Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules

Identification of antigens is important for vaccine production. We tested extraction protocols using cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) to formulate surfactant vesicles (SVs) containing components from Neisseria gonorrhoeae. Carbohydrate and protein assays demonstrated that protein and carbohydrates were incorporated into the vesicle leaflet. Depending on the extraction protocol utilized, 100–400 µg of protein/mL of SVs solution was obtained. Gel electrophoresis followed by silver staining demonstrated that SV extracts contained lipooligosaccharide and a subset of bacterial proteins and lipoproteins. Western blotting and mass spectral analysis indicated that the majority of the proteins were derived from the outer membrane. Mass spectrometric and bioinformatics analysis of SVs identified 29 membrane proteins, including porin and opacity-associated protein. Proteins embedded in the SVs leaflet could be degraded by the addition of trypsin or proteinase K. Our data showed that the incorporation of CTAT and SDBS into vesicles eliminated their toxicity as measured by a THP-1 killing assay. Incorporation of gonococcal cell surface components into SVs reduced toxicity as compared to the whole cell extracts, as measured by cytokine induction, while retaining the immunogenicity. This process constitutes a general method for extracting bacterial surface components and identification of antigens that might be included in vaccines.

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Degradation of zearalenone in swine feed and feed ingredients by Bacillus subtilis ANSB01G

World Mycotoxin Journal ◽

10.3920/wmj2013.1623 ◽

2014 ◽

Vol 7 (2) ◽

pp. 143-151 ◽

Cited By ~ 18

Author(s):

Y.P. Lei ◽

L.H. Zhao ◽

Q.G. Ma ◽

J.Y. Zhang ◽

T. Zhou ◽

...

Keyword(s):

Bacillus Subtilis ◽

Culture Supernatant ◽

Antimicrobial Activities ◽

Proteinase K ◽

Rrna Gene ◽

Distillers Grains With Solubles ◽

Dried Distillers Grains ◽

Cell Extracts ◽

Gene Sequence Analysis ◽

And Staphylococcus Aureus

Zearalenone (ZEA) and its derivatives are mycotoxins that can cause oestrogenic effects and impair the reproductive physiology of animals, especially in female swine. Strategies to reduce or eliminate ZEA contamination in foods and feeds are very much needed. Among 36 bacterial isolates obtained from a variety of animal intestinal chyme, mouldy foods and feeds, soils, etc., five isolates demonstrated the ability to reduce more than 50% of ZEA in a liquid medium; ANSB01G isolate taken from normal broiler intestinal chyme reduced ZEA the most, by 88.65%. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, the ANSB01G isolate was identified as Bacillus subtilis. Under simulated intestinal tract conditions, the ANSB01G B. subtilis isolate degraded 84.58, 66.34 and 83.04% of ZEA in naturally contaminated maize, dried distillers’ grains with solubles, and swine complete feed, respectively. The highest degradation of ZEA occurred when the mycotoxin was co-incubated with the whole bacterial culture, resulting in a reduction of 88.65%, followed by 75.60% using culture supernatant, 26.11% using cell extracts, and 15.06% using viable cells. Treatments consisting of both heating and addition of proteinase K significantly reduced the rate of ZEA degradation in the culture supernatant, indicating that the ZEA degradation might be enzymatic. B. subtilis ANSB01G displayed resistance to simulated gastrointestinal tract environments and antimicrobial activities against several common bacterial pathogens, including Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. These properties of B. subtilis ANSB01G suggest the possibility of its potential to effectively degrade ZEA in feed and to develop functional feed products for livestock industries.

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Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2382-2391.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2382-2391

Author(s):

C A Kaiser ◽

D Botstein

Keyword(s):

Saccharomyces Cerevisiae ◽

Secretory Pathway ◽

Signal Sequence ◽

Mutant Gene ◽

Yeast Cells ◽

Molecular Weight Characteristic ◽

Cell Extracts ◽

Substitution Mutations ◽

Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

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A role for sulfite in inducing surface changes In Neisseria gonorrhoeae

Canadian Journal of Microbiology ◽

10.1139/m84-207 ◽

1984 ◽

Vol 30 (10) ◽

pp. 1297-1301 ◽

Cited By ~ 1

Author(s):

E. Pinina Norrod

Keyword(s):

Neisseria Gonorrhoeae ◽

Human Serum ◽

Growth Medium ◽

Proteinase K ◽

Increased Sensitivity ◽

Normal Human ◽

Induced Changes ◽

Inducing Surface ◽

Surface Changes

Addition of pyruvate to growth medium failed to induce the changes in Neisseria gonorrhoeae that have been reported previously. Addition of 3.8 mM sulfite or 1.3 mM sulfite plus 14 mM pyruvate restored the medium's reactivity in a test for cysteine and its ability to induce changes in N. gonorrhoeae. The induced changes that were restored were (i) increased colonial opacity and roughness, (ii) increased sensitivity to killing by normal human serum, and (iii) electrophoretic changes that may represent changes in lipopolysaccharide. Further characterization of the electrophoretic changes showed that the bands were resistant to treatment with proteinase K, that they were not affected by EDTA and urea, and that they were not dependent upon the stage of growth.

