scholarly journals Comparison of serological specificity of anti-endotoxin sera directed against whole bacterial cells and core oligosaccharide of Escherichia coli J5-tetanus toxoid conjugate.

2002 ◽  
Vol 49 (3) ◽  
pp. 721-734 ◽  
Author(s):  
Jolanta Lukasiewicz ◽  
Wojciech Jachymek ◽  
Tomasz Niedziela ◽  
Malgorzta Malik-Gebicka ◽  
Monika Dzieciatkowska ◽  
...  
Keyword(s):  
Tetanus Toxoid ◽  
Surface Antigens ◽  
Immune Serum ◽  
Bacterial Cells ◽  
Core Oligosaccharide ◽  
Cell Surface Antigens ◽  
Serological Tests ◽  
Gram Negative ◽  
Bacterial Surface ◽  
E Coli

The rough mutants of Gram-negative bacteria are widely used to induce protective antisera but the nature of the target epitope for such antibodies is not precisely defined. Endotoxin is one of several antigens present on the surface of bacterial cells, which are able to elicit specific antibodies. We studied the specificity of antibodies produced against a conjugate of E. coli J5 endotoxin core oligosaccharide with tetanus toxoid. The use of chemically defined antigen for immunisation excludes the possibility of production of antibodies against other cell surface antigens. A comparison of this monospecific anti-endotoxin serum with antiserum against E. coli J5 whole cells was performed in order to distinguish the role that endotoxin core oligosaccharide plays in the interaction with humoral host defences from that of other potentially important Gram-negative bacterial surface antigens. The reactivity of both sera with smooth and rough lipopolysaccharides was determined in ELISA, immunoblotting and by flow cytometry. Both antisera reacted with similar specificity with most lipopolysaccharides of identical or related core type. Less distinct reactions with endotoxins of the antibacterial serum in comparison with the anti-conjugate serum were found in all serological tests. LPS of E. coli O100 that showed the strongest reactions with both sera was used to stimulate IL-6, TNFalpha and nitric oxide production by the J-774A.1 cell line. Both sera were used to inhibit that stimulation and no inhibitory effects of the examined sera in comparison with non-immune serum were observed.

scholarly journals Targeting of Pseudomonas aeruginosa cell surface via GP12, an Escherichia coli specific bacteriophage protein

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
George M. Ongwae ◽  
Mahendra D. Chordia ◽  
Jennie L. Cawley ◽  
Brianna E. Dalesandro ◽  
Nathan J. Wittenberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Pathogenic Bacteria ◽  
Inner Core ◽  
Tail Fiber ◽  
Bacterial Cells ◽  
Core Oligosaccharide ◽  
Gram Negative ◽  
E Coli

AbstractBacteriophages are highly abundant molecular machines that have evolved proteins to target the surface of host bacterial cells. Given the ubiquity of lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria, we reasoned that targeting proteins from bacteriophages could be leveraged to target the surface of Gram-negative pathogens for biotechnological applications. To this end, a short tail fiber (GP12) from the T4 bacteriophage, which infects Escherichia coli (E. coli), was isolated and tested for the ability to adhere to whole bacterial cells. We found that, surprisingly, GP12 effectively bound the surface of Pseudomonas aeruginosa cells despite the established preferred host of T4 for E. coli. In efforts to elucidate why this binding pattern was observed, it was determined that the absence of the O-antigen region of LPS on E. coli improved cell surface tagging. This indicated that O-antigens play a significant role in controlling cell adhesion by T4. Probing GP12 and LPS interactions further using deletions of the enzymes involved in the biosynthetic pathway of LPS revealed the inner core oligosaccharide as a possible main target of GP12. Finally, we demonstrated the potential utility of GP12 for biomedical applications by showing that GP12-modified agarose beads resulted in the depletion of pathogenic bacteria from solution.

scholarly journals Antigenic modulation of Friend virus erythroleukemic cells in vitro by serum from mice with dormant erythroleukemia.

1977 ◽  
Vol 146 (2) ◽  
pp. 520-534 ◽  
Author(s):  
E V Genovesi ◽  
P A Marx ◽  
E F Wheelock
Keyword(s):  
Cell Surface ◽  
Leukemia Virus ◽  
Surface Antigens ◽  
Immune Serum ◽  
Mouse Serum ◽  
Cell Surface Antigens ◽  
Antigenic Modulation ◽  
In Cells ◽  
Erythroleukemic Cells

