scholarly journals An “Uncommon” Role for Cyclic Enterobacterial Common Antigen in Maintaining Outer Membrane Integrity

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Corey S. Westfall
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Membrane Integrity ◽  
Physiological Role ◽  
Permeability Barrier ◽  
Common Antigen ◽  
Enterobacterial Common Antigen ◽  
Inner Membrane Protein ◽  
Outer Membrane Permeability

ABSTRACTAlthough discovered over 50 years ago, the physiological role of enterobacterial common antigen, a surface antigen produced by all members of theEnterobacteriaceae, has been poorly understood. In the work of Mitchell et al. (mBio 9:e01321-18, 2018,https://doi.org/10.1128/mBio.01321-18), the cyclized version of enterobacterial common antigen has been shown to play a role in maintaining the outer membrane permeability barrier, possibly through the inner membrane protein YhdP. This work also provides the tests needed to separate true effects from the numerous possible artifacts possible with mutations in enterobacterial common antigen synthesis.

scholarly journals Cyclic Enterobacterial Common Antigen Maintains the Outer Membrane Permeability Barrier ofEscherichia coliin a Manner Controlled by YhdP

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Angela M. Mitchell ◽  
Tharan Srikumar ◽  
Thomas J. Silhavy
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Unknown Function ◽  
Permeability Barrier ◽  
Dependent Manner ◽  
Common Antigen ◽  
Gram Negative ◽  
Enterobacterial Common Antigen ◽  
K 12 ◽  
Outer Membrane Permeability

ABSTRACTGram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. InEnterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughoutEnterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes inEscherichia coliK-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these,yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants ofyhdPwith loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability fromyhdPdeletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore,yhdPdeletion changed cellular ECA levels andyhdPwas found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability withyhdPdeletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found withyhdPdeletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP.IMPORTANCEEnterobacterial common antigen (ECA) is a surface antigen made by all members ofEnterobacteriaceae, including many clinically relevant genera (e.g.,Escherichia,Klebsiella,Yersinia). Although this surface-exposed molecule is conserved throughoutEnterobacteriaceae, very few functions have been ascribed to it. Here, we have determined that the periplasmic form of ECA, cyclic ECA, plays a role in maintaining the outer membrane permeability barrier. This activity is controlled by a protein of unknown function, YhdP, and deletion ofyhdPdamages the OM permeability barrier in a cyclic ECA-dependent manner, allowing harmful molecules such as antibiotics into the cell. This role in maintenance of the envelope permeability barrier is the first time a phenotype has been described for cyclic ECA. As the Gram-negative envelope is generally impermeable to antibiotics, understanding the mechanisms through which the barrier is maintained and antibiotics are excluded may lead to improved antibiotic delivery.

scholarly journals Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli

2015 ◽  
Vol 197 (15) ◽  
pp. 2479-2488 ◽  
Author(s):  
Rajeev Misra ◽  
Keith D. Morrison ◽  
Hyun Jae Cho ◽  
Thanh Khuu
Keyword(s):  
Escherichia Coli ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Real Time ◽  
Efflux Pump ◽  
Membrane Integrity ◽  
Efflux Pumps ◽  
Permeability Barrier ◽  
Multidrug Efflux ◽  
Outer Membrane Permeability

ABSTRACTThe constitutively expressed AcrAB multidrug efflux system ofEscherichia colishows a high degree of homology with the normally silent AcrEF system. Exposure of a strain withacrABdeleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN.IMPORTANCEThe increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds.

scholarly journals ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen

mBio ◽  
2021 ◽  
Author(s):  
Ashutosh K. Rai ◽  
Joseph F. Carr ◽  
David E. Bautista ◽  
Wei Wang ◽  
Angela M. Mitchell
Keyword(s):  
Klebsiella Pneumoniae ◽  
Yersinia Pestis ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Pathogenic Bacteria ◽  
Permeability Barrier ◽  
Common Antigen ◽  
Content Type ◽  
Enterobacterial Common Antigen ◽  
The Many

Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales , including Klebsiella pneumoniae , Salmonella enterica , and Yersinia pestis . As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery.

scholarly journals Overcoming Iron Deficiency of anEscherichia colitonBMutant by Increasing Outer Membrane Permeability

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Nan Qiu ◽  
Rajeev Misra
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Membrane Integrity ◽  
Receptor Protein ◽  
Permeability Barrier ◽  
Prolonged Incubation ◽  
Content Type ◽  
E Coli ◽  
Outer Membrane Permeability

