions. The medium-adapted strains showed no virulence attenuation. Continuous exposure of medium-adapted strains to increasing concentrations of nafcillin led to medium-specific evolutionary strategies. Key reproducibly occurring mutations were specific for nafcillin adaptation in each medium type and did not confer resistance in the other medium environment. Only the vraRST operon, a regulator of membrane- and cell wall-related genes, showed mutations in both CA-MHB- and RPMI-evolved strains. Collectively, these results demonstrate the medium-specific genetic adaptive responses of MRSA and establish adaptive laboratory evolution as a platform to study clinically relevant resistance mechanisms. IMPORTANCE The ability of pathogens such as Staphylococcus aureus to evolve resistance to antibiotics used in the treatment of infections has been an important concern in the last decades. Resistant acquisition usually translates into treatment failure and puts patients at risk of unfavorable outcomes. Furthermore, the laboratory testing of antibiotic resistance does not account for the different environment the bacteria experiences within the human body, leading to results that do not translate into the clinic. In this study, we forced methicillin-resistant S. aureus to develop nafcillin resistance in two different environments, a laboratory environment and a physiologically more relevant environment. This allowed us to identify genetic changes that led to nafcillin resistance under both conditions. We concluded that not only does the environment dictate the evolutionary strategy of S. aureus to nafcillin but also that the evolutionary strategy is specific to that given environment.

scholarly journals Susceptibility of Colistin-Resistant, Gram-Negative Bacteria to Antimicrobial Peptides and Ceragenins

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Marjan M. Hashemi ◽  
John Rovig ◽  
Scott Weber ◽  
Brian Hilton ◽  
Mehdi M. Forouzan ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Antimicrobial Peptides ◽  
Lipid A ◽  
Resistance Mechanisms ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type

ABSTRACT The susceptibility of colistin-resistant clinical isolates of Klebsiella pneumoniae to ceragenins and antimicrobial peptides (AMPs) suggests that there is little to no cross-resistance between colistin and ceragenins/AMPs and that lipid A modifications are found in bacteria with modest changes in susceptibility to ceragenins and with high levels of resistance to colistin. These results suggest that there are differences in the resistance mechanisms to colistin and ceragenins/AMPs.

scholarly journals β-Lactamase Characterization of Gram-Negative Pathogens Recovered from Patients Enrolled in the Phase 2 Trials for Ceftazidime-Avibactam: Clinical Efficacies Analyzed against Subsets of Molecularly Characterized Isolates

2015 ◽  
Vol 60 (3) ◽  
pp. 1328-1335 ◽  
Author(s):  
Rodrigo E. Mendes ◽  
Mariana Castanheira ◽  
Leanne Gasink ◽  
Gregory G. Stone ◽  
Wright W. Nichols ◽  
...  
Keyword(s):  
Complicated Urinary Tract Infection ◽  
Resistance Mechanisms ◽  
Phase 2 ◽  
Screening Criteria ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Baseline Visit ◽  
Intra Abdominal Infection

The correlation of the clinical efficacies of ceftazidime-avibactam and comparators (carbapenems) was evaluated against baseline Gram-negative isolates having characterized β-lactam resistance mechanisms from complicated urinary tract infection (cUTI) and complicated intra-abdominal infection (cIAI) phase 2 trials.Enterobacteriaceaedisplaying ceftriaxone and/or ceftazidime MICs of ≥2 μg/ml (69 isolates) and nonfermentative Gram-negative bacilli (NF-GNB [three isolates]) with ceftazidime MICs of ≥16 μg/ml were characterized for their narrow- and extended-spectrum β-lactamase (ESBL) content.Enterobacteriaceae(one isolate) and NF-GNB (three isolates) with imipenem/meropenem MICs of ≥2 and ≥16 μg/ml, respectively, were tested for carbapenemases. All cUTIE. colihad the lineage background investigated (ST131-like versus non-ST131-like). The primary efficacy endpoint was microbiological response (eradication) at test of cure (TOC) for cUTI and clinical response (inferred microbiological eradication) at TOC for cIAI. A total of 34.1% of baseline cUTI (36.4%) and cIAI (33.1%) pathogens met the MIC-based screening criteria (screen positive). All screen-positive cUTI pathogens were CTX-M-producingE. coli, except for oneE. cloacaeisolate with AmpC overexpression. The majority (66.7%) of screen-positive cIAI isolates produced CTX-M-type coupled with a diverse array of other β-lactamases. Similar favorable responses were observed with ceftazidime-avibactam (93.3%) and carbapenems (90.9%), when a non-ESBLEnterobacteriaceaeisolate was recovered at the baseline visit. When an ESBLEnterobacteriaceaeisolate was present, the favorable responses were 85.7% and 80.0% with ceftazidime-avibactam and carbapenems, respectively. Higher favorable responses were observed with ceftazidime-avibactam (75.0%) than with carbapenems (66.7%) when an ST131-likeE. coliisolate was recovered at baseline, as when a non-ST131-like isolate was present (93.8% versus 86.7%, respectively). The efficacy of ceftazidime-avibactam was similar to that of carbapenems for treatment of cUTI and cIAI caused by ESBL organisms.

