Isolation and partial characterization of the reduction-modifiable protein of Neisseria gonorrhoeae.

Lytton, E. J.; Blake, M. S.

doi:10.1084/jem.164.5.1749

Cited by 28 publications

(24 citation statements)

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“…Approximately 60% of the outer membrane protein content of pathogenic Gram-negative Neisseria consists of porin [2]. In the gonococcus these are termed protein IA (PIA) or IB (PIB), and for the meningococcus PorA (class 1 protein) or PorB (class 2 or 3 protein) [2].…”

Section: Neisserial Porins and Immune Adjuvanticitymentioning

confidence: 99%

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Innate Immune Function of the Neisserial Porins and the Relationship to Vaccine Adjuvant Activity

Wetzler¹

2010

Future Microbiol.

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Neisseria meningitidis is a Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric β-barrel structure. Neisserial porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells. Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of antigen-presenting cells, an effect that is dependent on Toll-like receptor (TLR)2 and MyD88, through a cascade of signal transduction events mediated by direct binding of the porin to the TLR2–TLR1 heterodimer. This article summarizes work carried out investigating the mechanisms of the immune stimulating capacity of the neisserial porins (specifically meningococcal PorB), emphasizing cellular events involved in antigen-presenting cell activation and induction of expression of cell surface molecules involved in the immune response.

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Section: Neisserial Porins and Immune Adjuvanticitymentioning

confidence: 99%

“…In the gonococcus these are termed protein IA (PIA) or IB (PIB), and for the meningococcus PorA (class 1 protein) or PorB (class 2 or 3 protein) [2]. There is significant structural and functional homology amongst Neisserial porins [3–8] and amongst other Gram-negative porins [8,9].…”

Section: Neisserial Porins and Immune Adjuvanticitymentioning

confidence: 99%

Innate Immune Function of the Neisserial Porins and the Relationship to Vaccine Adjuvant Activity

Wetzler¹

2010

Future Microbiol.

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“…It is worth noting that, in contrast to PorB and Opa, which undergo, respectively, antigenic and phase variation, PIII is highly conserved and expressed by all pathogenic Neisseriae [6]. This characteristic inter- and intra-strain homogeneity is unique among all the main outer membrane constituents, which, contrary to PIII, evolved a strong variability to escape the immune pressure of the host [7,8].…”

Section: Introductionmentioning

confidence: 99%

Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells

et al. 2013

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BackgroundProtein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated.ResultsWe generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain.We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells.ConclusionHere we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII in the virulence of gonococcus and suggests that the structural homology to OmpA is conserved also at functional level.

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“…It was completely released with minimal PI contamination. The PIII was then purified by cation-exchange chromatography on CM-Sepharose, followed by gel filtration on Sephacryl-200 (13). The resulting product retained all the biochemical and immunochemical characteristics of the native PIII, except that, unlike the native molecule, purified PIII was highly susceptible to * Corresponding author.…”

Section: Purificationmentioning

confidence: 99%

“…rum ( E) with purified OmpA. The basis of this ELISA has been described previously (13). Microdilution plates were coated with purified OmpA.…”

Section: Cloning Of the Structural Genementioning

confidence: 99%

Protein III: structure, function, and genetics

et al. 1989

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Protein III (PIII) was originally described by McDade and Johnston (14). In their studies of the gonococcal outer membrane, they observed that all gonococcal strains contained a similar protein whose apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis increased upon treatment with a reducing agent. Furthermore, they and others (14, 17) provided evidence, using bifunctional cross-linking reagents, that PIII was closely associated with the protein I (PI) porin within the gonococcal outer membrane. It was further shown that PIII was structurally and immunologically conserved among several strains of gonococci (11,12). In studies in which several proteolytic enzymes were used to investigate the surface orientation of gonococcal outer membrane proteins, PIII was found to be resistant to protease treatment (2). However, Shafer and Morse, using lysosomal cathepsin G, showed that PIII in gonococcal outer membranes could be cleaved by this enzyme (20). In addition, the rate at which PIII was degraded by cathepsin G could be correlated to the lipooligosaccharide expressed by a particular strain of gonococcus; i.e., the PIII associated with low-molecular-weight lipooligosaccharide was cleaved more rapidly than was the PIII associated with a higher-molecular-weight lipooligosaccharide. Surface-labeling experiments and the ability of PIII to react in vivo with a monoclonal antibody also indicated that PIII had surface-exposed domains (22). Our interest was piqued by several unanswered questions. We wanted to know (i) how this highly conserved, surface-exposed protein could exist in an organism that expended a great deal of its genome and energy in antigenic variation; (ii) how, if at all, PIII affected the functions of PI; and (iii) how PIII could be eliminated as a contaminant in preparations of PI. PURIFICATIONTo study PIII more directly, we developed a method for purifying it (13) and began to examine its biochemical and immunochemical characteristics. Early on, we observed that to purify PI, it was essential to separate PI from PIII in the initial extraction step. Otherwise, PIII remained tightly associated with PI throughout the subsequent steps of purification. We found that if the pH of the extraction buffer was altered, different proteins would be solubilized. If the extraction was performed at pH s 4, PI and PII were quantitatively released, but PIII remained with the cell debris (1). PIII was solubilized in rather pure form by reextraction of the cell debris with a pH 10.5 buffer. It was completely released with minimal PI contamination. The PIII was then purified by cation-exchange chromatography on CM-Sepharose, followed by gel filtration on Sephacryl-200 (13). The resulting product retained all the biochemical and immunochemical characteristics of the native PIII, except that, unlike the native molecule, purified PIII was highly susceptible to * Corresponding author.proteases. The amino acid composition and amino-terminal sequences of Pill isolated from several strains of Neis...

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Isolation and partial characterization of the reduction-modifiable protein of Neisseria gonorrhoeae.

Cited by 28 publications

References 14 publications

Innate Immune Function of the Neisserial Porins and the Relationship to Vaccine Adjuvant Activity

Innate Immune Function of the Neisserial Porins and the Relationship to Vaccine Adjuvant Activity

Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells

Protein III: structure, function, and genetics

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