Friend leukemia virus (FLV) erythroleukemic cells cultured in medium containing FLV-immune serum from dormant FLV-infected mice undergo modulation of FLV cell surface antigens. Modulation was determined by an increased resistance to FLV antibody-mediated complement-dependent lysis and was associated temporally with the capping of FLV-immune complexes at the cell surface. Modulated cells regained their susceptibility to FLV antibody-mediated complement-dependent lysis when transferred to medium containing normal mouse serum. After 48 h of culture in FLV-immune serum, 26% of the FLV erythroleukemic cells were devoid of FLV cell surface antigens as demonstrated by immunofluoresence. Antigenic modulation occurred to a greater extent in cells maintained in logarithmic growth than in cells in GO or resting phase. FLV-antigenic modulation is discussed as a possible mechanism by which antibody induces and maintains FLV-transformed cells in a dormant state.

scholarly journals Chimeric Analysis of the Multicomponent Multidrug Efflux Transporters from Gram-Negative Bacteria

2002 ◽  
Vol 184 (23) ◽  
pp. 6499-6507 ◽  
Author(s):  
Elena B. Tikhonova ◽  
Quiju Wang ◽  
Helen I. Zgurskaya
Keyword(s):  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Multidrug Efflux ◽  
Structural Determinants ◽  
Gram Negative ◽  
E Coli ◽  
Multidrug Transporters ◽  
Cytoplasmic Membranes ◽  
Hybrid Genes ◽  
Rnd Transporters

ABSTRACT Many multidrug transporters from gram-negative bacteria belong to the resistance-nodulation-cell division (RND) superfamily of transporters. RND-type multidrug transporters have an extremely broad substrate specificity and protect bacterial cells from the actions of antibiotics on both sides of the cytoplasmic membrane. They usually function as three-component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of gram-negative bacteria. The structural determinants of RND transporters responsible for multidrug recognition and complex assembly remain unknown. We constructed chimeric RND transporters composed of N-terminal residues of AcrB and C-terminal residues of MexB, the major RND-type transporters from Escherichia coli and Pseudomonas aeruginosa, respectively. The assembly of complexes and multidrug efflux activities of chimeric transporters were determined by coexpression of hybrid genes either with AcrA, the periplasmic component of the AcrAB transporter from E. coli, or with MexA and OprM, the accessory proteins of the MexAB-OprM pump from P. aeruginosa. We found that the specificity of interaction with the corresponding periplasmic component is encoded in the T60-V612 region of transporters. Our results also suggest that the large periplasmic loops of RND-type transporters are involved in multidrug recognition and efflux.

Transfer and expression of the genetic determinants for O and K antigen synthesis in Escherichia coli O9:K(A)30 and Klebsiella sp. Ol:K20, in Escherichia coli K12

1988 ◽  
Vol 34 (8) ◽  
pp. 987-992 ◽  
Author(s):  
Dayle H. Laakso ◽  
Mary K. Homonylo ◽  
Sheila J. Wilmot ◽  
Chris Whitfield
Keyword(s):  
Escherichia Coli ◽  
Surface Antigens ◽  
Capsular Polysaccharides ◽  
Genetic Determinants ◽  
Cell Surface Antigens ◽  
E Coli ◽  
Close Proximity ◽  
Morphological Differences ◽  
Genetically Determined ◽  
K Antigen

Escherichia coli serotype O9:K(A)30 and Klebsiella O1:K20 produce thermostable capsular polysaccharides or K antigens, which are chemically and serologically indistinguishable. Plasmid pULB113 (RP4::mini-Mu) has been used to mediate chromosomal transfer from E. coli O9:K30 and Klebsiella O1:K20 to a multiply marked, unencapsulated, E. coli K12 recipient. Analysis of the cell surface antigens of the transconjugants confirmed previous reports that the genetic determinants for the E. coli K(A) antigens are located near the his and rfb (O antigen) loci on the E. coli linkage map. The Klebsiella K20 capsule genes were also found to be in close proximity to the his and rfb loci. Electron microscopy revealed significant differences in the structural organization of capsular polysaccharides in these two microorganisms and the morphological differences were also readily apparent in transconjugants expressing the respective K antigens. These results are consistent with the interpretation that at least some of the organizational properties of capsular polysaccharides may be genetically determined, rather than being a function of the outer membrane to which the capsular polysaccharides are ultimately attached.

scholarly journals Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator inPseudomonas aeruginosa

2018 ◽  
Vol 115 (12) ◽  
pp. 3150-3155 ◽  
Author(s):  
Neil G. Greene ◽  
Coralie Fumeaux ◽  
Thomas G. Bernhardt
Keyword(s):  
Cell Wall ◽  
Drug Targets ◽  
Bacterial Cells ◽  
Acinetobacter Baylyi ◽  
Gram Negative ◽  
E Coli ◽  
Outer Membrane Lipoprotein ◽  
Synthase Regulation

