scholarly journals Correct Sorting of Lipoproteins into the Inner and Outer Membranes ofPseudomonas aeruginosaby theEscherichia coliLolCDE Transport System

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Christian Lorenz ◽  
Thomas J. Dougherty ◽  
Stephen Lory
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Transport Systems ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Outer Membranes

ABSTRACTBiogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. InEnterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that inPseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those fromEscherichia coli. TheP. aeruginosalipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either theE. coliorP. aeruginosaLolCDE. We further demonstrate that an inhibitor ofE. coliLolCDE is active againstP. aeruginosaonly when expressing theE. coliorthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCEGram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways ofEscherichia coliandPseudomonas aeruginosaare interchangeable, with theE. coliorthologues correctly sorting theP. aeruginosalipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified inE. colias aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

scholarly journals YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Randi L. Guest ◽  
Daniel Samé Guerra ◽  
Maria Wissler ◽  
Jacqueline Grimm ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Essential Protein ◽  
Ftsh Protease ◽  
Acyl Chains ◽  
Lipid Balance

ABSTRACT Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

scholarly journals National Surveillance of Antimicrobial Susceptibility of Bacteremic Gram-Negative Bacteria with Emphasis on Community-Acquired Resistant Isolates: Report from the 2019 Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART)

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Po-Yu Liu ◽  
Yu-Lin Lee ◽  
Min-Chi Lu ◽  
Pei-Lan Shao ◽  
Po-Liang Lu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Gram Negative Bacteria ◽  
National Surveillance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Carbapenem Resistant

ABSTRACT A multicenter collection of bacteremic isolates of Escherichia coli (n = 423), Klebsiella pneumoniae (n = 372), Pseudomonas aeruginosa (n = 300), and Acinetobacter baumannii complex (n = 199) was analyzed for susceptibility. Xpert Carba-R assay and sequencing for mcr genes were performed for carbapenem- or colistin-resistant isolates. Nineteen (67.8%) carbapenem-resistant K. pneumoniae (n = 28) and one (20%) carbapenem-resistant E. coli (n = 5) isolate harbored blaKPC (n = 17), blaOXA-48 (n = 2), and blaVIM (n = 1) genes.

scholarly journals Structure of LetB reveals a tunnel for lipid transport across the bacterial envelope

2019 ◽  
Author(s):  
Georgia L. Isom ◽  
Nicolas Coudray ◽  
Mark R. MacRae ◽  
Collin T. McManus ◽  
Damian C. Ekiert ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid Transport ◽  
Transport Systems ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Outer Membranes ◽  
Hydrophobic Tunnel ◽  
Outer Membrane Permeability

Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide (LPS), which acts as a barrier to the environment and contributes to antibiotic resistance. While mechanisms of LPS transport have been well characterised, systems that translocate phospholipids across the periplasm, such as MCE (Mammalian Cell Entry) transport systems, are less well understood. Here we show that E. coli MCE protein LetB (formerly YebT), forms a ∼0.6 megadalton complex in the periplasm. Our cryo-EM structure reveals that LetB consists of a stack of seven modular rings, creating a long hydrophobic tunnel through the centre of the complex. LetB is sufficiently large to span the gap between the inner and outer membranes, and mutations that shorten the tunnel abolish function. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Together, our results support a model in which LetB establishes a physical link between the bacterial inner and outer membranes, and creates a hydrophobic pathway for the translocation of lipids across the periplasm, to maintain the integrity of the outer membrane permeability barrier.

scholarly journals Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Hannah M. Behrens ◽  
Edward D. Lowe ◽  
Joseph Gault ◽  
Nicholas G. Housden ◽  
Renata Kaminska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Animal Infection ◽  
Inner Membrane Protein ◽  
Biochemical Analyses ◽  
Functional Analyses

ABSTRACT Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria. IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting 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 Preservation of Acquired Colistin Resistance in Gram-Negative Bacteria

2015 ◽  
Vol 60 (1) ◽  
pp. 609-612 ◽  
Author(s):  
Ji-Young Lee ◽  
Myung-Jin Choi ◽  
Hyeon Jin Choi ◽  
Kwan Soo Ko
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Gram Negative Bacteria ◽  
Free Medium ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Resistant Mutants ◽  
The Stability

ABSTRACTColistin-resistant mutants were obtained from 17 colistin-susceptible strains ofAcinetobacter baumannii,Pseudomonas aeruginosa,Klebsiella pneumoniae, andEscherichia coli. The stability of colistin resistance in these mutants was investigated. Three of four colistin-resistantP. aeruginosamutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each ofA. baumanniiandE. coli. No susceptible revertants were obtained fromK. pneumoniaemutants.

scholarly journals Characterization of lptA and lptB, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of Escherichia coli

2006 ◽  
Vol 189 (1) ◽  
pp. 244-253 ◽  
Author(s):  
Paola Sperandeo ◽  
Rachele Cescutti ◽  
Riccardo Villa ◽  
Cristiano Di Benedetto ◽  
Daniela Candia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
De Novo ◽  
Essential Genes ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
E Coli ◽  
Σ Factor

ABSTRACT The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals Heterogeneous and Flexible Transmission ofmcr-1in Hospital-AssociatedEscherichia coli

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Yingbo Shen ◽  
Zuowei Wu ◽  
Yang Wang ◽  
Rong Zhang ◽  
Hong-Wei Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Analysis ◽  
Multidrug Resistant ◽  
Fluoroquinolone Resistance ◽  
Gram Negative Bacteria ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

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