WSJM, a simple chemically defined medium for growth of Neisseria gonorrhoeae

Wong, Tung Po; Shockley, Robert K.; Johnston, Kenneth H.

doi:10.1128/jcm.11.4.363-369.1980

Cited by 24 publications

(15 citation statements)

References 17 publications

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“…The P.I of JS1 has Mr 37,500, which is very similar to that of band 1. To determine whether or not this higher Mr form was related to P.I, strain JS3 (P.I of 34,000 M 0 was first extrinsically labeled with 125I (P.I has been shown [5] to be the most prominently labeled band in the 125I-labeled whole-cell lysate), and used in a similar electrophoresis and immunoblotting experiment. A very small amount, equivalent to 5 ~g of protein, of whole-cell lysate was used so that the radioemitting signal from the 125I-label would not obscure the mAb 10-antigen-~25I-protein A complexes; because of this, only band 2 is shown; increased exposure time permits visualization of bands 3 and 4.…”

Section: Resultsmentioning

confidence: 99%

“…Bacteria were grown on solid clear-typing medium (3,4), at 37°C in a humidified 5% CO2 atmosphere. Intrinsic labeling of gonococci with 14C-labeled amino acids (New England Nuclear, Boston, MA) was done using Wong-Shockley-Johnston's medium (5), both in the presence and absence of glucose as a carbon source. Radioactive amino acids (sp act 0.025 mCi) were added to the medium at the time of inoculation and during the log phase (t = 4 h) of growth.…”

Section: Culturementioning

confidence: 99%

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Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics.

Hitchcock¹,

Hayes²,

Mayer³

et al. 1985

The Journal of Experimental Medicine

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The H8 protein is a surface-exposed antigen that is found, among members of the Neisseria genus, primarily on pathogenic species. In this study, the surface exposure of H8 was reassessed by four techniques. Results of slide agglutination, indirect fluorescent antibody binding, absorption of sera with whole gonococci, and immune electron microscopy all confirmed the presence of H8 in the outer membrane. The degree to which protein A-gold-labeled monoclonal antibodies bound to H8 was marked, and suggested that this antigen was present in abundant amounts in the outer membrane. Also in this study, the electrophoretic heterogeneity of this common surface antigen was examined. Because H8 stains poorly, electrophoretic mobility was assessed using polyclonal antibodies and a monoclonal antibody that recognizes a common H8 epitope. H8 was analyzed with respect to protein I, lipopolysaccharide (LPS), and pilus and opacity phenotypic variation; results confirmed that heterogeneity of Mr was the rule among strains (21 were examined), however, the variability in Mr was independent of protein I or LPS Mr. In one strain (FA1090), the heterogeneity of H8 was examined among 10 piliation/opacity variants; the H8 (and LPS) Mr was identical in all variants; similar data were generated in strains JS3 and JS1. The electrophoretic mobility of H8 was altered in serum-resistant and neutrophil enzyme-resistant gonococci compared to the sensitive gonococci. Some of the unusual electrophoretic migration characteristics of the antigen were also examined. H8 formed a unique mushroom-shaped band in one-dimensional gels; in a two-dimensional electrophoresis system, the antigen migrated aberrantly, very similarly to LPS. Also seen in the two-dimensional electrophoresis profile were multimers of the H8 antigen; in strain JS3 (Mr 23,500), these migrated at 43,600, 86,000, and greater than 150,000. In other strains, the Mr of the multimers differed depending upon the Mr of the monomer. The two-dimensional migration characteristics (as measured by antigenicity) were completely destroyed by proteinase K digestion. Activity of H8 polyclonal antibodies to the antigens in two-dimensional gels was completely removed by adsorption of formalin-fixed whole cells, but was not affected by adsorption with LPS. These electrophoretic characteristics may reflect the close association of some nonprotein constituent, perhaps lipid or carbohydrate or both.

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Section: Resultsmentioning

confidence: 99%

Section: Culturementioning

confidence: 99%

Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics.

