Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa

Lam, Joseph S.; MacDonald, Leslie A.; Lam, M. Y. C.; Duchesne, Lorraine G. M.; Southam, Gordon

doi:10.1128/iai.55.5.1051-1057.1987

Cited by 96 publications

(48 citation statements)

References 51 publications

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“…The lack of A-band in the double mutant, however, indicates that, in spite of possible polar effects on wzm and/or wzt, wbpW was indeed deleted and is not able to contribute to GDP-D-Man synthesis. As a control B-band LPS was analysed by Western blot using the B-band-specific monoclonal antibody MF15-4 (Lam et al, 1987). There were no differences in B-band LPS between the strains shown in Fig.…”

Section: Pslb Is the Pmi/gmp That Promotes A-band Lps Production In Amentioning

confidence: 98%

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Byrd

Sadovskaya

Vinogradov

et al. 2009

Molecular Microbiology

342

403

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Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in nonmucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that eleven psl genes, pslACDEFGHIJKL, are required for Psl production and surface attachment. We also present the first structural analysis of the psl-dependent polysaccharide, which consists of a repeating pentasaccharide containing d-mannose, d-glucose, and l-rhamnose: false[→3false)−normalβ−normalD−Manp2↑1α−D−Manp−false(1→3false)−normalβ−normalD−Manp−false(1→3false)−normalα−normalL−Rhap−false(1→3false)−normalβ−normalD−Glcp−false(1→false]normaln− In addition we identified the sugar nucleotide precursors involved in Psl generation and demonstrated the requirement for GDP-d-mannose, UDP-d-glucose, and dTDP-l-rhamnose in Psl production and surface attachment. Finally, genetic analyses revealed that wbpW restored Psl production in a pslB mutant and pslB promoted A-band LPS synthesis in a wbpW mutant, indicating functional redundancy and overlapping roles for these two enzymes. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and pathogenesis of P. aeruginosa.

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Section: Pslb Is the Pmi/gmp That Promotes A-band Lps Production In Amentioning

confidence: 98%

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Byrd

Sadovskaya

Vinogradov

et al. 2009

Molecular Microbiology

342

403

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“…Mutation of wzx results in an abrogation of heteropolymeric O-antigen production compared with wild-type bacteria (Burrows and Lam, 1999). Blot was probed with the heteropolymeric O-Ag-specific mAb MF15-4 (Lam et al, 1987).…”

Section: Figmentioning

confidence: 99%

Wzx flippase‐mediated membrane translocation of sugar polymer precursors in bacteria

Islam

Lam

2012

Environmental Microbiology

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SummaryBacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/ Wzy-dependent pathway. Wzx is an integral IM protein found in Gram-negative and Gram-positive bacteria that mediates IM translocation of UndPP-linked sugar repeats from the cytoplasmic to the periplasmic leaflet; interaction of Wzx with other assembly proteins is indirectly supported by genetic evidence. Topological mapping has indicated 12 a-helical transmembrane segments (TMS), with the number of charged TMS residues fluctuating based on the mapping method used. A novel Wzx tertiary structure model has been built, allowing for substrate-binding or energy-coupling roles to be proposed for functionally important charged and aromatic TMS residues. It has also led to a proposed antiport-like mechanism of Wzx function. Exquisite substrate specificity of Wzx proteins was recently revealed in distinguishing between UndPP-linked substrates with identical main-chain sugar repeats, but differing in the chemical composition of a terminal sugar side-branch cap. The objective of this review is to synthesize the most up-to-date knowledge concerning Wzx flippases and to provide perspective for future investigations in this burgeoning field.

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“…Hybridomas were produced as described by Oi & Herzenberg (1980) with some modifications (Lam, Macdonald, Lam, Duchesne &: Southam 1987). Briefly, mouse spleen cells were fused with NS-1 myeloma cells in a 10:1 ratio.…”

Section: Hybridoma Productionmentioning

confidence: 99%

Characterization of a monoclonal antibody against the 47‐ kDa antigen of Cryptobia salmositica Katz and its use in an antigen‐capture enzyme‐linked immunosorbent assay for detection of parasite antigen in infected rainbow trout, Oncorhynchus mykiss (Walbaum)

Verity

Woo

1996

Journal of Fish Diseases

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A monoclonal antibody (designated MAb-007) was produced against the pathogenic haemoflagellate Cryptobia salmositica Katz. This IgG3 antibody recognized the 47-kDa antigenic polypeptide of C salmositica (SDS-PAGE and Western immunoblotting). The antibody did not agglutinate live parasites, and there was no change in the staining intensity of the 47-kDa band on Western immunoblots after immnnoabsorption of MAb-007 with live intact parasites. The 47-kDa antigen recognized by MAb-007 was localized in the cytoplasm of the parasite (immunogold labelling and electron microscopy). The monoclonal antibody crossreacted with the 47-kDa polypeptides of C. bullocki Strout and C catostomi Bower & Woo. It was used in an antigen-capture ELISA for the detection of parasite antigen in the plasma of rainbow trout inoculated with the parasite, or with an attenuated vaccine strain of C. salmositica. All pre-infection plasma were negative while all infected fish with detectable parasitaemias were positive for antigen at 1-9 weeks after infection. Parasite antigen was even detected in vaccinated fish that were negative for parasites using the wet mount microscopic technique. The antigen-capture ELISA detected C. salmositica antigen in whole cell lysate preparations at concentrations as low as 0-5 /^ ml"'. Fifty microlitres of fish plasma was required in the antigen-capture ELISA, and the use of a plate reader and 96-well plates facilitated rapid analysis of a large number of plasma samples. The sensitivity of the assay makes it a potentially useful tool for detection of Cryptobia infections.

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Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa

Cited by 96 publications

References 51 publications

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Wzx flippase‐mediated membrane translocation of sugar polymer precursors in bacteria

Characterization of a monoclonal antibody against the 47‐ kDa antigen of Cryptobia salmositica Katz and its use in an antigen‐capture enzyme‐linked immunosorbent assay for detection of parasite antigen in infected rainbow trout, Oncorhynchus mykiss (Walbaum)

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