scholarly journals Interplay ofKlebsiella pneumoniaefabZandlpxCMutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Mina Mostafavi ◽  
Lisha Wang ◽  
Lili Xie ◽  
Kenneth T. Takeoka ◽  
Daryl L. Richie ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Resistance Mutations ◽  
Cellular Mechanisms ◽  
Gram Negative ◽  
Content Type ◽  
Outer Leaflet ◽  
Chemical Inhibition ◽  
Dependent Growth

ABSTRACTTight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging ofKlebsiella pneumoniaein increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations infabZandlpxCoccurring together (encoding either FabZR121L/LpxCV37Gor FabZF51L/LpxCV37G).K. pneumoniaemutants having only LpxCV37Gor LpxCV37Aor various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37Gaccumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37Gindicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37Gwas overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning.IMPORTANCEEmergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation ofK. pneumoniaeresistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process inK. pneumoniaeand how it may be linked to novel biosynthetic pathway inhibitors.

scholarly journals Robust Suppression of Lipopolysaccharide Deficiency in Acinetobacter baumannii by Growth in Minimal Medium

2019 ◽  
Vol 201 (22) ◽  
Author(s):  
Emma Nagy ◽  
Richard Losick ◽  
Daniel Kahne
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Minimal Medium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Growth Defects ◽  
Outer Leaflet ◽  
Morphological Defects ◽  
Outer Membrane Biogenesis

ABSTRACT Lipopolysaccharide (LPS) is normally considered to be essential for viability in Gram-negative bacteria but can be removed in Acinetobacter baumannii. Mutant cells lacking this component of the outer membrane show growth and morphological defects. Here, we report that growth rates equivalent to the wild type can be achieved simply by propagation in minimal medium. The loss of LPS requires that cells rely on phospholipids for both leaflets of the outer membrane. We show that growth rate in the absence of LPS is not limited by nutrient availability but by the rate of outer membrane biogenesis. We hypothesize that because cells grow more slowly, outer membrane synthesis ceases to be rate limiting in minimal medium. IMPORTANCE Gram-negative bacteria are defined by their asymmetric outer membrane that consists of phospholipids on the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS is essential in all but a few Gram-negative species; the reason for this differential essentiality is not well understood. One species that can survive without LPS, Acinetobacter baumannii, shows characteristic growth and morphology phenotypes. We show that these phenotypes can be suppressed under conditions of slow growth and describe how LPS loss is connected to the growth defects. In addition to better defining the challenges A. baumannii cells face in the absence of LPS, we provide a new hypothesis that may explain the species-dependent conditional essentiality.

scholarly journals Characterization of an Acinetobacter baumanniilptDDeletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

2015 ◽  
Vol 198 (4) ◽  
pp. 731-741 ◽  
Author(s):  
Jade Bojkovic ◽  
Daryl L. Richie ◽  
David A. Six ◽  
Christopher M. Rath ◽  
William S. Sawyer ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bacterial Infections ◽  
Lipid A ◽  
Fatty Acid Biosynthesis ◽  
Cell Permeability ◽  
Growth Defect ◽  
Gram Negative ◽  
Content Type ◽  
Chemical Inhibition

ABSTRACTLipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. SomeAcinetobacter baumanniistrains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such aslpxC. Here, we characterized a mutant deleted forlptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lackinglptDhad a growth defect comparable to that of anlpxCdeletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage andN-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact oflptDdeletion than oflpxCdeletion on inner and outer membrane permeability, respectively. Targeted liquid chromatography-mass spectrometry (LCMS) analysis of LPS intermediates from UDP-3-O-R-3-hydroxylauroyl-N-acetyl-α-d-glucosamine through lipid IVAshowed that the loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulation or mislocalization caused by the blockage of later LPS pathway steps impacts envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes, including LpxC and FabB/F, in thelptDdeletion strain partially rescued growth and permeability defects.IMPORTANCENew antibiotics to treat Gram-negative bacterial infections are urgently needed. Inhibition of LPS biosynthesis is attractive because this would impact viability and cell permeability. Therefore, a better understanding of this pathway is important, especially in strains such asA. baumanniiATCC 19606, where LPS biosynthesis is not essentialin vitro. We show that ATCC 19606 also survives the loss of the final translocation of LPS into the OM (lptDdeletion). Intriguingly, this impaired cell envelope integrity more than the loss of LPS biosynthesis (lpxCdeletion), presumably due to the accumulation of toxic intermediates. Supporting this, chemical inhibition of LPS biosynthesis partially reversed this permeability defect. This extends our understanding of the LPS machinery and provides insights into potential interrelationships of the target steps along this important pathway.