Hitchcock¹,

Hayes²,

Mayer³

et al. 1985

The Journal of Experimental Medicine

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“…was observed compared with organisms incubated in buffer alone. The addition of alphachymotrypsin at an early log phase of growth of gonococci in defined liquid medium (20) and the subsequent growth of the gonococci, as followed by optical density, revealed that regardless of the species of protein I, the gonococci continued to multiply and reached the same optical density as gonococci grown in the absence of the enzyme. An SDS-PAGE analysis of the organisms grown in the presence of alpha-chymotrypsin revealed that those organisms expressing 34K or 33K…”

Section: P I _omentioning

confidence: 99%

Effects of proteolytic enzymes on the outer membrane proteins of Neisseria gonorrhoeae

Blake¹,

Gotschlich²,

Swanson³

1981

Infect Immun

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Proteolytic enzymes inhibit the growth of some strains and opacity variants of Neisseria gonorrhoeae. To understand the inhibitory effects of these enzymes, we examined several strains to determine the actions of proteases on the three predominant proteins in gonococcal outer membranes. namely, the major outer membrane protein (protein I), the sometimes-expressed opaque protein (protein II), and protein III. In a comparison of the protein I species expressed by different strains, we observed a pattern based on subunit molecular weight and susceptibility to enzymatic degradation. Protein I species having molecular weights of 34,000 were more susceptible to proteolysis, whereas protein I species having molecular weights of 33,000 were less susceptible, and protein I species having molecular weights of 32,000 were resistant. This pattern was observed both in intact cells and in purified outer membranes. All of the enzymes degraded protein II, but this susceptibility appeared to be influenced in part by the species of protein I present. Protein III was resistant to all of the proteolytic enzymes tested. Based on the resulting fragments from each proteolytic cleavage of proteins I and II and their membrane associations, we suggest how these proteins may be arranged in intact membranes. Our data suggested the presence of an endogenous gonococcal enzyme. This enzyme appeared to degrade proteins I and II into fragments resembling the fragments resulting from the action of chymotrypsin.

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“…Chemically defined liquid media for N. gonorrhoeae are usually minimal media and none has been demonstrated to support the growth of low (e.g. o 10 3 CFU mL À1 ) inocula (Welton et al, 1944;Kenny et al, 1967;Hunter & McVeigh, 1970;Bacigalupi & Lawson, 1973;Catlin, 1973;La Scolea & Young, 1974;Elmros et al, 1976;Manchee et al, 1980;Morse & Bartenstein, 1980;Wong et al, 1980). Currently, therefore, there are no available liquid media, defined or undefined, that permit study of the growth of N. gonorrhoeae from low inocula (Reinhold et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

A fully defined, clear and protein-free liquid medium permitting dense growth ofNeisseria gonorrhoeaefrom very low inocula

Wade

Graver

2007

FEMS Microbiology Letters

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Neisseria gonorrhoeae is difficult to cultivate in liquid medium. Currently there are no liquid media, defined or undefined, that reliably permit growth of this bacterium from low inocula. Standard clinical laboratory broths may allow multiplication of some strains of gonococci from large inocula, but such media incorporate infusates, extracts or digests and are therefore undefined. In this study, 20 gonococci of ten auxotypes were tested in various experimental media in the development of an easily prepared chemically defined, clear and protein-free liquid medium. The final medium - GW medium - allowed the growth of three clinical isolates of gonococci from inocula of <10(3) CFU mL(-1) to >10(8) CFU mL(-1) by 24 h. None of four commercially-available broths (nutrient broth, brain heart infusion, tryptone soya broth, and Mueller-Hinton broth) tested in parallel reliably supported growth of these isolates to the same extent. GW medium should be useful for studies of the growth of gonococci under different conditions and, as the medium is clear and colorless, this can be monitored turbidometrically. GW medium may be suitable as a basal medium for biochemical identification tests, antimicrobial susceptibility determinations and antimicrobial synergy studies.

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WSJM, a simple chemically defined medium for growth of Neisseria gonorrhoeae

Cited by 24 publications

References 17 publications

Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics.

Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics.

Effects of proteolytic enzymes on the outer membrane proteins of Neisseria gonorrhoeae

A fully defined, clear and protein-free liquid medium permitting dense growth ofNeisseria gonorrhoeaefrom very low inocula

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