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Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2382 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2382-2391 ◽

Cited By ~ 47

Author(s):

C A Kaiser ◽

D Botstein

Keyword(s):

Saccharomyces Cerevisiae ◽

Secretory Pathway ◽

Signal Sequence ◽

Mutant Gene ◽

Yeast Cells ◽

Molecular Weight Characteristic ◽

Cell Extracts ◽

Substitution Mutations ◽

Membrane Components

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Cysteine proteinase SpeB from Streptococcus pyogenes – a potent modifier of immunologically important host and bacterial proteins

Biological Chemistry ◽

10.1515/bc.2011.208 ◽

2011 ◽

Vol 392 (12) ◽

pp. 1077-1088 ◽

Cited By ~ 83

Author(s):

Daniel C. Nelson ◽

Julia Garbe ◽

Mattias Collin

Keyword(s):

Streptococcus Pyogenes ◽

Group A Streptococcus ◽

Wide Spectrum ◽

Cysteine Proteinase ◽

Complement Components ◽

Bacterial Surface ◽

Bacterial Proteins ◽

Disease States ◽

Group A ◽

Important Host

AbstractGroup A streptococcus (Streptococcus pyogenes) is an exclusively human pathogen that causes a wide spectrum of diseases ranging from pharyngitis, to impetigo, to toxic shock, to necrotizing fasciitis. The diversity of these disease states necessitates thatS. pyogenespossess the ability to modulate both the innate and adaptive immune responses. SpeB, a cysteine proteinase, is the predominant secreted protein fromS. pyogenes. Because of its relatively indiscriminant specificity, this enzyme has been shown to degrade the extracellular matrix, cytokines, chemokines, complement components, immunoglobulins, and serum protease inhibitors, to name but a few of the known substrates. Additionally, SpeB regulates other streptococcal proteins by degrading them or releasing them from the bacterial surface. Despite the wealth of literature on putative SpeB functions, there remains much controversy about this enzyme because many of reported activities would produce contradictory physiological results. Here we review all known host and bacterial protein substrates for SpeB, their cleavage sites, and discuss the role of this enzyme in streptococcal pathogenesis based on the current literature.

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Stick to Your Gums

Journal of Dental Research ◽

10.1177/0022034511399096 ◽

2011 ◽

Vol 90 (11) ◽

pp. 1271-1278 ◽

Cited By ~ 76

Author(s):

A.H. Nobbs ◽

H.F. Jenkinson ◽

N.S. Jakubovics

Keyword(s):

Microbial Communities ◽

Oral Bacteria ◽

Surface Proteins ◽

Major Focus ◽

Growth And Survival ◽

Bacterial Surface ◽

Bacterial Proteins ◽

Functional Regions ◽

Binding Pockets ◽

Microbiology Research

Studies on the adherence properties of oral bacteria have been a major focus in microbiology research for several decades. The ability of bacteria to adhere to the variety of surfaces present in the oral cavity, and to become integrated within the resident microbial communities, confers growth and survival properties. Molecular analyses have revealed several families of Gram-positive bacterial surface proteins, including serine-rich repeat, antigen I/II, and pilus families, that mediate adherence to a variety of salivary and oral bacterial receptors. In Gram-negative bacteria, pili, auto-transporters, and extracellular matrix-binding proteins provide components for host tissue recognition and building of complex microbial communities. Future studies will reveal in greater detail the binding pockets for these adhesin families and their receptors. This information will be crucial for the development of new inhibitors or vaccines that target the functional regions of bacterial proteins that are involved in colonization and pathogenesis.