scholarly journals A Bilayer-Couple Model of Bacterial Outer Membrane Vesicle Biogenesis

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Jeffrey W. Schertzer ◽  
Marvin Whiteley
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Curvature ◽  
Couple Model ◽  
Gram Negative Bacteria ◽  
Outer Membrane Vesicle ◽  
Gram Negative ◽  
Content Type ◽  
Outer Leaflet ◽  
Bilayer Couple

ABSTRACTGram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated. We recently demonstrated that the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) is required for OMV biogenesis inP. aeruginosa. We hypothesized that PQS stimulates OMV formation through direct interaction with the outer leaflet of the outer membrane. To test this hypothesis, we employed a red blood cell (RBC) model that has been used extensively to study small-molecule–membrane interactions. Our results revealed that addition of PQS to RBCs induced membrane curvature, resulting in the formation of membrane spicules (spikes), consistent with small molecules that are inserted stably into the outer leaflet of the membrane. Radiotracer experiments demonstrated that sufficient PQS was inserted into the membrane to account for this curvature and that curvature induction was specific to PQS structure. These data suggest that a low rate of interleaflet flip-flop forces PQS to accumulate in and expand the outer leaflet relative to the inner leaflet, thus inducing membrane curvature. In support of PQS-mediated outer leaflet expansion, the PQS effect was antagonized by chlorpromazine, a molecule known to be preferentially inserted into the inner leaflet. Based on these data, we propose a bilayer-couple model to describeP. aeruginosaOMV biogenesis and suggest that this is a general mechanism for bacterial OMV formation.IMPORTANCEDespite the ubiquity and importance of outer membrane vesicle (OMV) production in Gram-negative bacteria, the molecular details of OMV biogenesis are not fully understood. Early experiments showed that 2-heptyl-3-hydroxy-4-quinolone (PQS) induces OMV formation through physical interaction with the membrane but did not elucidate the mechanism. The present study demonstrates that PQS specifically and reversibly promotes blebbing of model membranes dependent upon the same properties that are required for OMV formation inP. aeruginosa. These results are consistent with a mechanism where expansion of the outer leaflet relative to the inner leaflet induces localized membrane curvature. This “bilayer-couple” model can account for OMV formation under all conditions and is easily generalized to other Gram-negative bacteria. The model therefore raises the possibility of a universal paradigm for vesicle production in prokaryotes with features strikingly different from what is known in eukaryotes.

scholarly journals Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Federica A. Falchi ◽  
Elisa A. Maccagni ◽  
Simone Puccio ◽  
Clelia Peano ◽  
Cristina De Castro ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Antibiotic Sensitivity ◽  
Major Constituent ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Increased Sensitivity

ABSTRACTIn Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction oflptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting thatlptA41affects LPS transport. Indeed,lptA41is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors oflptA41. One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion inmlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation inopgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCELipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective inlptAthat exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

scholarly journals Structure-guided enzymology of the lipid A acyltransferase LpxM reveals a dual activity mechanism

2016 ◽  
Vol 113 (41) ◽  
pp. E6064-E6071 ◽  
Author(s):  
Dustin Dovala ◽  
Christopher M. Rath ◽  
Qijun Hu ◽  
William S. Sawyer ◽  
Steven Shia ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Structural Information ◽  
Mechanistic Model ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Label Free ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Domains Of Life

Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and “reset” mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases’ structure–mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins.

scholarly journals Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Mary Kate Alexander ◽  
Anh Miu ◽  
Angela Oh ◽  
Mike Reichelt ◽  
Hoangdung Ho ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Drug Targets ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Transport Pathway ◽  
Gram Negative ◽  
Content Type ◽  
Outer Leaflet ◽  
New Antibiotics ◽  
Outer Membrane Biogenesis

ABSTRACTThere is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that killEscherichia colithrough inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

scholarly journals Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in Pseudomonas aeruginosa PAO1