ABSTRACTThe intake of certain nutrients, including ferric ion, is facilitated by the outer membrane-localized transporters. Due to ferric insolubility at physiological pH,Escherichia colisecretes a chelator, enterobactin, outside the cell and then transports back the enterobactin-ferric complex via an outer membrane receptor protein, FepA, whose activity is dependent on the proton motive force energy transduced by the TonB-ExbBD complex of the inner membrane. Consequently, ΔtonBmutant cells grow poorly on a medium low in iron. Prolonged incubation of ΔtonBcells on low-iron medium yields faster-growing colonies that acquired suppressor mutations in theyejM(pbgA) gene, which codes for a putative inner-to-outer membrane cardiolipin transporter. Further characterization of suppressors revealed that they display hypersusceptibility to vancomycin, a large hydrophilic antibiotic normally precluded from enteringE. colicells, and leak periplasmic proteins into the culture supernatant, indicating a compromised outer membrane permeability barrier. All phenotypes were reversed by supplying the wild-type copy ofyejMon a plasmid, suggesting thatyejMmutations are solely responsible for the observed phenotypes. The deletion of all known cardiolipin synthase genes (clsABC) did not produce the phenotypes similar to mutations in theyejMgene, suggesting that the absence of cardiolipin from the outer membraneper seis not responsible for increased outer membrane permeability. Elevated lysophosphatidylethanolamine levels and the synthetic growth phenotype withoutpldAindicated that defective lipid homeostasis in theyejMmutant compromises outer membrane lipid asymmetry and permeability barrier to allow enterobactin intake, and that YejM has additional roles other than transporting cardiolipin.IMPORTANCEThe work presented here describes a positive genetic selection strategy for isolating mutations that destabilize the outer membrane permeability barrier ofE. coli. Given the importance of the outer membrane in restricting the entry of antibiotics, characterization of the genes and their products that affect outer membrane integrity will enhance the understanding of bacterial membranes and the development of strategies to bypass the outer membrane barrier for improved drug efficacy.

scholarly journals Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 571-582 ◽  
Author(s):  
Amal O Amer ◽  
Miguel A Valvano
Keyword(s):  
Membrane Protein ◽  
Aspartic Acid ◽  
Integral Membrane Protein ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Common Antigen ◽  
Phosphodiester Bond ◽  
Enterobacterial Common Antigen ◽  
Inner Membrane Protein ◽  
Cytoplasmic Loops

The integral membrane protein WecA mediates the transfer of N-acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner-membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme.

scholarly journals Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity

2002 ◽  
Vol 184 (3) ◽  
pp. 754-759 ◽  
Author(s):  
Eric Cascales ◽  
Alain Bernadac ◽  
Marthe Gavioli ◽  
Jean-Claude Lazzaroni ◽  
Roland Lloubes
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Permeability Barrier ◽  
Oligomeric State ◽  
Periplasmic Proteins ◽  
Outer Membrane Permeability

ABSTRACT The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.

scholarly journals Role of Porins for Uptake of Antibiotics by Mycobacterium smegmatis

2008 ◽  
Vol 52 (9) ◽  
pp. 3127-3134 ◽  
Author(s):  
Olga Danilchanka ◽  
Mikhail Pavlenok ◽  
Michael Niederweis
Keyword(s):  
Outer Membrane ◽  
Mycobacterium Smegmatis ◽  
Lipid Membrane ◽  
Model Organism ◽  
Permeability Barrier ◽  
Opportunistic Pathogens ◽  
Transport Pathways ◽  
Antibiotics Use ◽  
Outer Membrane Permeability

ABSTRACT The outer membrane of mycobacteria presents an effective permeability barrier for many antibiotics. Transport pathways across this membrane are unknown for most drugs. Here, we examined which antibiotics utilize the porin pathway across the outer membrane of the model organism Mycobacterium smegmatis. Deletion of the porins MspA and MspC drastically increased the resistance of M. smegmatis ML10 to β-lactam antibiotics, while its β-lactamase activity remained unchanged. These results are consistent with the ninefold-reduced outer membrane permeability of the M. smegmatis porin mutants for cephaloridine and strongly indicate that β-lactam antibiotics rely on the porin pathway. The porin mutant ML10 accumulated less chloramphenicol and norfloxacin and was less susceptible to these antibiotics than wild-type M. smegmatis. These results demonstrated that small and hydrophilic antibiotics use the Msp porins for entering the cell. In contrast to norfloxacin, the hydrophobic moxifloxacin was 32-fold more effective in inhibiting the growth of M. smegmatis, presumably because it was able to diffuse through the lipid membrane. Structural models indicated that erythromycin, kanamycin, and vancomycin are too large to move through the MspA channel. This study presents the first experimental evidence that hydrophilic fluoroquinolones and chloramphenicol diffuse through porins in mycobacteria. Thus, mutations resulting in less efficient porins or lower porin expression levels are likely to represent a mechanism for the opportunistic pathogens M. avium, M. chelonae, and M. fortuitum, which have Msp-like porins, to acquire resistance to fluoroquinolones.