scholarly journals Biofilms Formed by Gram-Negative Bacteria Undergo Increased Lipid A Palmitoylation, Enhancing In Vivo Survival

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Sabina Chalabaev ◽  
Ashwini Chauhan ◽  
Alexey Novikov ◽  
Pavithra Iyer ◽  
Magdalena Szczesny ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Lipid A ◽  
Acyl Chain ◽  
Bacterial Biofilm ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Planktonic Bacteria ◽  
Biofilm Bacteria

ABSTRACTBacterial biofilm communities are associated with profound physiological changes that lead to novel properties compared to the properties of individual (planktonic) bacteria. The study of biofilm-associated phenotypes is an essential step toward control of deleterious effects of pathogenic biofilms. Here we investigated lipopolysaccharide (LPS) structural modifications in Escherichia coli biofilm bacteria, and we showed that all tested commensal and pathogenic E. coli biofilm bacteria display LPS modifications corresponding to an increased level of incorporation of palmitate acyl chain (palmitoylation) into lipid A compared to planktonic bacteria. Genetic analysis showed that lipid A palmitoylation in biofilms is mediated by the PagP enzyme, which is regulated by the histone-like protein repressor H-NS and the SlyA regulator. While lipid A palmitoylation does not influence bacterial adhesion, it weakens inflammatory response and enhances resistance to some antimicrobial peptides. Moreover, we showed that lipid A palmitoylation increasesin vivosurvival of biofilm bacteria in a clinically relevant model of catheter infection, potentially contributing to biofilm tolerance to host immune defenses. The widespread occurrence of increased lipid A palmitoylation in biofilms formed by all tested bacteria suggests that it constitutes a new biofilm-associated phenotype in Gram-negative bacteria.IMPORTANCEBacterial communities called biofilms display characteristic properties compared to isolated (planktonic) bacteria, suggesting that some molecules could be more particularly produced under biofilm conditions. We investigated biofilm-associated modifications occurring in the lipopolysaccharide (LPS), a major component of all Gram-negative bacterial outer membrane. We showed that all tested commensal and pathogenic biofilm bacteria display high incorporation of a palmitate acyl chain into the lipid A part of LPS. This lipid A palmitoylation is mediated by the PagP enzyme, whose expression in biofilm is controlled by the regulatory proteins H-NS and SlyA. We also showed that lipid A palmitoylation in biofilm bacteria reduces host inflammatory response and enhances their survival in an animal model of biofilm infections. While these results provide new insights into the biofilm lifestyle, they also suggest that the level of lipid A palmitoylation could be used as an indicator to monitor the development of biofilm infections on medical surfaces.

scholarly journals The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Jinshi Zhao ◽  
Jinsu An ◽  
Dohyeon Hwang ◽  
Qinglin Wu ◽  
Su Wang ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Lipid A ◽  
Host Immune System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Phosphatase Activities ◽  
O Antigen

ABSTRACT Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C55-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C55-PP, and rescues Escherichia coli deficient in PGP and C55-PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C55-PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C55-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope. IMPORTANCE Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.

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