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Polyamines Can Increase Resistance of Neisseria gonorrhoeae to Mediators of the Innate Human Host Defense

Infection and Immunity ◽

10.1128/iai.01301-09 ◽

2010 ◽

Vol 78 (7) ◽

pp. 3187-3195 ◽

Cited By ~ 29

Author(s):

Maira Goytia ◽

William M. Shafer

Keyword(s):

Neisseria Gonorrhoeae ◽

Antimicrobial Peptides ◽

Host Defense ◽

Polymyxin B ◽

Cellular Functions ◽

Cationic Antimicrobial Peptides ◽

Sexually Transmitted ◽

Bacterial Surface ◽

Bodily Fluids ◽

Mucosal Surfaces

ABSTRACT Polyamines are biogenic polycationic molecules involved in key cellular functions. Extracellular polyamines found in bodily fluids or laboratory media can be imported by bacteria or bind to negatively charged bacterial surface structures, where they can impair binding of antimicrobials. We hypothesized that the presence of polyamines in fluids that bathe urogenital mucosal surfaces could alter the susceptibility of the sexually transmitted strict human pathogen Neisseria gonorrhoeae to mediators of the innate host defense. Herein we report that polyamines can significantly increase gonococcal resistance to two structurally diverse cationic antimicrobial peptides (polymyxin B and LL-37) but not to antibiotics that exert activity in the cytosol or periplasm (e.g., ciprofloxacin, spectinomycin, or penicillin). The capacity of polyamines to increase gonococcal resistance to cationic antimicrobial peptides was dose dependent, correlated with the degree of cationicity, independent of a polyamine transport system involving the polyamine permeases PotH and PotI, and was reversible. In addition, we found that polyamines increase gonococcal resistance to complement-mediated killing by normal human serum. We propose that polyamines in genital mucosal fluids may enhance gonococcal survival during infection by reducing bacterial susceptibility to host-derived antimicrobials that function in innate host defense.

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Binding of Complement Factor H to Loop 5 of Porin Protein 1A: A Molecular Mechanism of Serum Resistance of Nonsialylated Neisseria gonorrhoeae

Journal of Experimental Medicine ◽

10.1084/jem.188.4.671 ◽

1998 ◽

Vol 188 (4) ◽

pp. 671-680 ◽

Cited By ~ 191

Author(s):

Sanjay Ram ◽

Daniel P. McQuillen ◽

Sunita Gulati ◽

Christopher Elkins ◽

Michael K. Pangburn ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Binding Site ◽

Molecular Basis ◽

Factor H ◽

Serum Resistance ◽

Complement Factor ◽

Bacterial Surface ◽

Exposed Region ◽

Porin Protein ◽

Serum Bactericidal Assay

Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N. gonorrhoeae.

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Lactobacillus jensenii Surface-Associated Proteins Inhibit Neisseria gonorrhoeae Adherence to Epithelial Cells

Infection and Immunity ◽

10.1128/iai.01200-09 ◽

2010 ◽

Vol 78 (7) ◽

pp. 3103-3111 ◽

Cited By ~ 45

Author(s):

Rachel R. Spurbeck ◽

Cindy Grove Arvidson

Keyword(s):

Epithelial Cells ◽

Neisseria Gonorrhoeae ◽

Size Exclusion ◽

Proteinase K ◽

Dependent Manner ◽

Inhibitory Protein ◽

Healthy Human ◽

Significant Similarity ◽

Lactobacillus Jensenii ◽

Vaginal Tract

ABSTRACT High numbers of lactobacilli in the vaginal tract have been correlated with a decreased risk of infection by the sexually transmitted pathogen Neisseria gonorrhoeae. We have previously shown that Lactobacillus jensenii, one of the most prevalent microorganisms in the healthy human vaginal tract, can inhibit gonococcal adherence to epithelial cells in culture. Here we examined the role of the epithelial cells and the components of L. jensenii involved in the inhibition of gonococcal adherence. L. jensenii inhibited the adherence of gonococci to glutaraldehyde-fixed epithelial cells like it inhibited the adherence of gonococci to live epithelial cells, suggesting that the epithelial cells do not need to be metabolically active for the inhibition to occur. In addition, methanol-fixed L. jensenii inhibited gonococcal adherence to live epithelial cells, indicating that L. jensenii uses a constitutive component to inhibit gonococcal interactions with epithelial cells. Proteinase K treatment of methanol-fixed lactobacilli eliminated the inhibitory effect, suggesting that the inhibitory component contains protein. Released surface components (RSC) isolated from L. jensenii were found to contain at least two inhibitory components, both of which are protease sensitive. Using anion-exchange and size exclusion chromatography, an inhibitory protein which exhibits significant similarity to the enzyme enolase was isolated. A recombinant His6-tagged version of this protein was subsequently produced and shown to inhibit gonococcal adherence to epithelial cells in a dose-dependent manner.

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