Neisseria gonorrhoeae Virulence Factor NG1686 Is a Bifunctional M23B Family Metallopeptidase That Influences Resistance to Hydrogen Peroxide and Colony Morphology

Stohl, Elizabeth A.; Chan, Yolande A.; Hackett, Kathleen T.; Kohler, Petra L.; Dillard, Joseph P.; Seifert, H. Steven

doi:10.1074/jbc.m111.338830

Cited by 29 publications

(40 citation statements)

References 56 publications

(58 reference statements)

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“…PG was purified from N. gonorrhoeae with modifications as described previously (12). Zymogram analysis was performed as described previously (12,20).…”

Section: Methodsmentioning

confidence: 99%

“…PG was purified from N. gonorrhoeae with modifications as described previously (12). Zymogram analysis was performed as described previously (12,20). E. coli BL21(DE3) cell extracts carrying pET/His-NlpD were electrophoresed on a 12% SDS gel containing 0.1% (wt/vol) N. gonorrhoeae murein sacculi using 75 V at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Mutation of the predicted M23 active sites revealed that only two of the four M23 active sites are important for Mpg-associated phenotypes and activities. Specifically, a mutation in the predicted catalytic histidine residue has no effect on phenotypes conferred by the Mpg protein (12). In this current work, we have characterized an Mpg paralog, NlpD (NG1056; E value, 2eϪ12).…”

mentioning

confidence: 97%

“…Finally, although E. coli has three redundant amidases, N. gonorrhoeae strain FA1090 is predicted to contain only the AmiC amidase, and a gonococcal amiC mutant shows a defect in cell separation (11). We previously showed that the mpg gene product of N. gonorrhoeae (also called NGO1686) is a LytM protein and a bifunctional M23 metallopeptidase, possessing and exhibiting endopeptidase as well as carboxypeptidase activities on PG (12). Although an mpg mutant has no defects in cell separation, we demonstrated that Mpg alters gonococcal resistance to polymorphonuclear leukocyte (PMN)-mediated oxidative and nonoxidative killing mechanisms through its activity in type IV pilus expression (13).…”

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confidence: 99%

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The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

et al. 2016

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Key steps in bacterial cell division are the synthesis and subsequent hydrolysis of septal peptidoglycan (PG), which allow efficient separation of daughter cells. Extensive studies in the Gram-negative, rod-shaped bacterium Escherichia coli have revealed that this hydrolysis is highly regulated spatially and temporally. Neisseria gonorrhoeae is an obligate Gram-negative, diplococcal pathogen and is the only causative agent of the sexually transmitted infection gonorrhea. We investigated how cell separation proceeds in this diplococcal organism. We demonstrated that deletion of the nlpD gene in strain FA1090 leads to poor growth and to an altered colony and cell morphology. An isopropyl-beta-D-galactopyranoside (IPTG)-regulated nlpD complemented construct can restore these defects only when IPTG is supplied in the growth medium. Thin-section transmission electron microscopy (TEM) revealed that the nlpD mutant strain grew in large clumps containing live and dead bacteria, which was consistent with deficient cell separation. Biochemical analyses of purified NlpD protein showed that it was able to bind purified PG. Finally, we showed that, although NlpD has no hydrolase activity itself, NlpD potentiates the hydrolytic activity of AmiC. These results indicate that N. gonorrhoeae NlpD is required for proper cell growth and division through its interactions with the amidase AmiC. IMPORTANCE N. gonorrhoeae is the sole causative agent of the sexually transmitted infection gonorrhea. The incidence of antibiotic-resistant gonococcal infections has risen sharply in recent years, and N. gonorrhoeae has been classified as a "superbug" by the CDC. Since there is a dearth of new antibiotics to combat gonococcal infections, elucidating the essential cellular process of N. gonorrhoeae may point to new targets for antimicrobial therapies. Cell division and separation is one such essential process. We identified and characterized the gonococcal nlpD gene and showed that it is essential for cell separation. In contrast to other pathogenic bacteria, the gonococcal system is streamlined and does not appear to have any redundancies.

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“…PG was purified from N. gonorrhoeae with modifications as described previously (12). Zymogram analysis was performed as described previously (12,20).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 97%

mentioning

confidence: 99%

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The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

et al. 2016

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“…N. gonorrhoeae may attach to the spermatozoa by pili as T1 gonococci or by direct contact as T4 gonococci (113). Gonococcal infection caused by N. gonorrhoeae triggers the flow of PMNs into the infected tissue (114). As mentioned previously, the presence of leucocytes in the genital tract can increase the level of reactive oxygen species which have harmful effects on spermatozoa (40,42).…”

Section: Escherichia Colimentioning

confidence: 96%