Variant Membrane Proteins

Wise, Kim S.; Kim, Mary F.; Watson-McKown, Robyn

doi:10.1016/b978-012583805-4/50024-8

Cited by 32 publications

(22 citation statements)

References 11 publications

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“…Cell agglomerations were reduced by resuspending the bacteria with a 27G syringe, and 200 ml TX-114 (10 %, Sigma) was added. Extraction of proteins was carried out as described elsewhere (Wise et al, 1995). Briefly, after incubation for 2 h at 4 uC, the suspensions were pelleted by centrifugation at 13 000 g (10 min, 4 uC).…”

Section: Methodsmentioning

confidence: 99%

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

2013

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The obligate pathogenic mycoplasma species Mycoplasma pneumoniae uses a limited but effective repertoire of virulence factors to infect and colonize the human respiratory tract. Besides the development of a unique adhesion complex and the expression of tissue-damaging factors, surface-located glycolytic enzymes and their capacity to bind to components of the human extracellular matrix (ECM) support pathogen-host interactions. Here, we demonstrated that the glycolytic enzymes enolase (Mpn606) and pyruvate dehydrogenase subunit B (Mpn392; PDHB) of M. pneumoniae show concentration-dependent binding to human plasminogen. Monospecific polyclonal antisera against both recombinant proteins reduced the binding to plasminogen significantly. The surface location of PDHB but not of enolase was demonstrated using Triton X fractionation of M. pneumoniae total protein content, membrane fractionation, colony blotting, mild proteolysis of mycoplasma cells, and immunofluorescence tests. To characterize the binding site of plasminogen in surface-displaced PDHB, the mycoplasmal protein was separated into four recombinant proteins followed by investigation of the binding behaviour of peptides that overlap the protein part interacting with plasminogen. Spot analysis resulted in a novel region of 12 amino acids (FPAMFQIFTHAA, position 91 to 102 of PDHB), which is responsible exclusively for binding of human plasminogen and also interacts in a dose-dependent manner with this host protein. The data indicate that the plasminogen-binding enzymes enolase and especially the surface-associated PDHB may contribute to the pathogenesis of M. pneumoniae infections.

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Section: Methodsmentioning

confidence: 99%

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

2013

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“…All cultures were incubated at 37 • C for 4 weeks and observed for color change (acid shift) or mycoplasma growth on plates. Isolates were analyzed by Triton X-114 phase partitioning [16] and SDS-PAGE [17] to determine phenotypic profiles.…”

Section: Re-isolation and Quantitation Of Mycoplasmasmentioning

confidence: 99%

A modified live Mycoplasma gallisepticum vaccine to protect chickens from respiratory disease

Papazisi

Silbart

Frasca

et al. 2002

Vaccine

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“…M. gallisepticum clonal variant RCL1, which has been previously described and shown to express the CrmA product (15), was subjected to Triton X-114 fractionation as previously described (27). Proteins that partitioned into the insoluble pellet were resolved by 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

Section: Methodsmentioning

confidence: 99%

“…Protein profile analyses of populations and clones used in this study were performed by SDS-PAGE followed by Coomassie blue staining and/or Western blotting using fractions obtained after Triton-X114 (Sigma-Aldrich, St. Louis, MO) partitioning as described elsewhere (27). The membranes were probed either with rabbit anti-GapA antibodies (13) or with rabbit anti-CrmA antibodies diluted 1:8,000.…”

Section: Sds-page and Western Blot Analysismentioning

confidence: 99%

Role of the GapA and CrmA Cytadhesins of Mycoplasma gallisepticum in Promoting Virulence and Host Colonization

Indikova

Much

Stipkovits³

et al. 2013

Infect Immun

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bMycoplasma gallisepticum is an important avian pathogen that commonly induces chronic respiratory disease in chicken. To better understand the mycoplasma factors involved in host colonization, chickens were infected via aerosol with two hemadsorption-negative (HA ؊ ) mutants, mHAD3 and RCL2, that were derived from a low passage of the pathogenic strain R (R low ) and are both deficient in the two major cytadhesins GapA and CrmA. After 9 days of infection, chickens were monitored for air sac lesions and for the presence of mycoplasmas in various organs. The data showed that mHAD3, in which the crmA gene has been disrupted, did not promote efficient colonization or significant air sac lesions. In contrast, the spontaneous HA ؊ RCL2 mutant, which contains a point mutation in the gapA structural gene, successfully colonized the respiratory tract and displayed an attenuated virulence compared to that of R low . It has previously been shown in vitro that the point mutation of RCL2 spontaneously reverts with a high frequency, resulting in on-and-off switching of the HA phenotype. Detailed analyses further revealed that such an event is not responsible for the observed in vivo outcome, since 98.4% of the mycoplasma populations recovered from RCL2-infected chickens still display the mutation and the associated phenotype. Unlike R low , however, RCL2 was unable to colonize inner organs. These findings demonstrate the major role played by the GapA and CrmA proteins in M. gallisepticum host colonization and virulence.

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Variant Membrane Proteins

Cited by 32 publications

References 11 publications

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

A modified live Mycoplasma gallisepticum vaccine to protect chickens from respiratory disease

Role of the GapA and CrmA Cytadhesins of Mycoplasma gallisepticum in Promoting Virulence and Host Colonization

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