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Neisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme

Neisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme
Neisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme
The bacterial pathogen Neisseria gonorrhoeae (Gc) infects mucosal sites rich in antimicrobial proteins, including the bacterial cell wall-degrading enzyme lysozyme. Certain Gram-negative bacteria produce protein inhibitors that bind to and inhibit lysozyme. Here, we identify Ng_1063 as a new inhibitor of lysozyme in Gc, and we define its functions in light of a second, recently identified lysozyme inhibitor, Ng_1981. In silico analyses indicated that Ng_1063 bears sequence and structural homology to MliC-type inhibitors of lysozyme. Recombinant Ng_1063 inhibited lysozyme-mediated killing of a susceptible mutant of Gc and the lysozyme-sensitive bacterium Micrococcus luteus. This inhibitory activity was dependent on serine 83 and lysine 103 of Ng_1063, which are predicted to interact with lysozyme’s active site residues. Lysozyme co-immunoprecipitated with Ng_1063 and Ng_1981 from intact Gc. Ng_1063 and Ng_1981 protein levels were also increased in Gc exposed to lysozyme. Gc lacking both ng1063 and ng1981 was significantly more sensitive to killing by lysozyme than wild-type or single mutant bacteria. When exposed to human tears or saliva, in which lysozyme is abundant, survival of Δ1981Δ1063 Gc was significantly reduced compared to wild-type, and survival was restored upon addition of recombinant Ng_1981. Δ1981Δ1063 mutant Gc survival was additionally reduced in the presence of human neutrophils, which produce lysozyme. We found that while Ng_1063 was exposed on the surface of Gc, Ng_1981 was both in an intracellular pool and extracellularly released from the bacteria, suggesting that Gc employs these two proteins at multiple spatial barriers to fully neutralize lysozyme activity. Together, these findings identify Ng_1063 and Ng_1981 as critical components for Gc defense against lysozyme. These proteins may be attractive targets for antimicrobial therapy aimed to render Gc susceptible to host defenses and/or for vaccine development, both of which are urgently needed against drug-resistant gonorrhea.
1553-7366
Ragland, Stephanie A.
2ef087d5-75ae-47c3-9778-5336ba9b163f
Humbert, Maria V.
82134d25-24b8-4fdd-bd1c-461683b5322e
Christodoulides, Myron
eba99148-620c-452a-a334-c1a52ba94078
Criss, Alison K.
a9b3b040-4b93-48b2-97e9-e7ccc8a947f8
Ragland, Stephanie A.
2ef087d5-75ae-47c3-9778-5336ba9b163f
Humbert, Maria V.
82134d25-24b8-4fdd-bd1c-461683b5322e
Christodoulides, Myron
eba99148-620c-452a-a334-c1a52ba94078
Criss, Alison K.
a9b3b040-4b93-48b2-97e9-e7ccc8a947f8

Ragland, Stephanie A., Humbert, Maria V., Christodoulides, Myron and Criss, Alison K. (2018) Neisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme. PLoS Pathogens, 14 (7), [e1007080]. (doi:10.1371/journal.ppat.1007080).

Record type: Article

Abstract

The bacterial pathogen Neisseria gonorrhoeae (Gc) infects mucosal sites rich in antimicrobial proteins, including the bacterial cell wall-degrading enzyme lysozyme. Certain Gram-negative bacteria produce protein inhibitors that bind to and inhibit lysozyme. Here, we identify Ng_1063 as a new inhibitor of lysozyme in Gc, and we define its functions in light of a second, recently identified lysozyme inhibitor, Ng_1981. In silico analyses indicated that Ng_1063 bears sequence and structural homology to MliC-type inhibitors of lysozyme. Recombinant Ng_1063 inhibited lysozyme-mediated killing of a susceptible mutant of Gc and the lysozyme-sensitive bacterium Micrococcus luteus. This inhibitory activity was dependent on serine 83 and lysine 103 of Ng_1063, which are predicted to interact with lysozyme’s active site residues. Lysozyme co-immunoprecipitated with Ng_1063 and Ng_1981 from intact Gc. Ng_1063 and Ng_1981 protein levels were also increased in Gc exposed to lysozyme. Gc lacking both ng1063 and ng1981 was significantly more sensitive to killing by lysozyme than wild-type or single mutant bacteria. When exposed to human tears or saliva, in which lysozyme is abundant, survival of Δ1981Δ1063 Gc was significantly reduced compared to wild-type, and survival was restored upon addition of recombinant Ng_1981. Δ1981Δ1063 mutant Gc survival was additionally reduced in the presence of human neutrophils, which produce lysozyme. We found that while Ng_1063 was exposed on the surface of Gc, Ng_1981 was both in an intracellular pool and extracellularly released from the bacteria, suggesting that Gc employs these two proteins at multiple spatial barriers to fully neutralize lysozyme activity. Together, these findings identify Ng_1063 and Ng_1981 as critical components for Gc defense against lysozyme. These proteins may be attractive targets for antimicrobial therapy aimed to render Gc susceptible to host defenses and/or for vaccine development, both of which are urgently needed against drug-resistant gonorrhea.

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More information

Accepted/In Press date: 8 May 2018
e-pub ahead of print date: 5 July 2018
Published date: 5 July 2018

Identifiers

Local EPrints ID: 423108
URI: http://eprints.soton.ac.uk/id/eprint/423108
ISSN: 1553-7366
PURE UUID: b9c6ef4a-7dab-4cbc-8db8-5fb443ce999c
ORCID for Maria V. Humbert: ORCID iD orcid.org/0000-0002-5728-6981
ORCID for Myron Christodoulides: ORCID iD orcid.org/0000-0002-9663-4731

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Date deposited: 14 Aug 2018 16:30
Last modified: 26 Nov 2021 03:02

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Contributors

Author: Stephanie A. Ragland
Author: Maria V. Humbert ORCID iD
Author: Alison K. Criss

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