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoproteinEcLpoB was identified as a critical activator ofEscherichia coliPBP1b (EcPBP1b), one of the major PG synthases of this organism. Activation ofEcPBP1b is mediated through the association ofEcLpoB with a regulatory domain onEcPBP1b called UB2H. Notably,Pseudomonas aeruginosaalso encodes PBP1b (PaPBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potentialPaPBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity ofPaPBP1b. We named this protein LpoP and found that it interacts directly withPaPBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated thatPaLpoP-PaPBP1b as well as an equivalent protein pair fromAcinetobacter baylyican fully substitute forEcLpoB-EcPBP1b inE. colifor PG synthesis. Furthermore, we show that amino acid changes inPaPBP1b that bypass thePaLpoP requirement map to similar locations in the protein as changes promotingEcLpoB bypass inEcPBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.

scholarly journals LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

2021 ◽  
Vol 12 ◽  
Author(s):  
Xudong Tian ◽  
Guillaume Manat ◽  
Elise Gasiorowski ◽  
Rodolphe Auger ◽  
Samia Hicham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Lipid A ◽  
Negative Charge ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
E Coli ◽  
Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

scholarly journals Exploration of Galectin Ligands Displayed on Gram-Negative Respiratory Bacterial Pathogens with Different Cell Surface Architectures

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 595
Author(s):  
María A. Campanero-Rhodes ◽  
Ioanna Kalograiaki ◽  
Begoña Euba ◽  
Enrique Llobet ◽  
Ana Ardá ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Wild Type ◽  
Binding Assays ◽  
Gram Negative ◽  
Bacterial Surface ◽  
Oligosaccharide Chain ◽  
Bacterial Surfaces ◽  
Mutant Cells

Galectins bind various pathogens through recognition of distinct carbohydrate structures. In this work, we examined the binding of four human galectins to the Gram-negative bacteria Klebsiella pneumoniae (Kpn) and non-typeable Haemophilus influenzae (NTHi), which display different surface glycans. In particular, Kpn cells are covered by a polysaccharide capsule and display an O-chain-containing lipopolysaccharide (LPS), whereas NTHi is not capsulated and its LPS, termed lipooligosacccharide (LOS), does not contain O-chain. Binding assays to microarray-printed bacteria revealed that galectins-3, -4, and -8, but not galectin-1, bind to Kpn and NTHi cells, and confocal microscopy attested binding to bacterial cells in suspension. The three galectins bound to array-printed Kpn LPS. Moreover, analysis of galectin binding to mutant Kpn cells evidenced that the O-chain is the docking point for galectins on wild type Kpn. Galectins-3, -4, and -8 also bound the NTHi LOS. Microarray-assisted comparison of the binding to full-length and truncated LOSs, as well as to wild type and mutant cells, supported LOS involvement in galectin binding to NTHi. However, deletion of the entire LOS oligosaccharide chain actually increased binding to NTHi cells, indicating the availability of other ligands on the bacterial surface, as similarly inferred for Kpn cells devoid of both O-chain and capsule. Altogether, the results illustrate galectins’ versatility for recognizing different bacterial structures, and point out the occurrence of so far overlooked galectin ligands on bacterial surfaces.

scholarly journals Identification of the Fucose Synthetase Gene in the Colanic Acid Gene Cluster of Escherichia coli K-12

1998 ◽  
Vol 180 (4) ◽  
pp. 998-1001 ◽  
Author(s):  
Kanella Andrianopoulos ◽  
Lei Wang ◽  
Peter R. Reeves
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Extracellular Polysaccharide ◽  
Surface Antigens ◽  
Bacterial Surface ◽  
E Coli ◽  
Colanic Acid ◽  
K 12

ABSTRACT GDP–l-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP–d-mannose.l-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced theE. coli colanic acid gene cluster and identified one of the GDP–l-fucose biosynthetic pathway genes, gmd. We report here the identification of the gene (fcl), located downstream of gmd, encoding the fucose synthetase.

scholarly journals The Hek Outer Membrane Protein of Escherichia coli Strain RS218 Binds to Proteoglycan and Utilizes a Single Extracellular Loop for Adherence, Invasion, and Autoaggregation

2007 ◽  
Vol 76 (3) ◽  
pp. 1135-1142 ◽  
Author(s):  
Robert P. Fagan ◽  
Matthew A. Lambert ◽  
Stephen G. J. Smith
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Membrane Protein ◽  
Molecular Level ◽  
Coli Strain ◽  
Bacterial Cells ◽  
Extracellular Loop ◽  
Gram Negative ◽  
E Coli

ABSTRACT Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the bloodstream, and eventually invasion of the meninges. The last two aspects have been well characterized at a molecular level. Less is known about the early stages of pathogenesis, i.e., adhesion to and invasion of epithelial cells. We have previously reported that the Hek protein causes autoaggregation and can mediate adherence to and invasion of epithelial cells. Here, we report that Hek-mediated adherence is dependent on binding to glycosoaminoglycan, in particular, heparin. The ability to hemagglutinate, autoaggregate, adhere, and invade is contingent on a putative 25-amino-acid loop that is exposed to the outside of the bacterial cells.

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