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Neisseria gonorrhoeae metalloprotease NGO1686 is required for full piliation, and piliation is required for resistance to H2O2- and neutrophil-mediated killing.

Stohl EA, Dale EM, Criss AK, Seifert HS - MBio (2013)

Bottom Line: The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan.Successful infectious agents need to overcome host defense systems to establish infection.We also show that to express the pilus, an enzyme needs to partially degrade the cell wall of the bacterium.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.

ABSTRACT

Unlabelled: The sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogen Neisseria gonorrhoeae. Type IV pili are an essential virulence factor uniformly expressed on clinical gonococcal isolates and are required for several aspects of gonococcal pathogenesis, including adherence to host tissues, autoagglutination, twitching motility, and the uptake of DNA during transformation. Symptomatic gonococcal infection is characterized by the influx of neutrophils or polymorphonuclear leukocytes (PMNs) to the site of infection. PMNs are a key component of gonococcal pathogenesis, mediating the innate immune response through the use of oxidative and nonoxidative killing mechanisms. The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan. We report that the effect of NGO1686 on survival after exposure to H2O2 and PMNs is mediated through its role in elaborating pili and that nonpiliated mutants of N. gonorrhoeae are less resistant to killing by H2O2, LL-37, and PMNs than the corresponding piliated strains. These findings add to the various virulence-associated functions attributable to gonococcal pili and may explain the selection basis for piliation in clinical isolates of N. gonorrhoeae.

Importance: Successful infectious agents need to overcome host defense systems to establish infection. We show that the Neisseria pilus, a major virulence factor of this organism, which causes gonorrhea, helps protect the bacterium from two major killing mechanisms used by the host to combat infections. We also show that to express the pilus, an enzyme needs to partially degrade the cell wall of the bacterium.

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Effect of pilT mutation on the RM11.2nvmpg (Δ1686) piliation state and H2O2 resistance. (A and B) Representative immunoelectron micrographs of immunogold-labeled pilus bundles on strain RM11.2nvmpg with regulatable pilT grown on solid medium in the presence (B) or absence (A) of IPTG and lifted onto Formvar-coated grids. (C) H2O2 resistance of the strain in the presence and absence of IPTG. Cells were treated with the indicated doses of H2O2 for 15 min, and the relative survival at each dose was calculated. Error bars represent the standard errors of the means of results from 2 to 8 experiments. *, P > 0.48.
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fig8: Effect of pilT mutation on the RM11.2nvmpg (Δ1686) piliation state and H2O2 resistance. (A and B) Representative immunoelectron micrographs of immunogold-labeled pilus bundles on strain RM11.2nvmpg with regulatable pilT grown on solid medium in the presence (B) or absence (A) of IPTG and lifted onto Formvar-coated grids. (C) H2O2 resistance of the strain in the presence and absence of IPTG. Cells were treated with the indicated doses of H2O2 for 15 min, and the relative survival at each dose was calculated. Error bars represent the standard errors of the means of results from 2 to 8 experiments. *, P > 0.48.

Mentions: The underpiliated phenotype of the mpg mutant was similar to phenotypes described for pathogenic Neisseria strains with mutations in pilus assembly genes. For a subset of these mutants, concomitant mutation of the retraction ATPase gene, pilT, restores piliation (4, 5, 38). We therefore introduced an IPTG-regulatable pilT (reg pilT) construct (37) into the FA1090 (RM11.2nv) mpg mutant background and used immunogold transmission EM (immunogold-TEM) to analyze piliation. In the presence of IPTG, strain RM11.2nvmpg reg pilT expressed an average of 0.6 pilus bundle per gonococcal cell (Fig. 8B), a number of bundles similar to that of strain RM11.2nvmpg (Fig. 2D to F). However, in the absence of IPTG, the strain expressed an average of 3.3 pilus bundles per gonococcal cell (Fig. 8A), a number of bundles nearly equivalent to that of strain RM11.2nv (Fig. 2A to C). By analogy with Neisseria pilus mutants having similar pilT-dependent phenotypes, this suggested a role for NGO1686 in pilus biogenesis.