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Angela M. DeLucia ◽  
David A. Six ◽  
Ruth E. Caughlan ◽  
Patricia Gee ◽  
Ian Hunt ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Lipid A ◽  
Inner Core ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Ms Analysis

ABSTRACTGram-negative outer membrane (OM) integrity is maintained in part by Mg2+cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria,waaP, encoding an inner-core kinase, could not be inactivated inPseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential inP. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), twol-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM.IMPORTANCEGram-negative bacteria have an outer membrane (OM) comprised of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The OM protects cells from toxic molecules and is important for survival during infection. The LPS core kinase genewaaPcan be deleted in several Gram-negative bacteria but not inPseudomonas aeruginosa. We used a controlled-expression system to deplete WaaP directly inP. aeruginosacells, which halted growth. WaaP depletion also caused gross changes in cell morphology and led to the accumulation of an aberrant LPS lacking several core sugars and all core phosphates. The aberrant LPS failed to reach the OM, suggesting that WaaP is essential inP. aeruginosabecause it is required to produce the full-length LPS that is recognized by the OM transport/assembly machinery in this organism. Therefore, WaaP may constitute a good target for the development of novel antipseudomonal agents.

scholarly journals PmrC (EptA) and CptA Negatively Affect Outer Membrane Vesicle Production inCitrobacter rodentium

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Anshul Sinha ◽  
Sammy Nyongesa ◽  
Charles Viau ◽  
Samantha Gruenheid ◽  
Frédéric J. Veyrier ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Membrane Vesicle ◽  
Enteric Bacteria ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Citrobacter Rodentium ◽  
Wild Type ◽  
Gram Negative ◽  
Content Type

ABSTRACTOuter membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-typeCitrobacter rodentium, the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline.C. rodentiumuses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, theC. rodentiumΔpmrABstrain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription ofpmrC(also known aseptA) andcptA. OMV production in strains lacking eitherpmrC(eptA) orcptAwas similarly increased in comparison to that of the wild type. Importantly, plasmid complementation ofC. rodentiumstrains with eitherpmrC(eptA) orcptAresulted in a drastic inhibition of OMV production. Finally, we showed that β-lactamase and CroP, two enzymes found in theC. rodentiumperiplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by whichC. rodentiumand possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genespmrC(eptA) andcptA.IMPORTANCEAlthough OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogenCitrobacter rodentium. In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron inC. rodentium. These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria.

scholarly journals Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics

2018 ◽  
Vol 115 (36) ◽  
pp. E8518-E8527 ◽  
Author(s):  
Matthew J. Powers ◽  
M. Stephen Trent
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Lipid A ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Two Factors ◽  
Evolution Experiment ◽  
Outer Membrane Permeability

The outer membrane of Gram-negative bacteria is a critical barrier that prevents entry of noxious compounds. Integral to this functionality is the presence of lipopolysaccharide (LPS) or lipooligosaccharide (LOS), a molecule that is located exclusively in the outer leaflet of the outer membrane. Its lipid anchor, lipid A, is a glycolipid whose hydrophobicity and net negative charge are primarily responsible for the robustness of the membrane. Because of this, lipid A is a hallmark of Gram-negative physiology and is generally essential for survival. Rare exceptions have been described, includingAcinetobacter baumannii, which can survive in the absence of lipid A, albeit with significant growth and membrane permeability defects. Here, we show by an evolution experiment that LOS-deficientA. baumanniican rapidly improve fitness over the course of only 120 generations. We identified two factors which negatively contribute to fitness in the absence of LOS, Mla and PldA. These proteins are involved in glycerophospholipid transport (Mla) and lipid degradation (PldA); both are active only on mislocalized, surface-exposed glycerophospholipids. Elimination of these two mechanisms was sufficient to cause a drastic fitness improvement in LOS-deficientA. baumannii. The LOS-deficient double mutant grows as robustly as LOS-positive wild-type bacteria while remaining resistant to the last-resort polymyxin antibiotics. These data provide strong biological evidence for the directionality of Mla-mediated glycerophospholipid transport in Gram-negative bacteria and furthers our knowledge of asymmetry-maintenance mechanisms in the context of the outer membrane barrier.

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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