scholarly journals Resuscitation-Promoting Factors Are Required for β-Lactam Tolerance and the Permeability Barrier in Mycobacterium tuberculosis

2011 ◽  
Vol 56 (3) ◽  
pp. 1591-1594 ◽  
Author(s):  
Carl N. Wivagg ◽  
Deborah T. Hung
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Outer Membrane ◽  
Membrane Integrity ◽  
Permeability Barrier ◽  
Content Type ◽  
Enhanced Sensitivity ◽  
Resuscitation Promoting Factors ◽  
Chemical Screen ◽  
Outer Membrane Permeability ◽  
Precise Role

ABSTRACTMycobacterial resuscitation-promoting factors (RPFs) have been of great interest since the discovery that they promote the growth of nonculturableMycobacterium tuberculosiscells. Yet, their precise role in mycobacterial survival and infection has remained elusive. We performed a chemical screen to identify molecules that show preferential killing of aMycobacterium tuberculosismutant lacking RPFs over wild-type bacilli and found that the mutant has enhanced sensitivity to the β-lactam class of antibiotics. By monitoring β-lactam diffusion across the mycobacterial outer membrane, we found that the RPFs are required to maintain the outer membrane integrity, as their deletion results in an increase in outer membrane permeability.

scholarly journals Lipopolysaccharide-Linked Enterobacterial Common Antigen (ECALPS) Occurs in Rough Strains of Escherichia coli R1, R2, and R4

2020 ◽  
Vol 21 (17) ◽  
pp. 6038
Author(s):  
Anna Maciejewska ◽  
Marta Kaszowska ◽  
Wojciech Jachymek ◽  
Czeslaw Lugowski ◽  
Jolanta Lukasiewicz
Keyword(s):  
Escherichia Coli ◽  
Surface Antigen ◽  
Structural Data ◽  
Outer Core ◽  
Permeability Barrier ◽  
Common Antigen ◽  
Enterobacterial Common Antigen ◽  
Specific Polysaccharide ◽  
K 12 ◽  
Outer Membrane Permeability

Enterobacterial common antigen (ECA) is a conserved surface antigen characteristic for Enterobacteriaceae. It is consisting of trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-β-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where prevailing forms include ECA linked to phosphatidylglycerol (ECAPG) and cyclic ECA (ECACYC). Lipopolysaccharide (LPS)-associated form (ECALPS) has been proved to date only for rough Shigella sonnei phase II. Depending on the structure organization, ECA constitutes surface antigen (ECAPG and ECALPS) or maintains the outer membrane permeability barrier (ECACYC). The existence of LPS was hypothesized in the 1960–80s on the basis of serological observations. Only a few Escherichia coli strains (i.e., R1, R2, R3, R4, and K-12) have led to the generation of anti-ECA antibodies upon immunization, excluding ECAPG as an immunogen and conjecturing ECALPS as the only immunogenic form. Here, we presented a structural survey of ECALPS in E. coli R1, R2, R3, and R4 to correlate previous serological observations with the presence of ECALPS. The low yields of ECALPS were identified in the R1, R2, and R4 strains, where ECA occupied outer core residues of LPS that used to be substituted by O-specific polysaccharide in the case of smooth LPS. Previously published observations and hypotheses regarding the immunogenicity and biosynthesis of ECALPS were discussed and correlated with presented herein structural data.

scholarly journals Molecular Basis of Bacterial Outer Membrane Permeability Revisited

2003 ◽  
Vol 67 (4) ◽  
pp. 593-656 ◽  
Author(s):  
Hiroshi Nikaido
Keyword(s):  
Outer Membrane ◽  
Pathogenic Bacteria ◽  
Structural Knowledge ◽  
Permeability Barrier ◽  
Lipophilic Compounds ◽  
Bacterial Outer Membrane ◽  
Membrane Components ◽  
Outer Membrane Permeability ◽  
Protein Channels

SUMMARY Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

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