Neisseria gonorrhoeae metalloprotease NGO1686 is required for full piliation, and piliation is required for resistance to H2O2- and neutrophil-mediated killing.

Stohl EA, Dale EM, Criss AK, Seifert HS - MBio (2013)

Effect of pilT mutation on the RM11.2nvmpg (Δ1686) piliation state and H2O2 resistance. (A and B) Representative immunoelectron micrographs of immunogold-labeled pilus bundles on strain RM11.2nvmpg with regulatable pilT grown on solid medium in the presence (B) or absence (A) of IPTG and lifted onto Formvar-coated grids. (C) H2O2 resistance of the strain in the presence and absence of IPTG. Cells were treated with the indicated doses of H2O2 for 15 min, and the relative survival at each dose was calculated. Error bars represent the standard errors of the means of results from 2 to 8 experiments. *, P > 0.48.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3735123&req=5

fig8: Effect of pilT mutation on the RM11.2nvmpg (Δ1686) piliation state and H2O2 resistance. (A and B) Representative immunoelectron micrographs of immunogold-labeled pilus bundles on strain RM11.2nvmpg with regulatable pilT grown on solid medium in the presence (B) or absence (A) of IPTG and lifted onto Formvar-coated grids. (C) H2O2 resistance of the strain in the presence and absence of IPTG. Cells were treated with the indicated doses of H2O2 for 15 min, and the relative survival at each dose was calculated. Error bars represent the standard errors of the means of results from 2 to 8 experiments. *, P > 0.48.
Mentions: The underpiliated phenotype of the mpg mutant was similar to phenotypes described for pathogenic Neisseria strains with mutations in pilus assembly genes. For a subset of these mutants, concomitant mutation of the retraction ATPase gene, pilT, restores piliation (4, 5, 38). We therefore introduced an IPTG-regulatable pilT (reg pilT) construct (37) into the FA1090 (RM11.2nv) mpg mutant background and used immunogold transmission EM (immunogold-TEM) to analyze piliation. In the presence of IPTG, strain RM11.2nvmpg reg pilT expressed an average of 0.6 pilus bundle per gonococcal cell (Fig. 8B), a number of bundles similar to that of strain RM11.2nvmpg (Fig. 2D to F). However, in the absence of IPTG, the strain expressed an average of 3.3 pilus bundles per gonococcal cell (Fig. 8A), a number of bundles nearly equivalent to that of strain RM11.2nv (Fig. 2A to C). By analogy with Neisseria pilus mutants having similar pilT-dependent phenotypes, this suggested a role for NGO1686 in pilus biogenesis.

Bottom Line: The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan.Successful infectious agents need to overcome host defense systems to establish infection.We also show that to express the pilus, an enzyme needs to partially degrade the cell wall of the bacterium.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.

ABSTRACT

Unlabelled: The sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogen Neisseria gonorrhoeae. Type IV pili are an essential virulence factor uniformly expressed on clinical gonococcal isolates and are required for several aspects of gonococcal pathogenesis, including adherence to host tissues, autoagglutination, twitching motility, and the uptake of DNA during transformation. Symptomatic gonococcal infection is characterized by the influx of neutrophils or polymorphonuclear leukocytes (PMNs) to the site of infection. PMNs are a key component of gonococcal pathogenesis, mediating the innate immune response through the use of oxidative and nonoxidative killing mechanisms. The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan. We report that the effect of NGO1686 on survival after exposure to H2O2 and PMNs is mediated through its role in elaborating pili and that nonpiliated mutants of N. gonorrhoeae are less resistant to killing by H2O2, LL-37, and PMNs than the corresponding piliated strains. These findings add to the various virulence-associated functions attributable to gonococcal pili and may explain the selection basis for piliation in clinical isolates of N. gonorrhoeae.

Importance: Successful infectious agents need to overcome host defense systems to establish infection. We show that the Neisseria pilus, a major virulence factor of this organism, which causes gonorrhea, helps protect the bacterium from two major killing mechanisms used by the host to combat infections. We also show that to express the pilus, an enzyme needs to partially degrade the cell wall of the bacterium.

Show MeSH
Related in: MedlinePlus