Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA
Lipid-modified cupredoxin azurin (Laz) is involved in electron transport in Neisseria and proposed to act as an electron donor to the surface-displayed nitrite reductase AniA. We identified Laz in Neisseria gonorrhoeae cell envelopes and naturally elaborated membrane vesicles in proteomic investigat...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2018-11-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2018.02915/full |
| _version_ | 1828524812869304320 |
|---|---|
| author | Benjamin I. Baarda Ryszard A. Zielke Ann E. Jerse Aleksandra E. Sikora Aleksandra E. Sikora |
| author_facet | Benjamin I. Baarda Ryszard A. Zielke Ann E. Jerse Aleksandra E. Sikora Aleksandra E. Sikora |
| author_sort | Benjamin I. Baarda |
| collection | DOAJ |
| description | Lipid-modified cupredoxin azurin (Laz) is involved in electron transport in Neisseria and proposed to act as an electron donor to the surface-displayed nitrite reductase AniA. We identified Laz in Neisseria gonorrhoeae cell envelopes and naturally elaborated membrane vesicles in proteomic investigations focused on discovering new vaccine and therapeutic targets for this increasingly difficult to treat pathogen. Its surface exposure in N. meningitidis suggested Laz could be a vaccine candidate for N. gonorrhoeae. Here we characterized the localization, expression, and role of Laz within the gonococcal cell envelope and challenged the hypothesis that Laz and AniA interact. While we demonstrate that Laz indeed shows some good features of a vaccine antigen, such as stable expression, high conservation, and ability to elicit antibodies that cross-react with a diverse panel of Neisseria, it is not a surface-displayed lipoprotein in the gonococcus. This discovery eliminates Laz as a gonorrhea vaccine candidate, further highlighting the necessity of examining homologous protein localization between closely related species. Absence of Laz slightly altered cell envelope integrity but was not associated with growth defects in vitro, including during anoxia, implicating the presence of other electron pathways to AniA. To further dissect the implied AniA-Laz interaction, we utilized biolayer interferometry and optimized and executed chemical cross-linking coupled with immunoblotting to covalently link interacting protein partners in living gonococci. This method, applied for the first time in N. gonorrhoeae research to interrogate protein complexes, was validated by the appearance of the trimer form of AniA, as well as by increased formation of the β-barrel assembly machinery complex, in the presence of cross-linker. We conclude that Laz is not an electron donor to AniA based on their distinct subcellular localization, discordant expression during infection of the female mouse lower genital tract, and lack of interaction in vivo and in vitro. |
| first_indexed | 2024-12-11T20:54:37Z |
| format | Article |
| id | doaj.art-2666fd5fd9e340b59db1f73888b72900 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-11T20:54:37Z |
| publishDate | 2018-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-2666fd5fd9e340b59db1f73888b729002022-12-22T00:51:08ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2018-11-01910.3389/fmicb.2018.02915414537Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniABenjamin I. Baarda0Ryszard A. Zielke1Ann E. Jerse2Aleksandra E. Sikora3Aleksandra E. Sikora4Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, United StatesDepartment of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, United StatesDepartment of Microbiology and Immunology, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, United StatesDepartment of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, United StatesVaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, United StatesLipid-modified cupredoxin azurin (Laz) is involved in electron transport in Neisseria and proposed to act as an electron donor to the surface-displayed nitrite reductase AniA. We identified Laz in Neisseria gonorrhoeae cell envelopes and naturally elaborated membrane vesicles in proteomic investigations focused on discovering new vaccine and therapeutic targets for this increasingly difficult to treat pathogen. Its surface exposure in N. meningitidis suggested Laz could be a vaccine candidate for N. gonorrhoeae. Here we characterized the localization, expression, and role of Laz within the gonococcal cell envelope and challenged the hypothesis that Laz and AniA interact. While we demonstrate that Laz indeed shows some good features of a vaccine antigen, such as stable expression, high conservation, and ability to elicit antibodies that cross-react with a diverse panel of Neisseria, it is not a surface-displayed lipoprotein in the gonococcus. This discovery eliminates Laz as a gonorrhea vaccine candidate, further highlighting the necessity of examining homologous protein localization between closely related species. Absence of Laz slightly altered cell envelope integrity but was not associated with growth defects in vitro, including during anoxia, implicating the presence of other electron pathways to AniA. To further dissect the implied AniA-Laz interaction, we utilized biolayer interferometry and optimized and executed chemical cross-linking coupled with immunoblotting to covalently link interacting protein partners in living gonococci. This method, applied for the first time in N. gonorrhoeae research to interrogate protein complexes, was validated by the appearance of the trimer form of AniA, as well as by increased formation of the β-barrel assembly machinery complex, in the presence of cross-linker. We conclude that Laz is not an electron donor to AniA based on their distinct subcellular localization, discordant expression during infection of the female mouse lower genital tract, and lack of interaction in vivo and in vitro.https://www.frontiersin.org/article/10.3389/fmicb.2018.02915/fullNeisseria gonorrhoeaeLazcell envelopeanaerobic respirationvaccinemouse model |
| spellingShingle | Benjamin I. Baarda Ryszard A. Zielke Ann E. Jerse Aleksandra E. Sikora Aleksandra E. Sikora Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA Frontiers in Microbiology Neisseria gonorrhoeae Laz cell envelope anaerobic respiration vaccine mouse model |
| title | Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA |
| title_full | Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA |
| title_fullStr | Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA |
| title_full_unstemmed | Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA |
| title_short | Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA |
| title_sort | lipid modified azurin of neisseria gonorrhoeae is not surface exposed and does not interact with the nitrite reductase ania |
| topic | Neisseria gonorrhoeae Laz cell envelope anaerobic respiration vaccine mouse model |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2018.02915/full |
| work_keys_str_mv | AT benjaminibaarda lipidmodifiedazurinofneisseriagonorrhoeaeisnotsurfaceexposedanddoesnotinteractwiththenitritereductaseania AT ryszardazielke lipidmodifiedazurinofneisseriagonorrhoeaeisnotsurfaceexposedanddoesnotinteractwiththenitritereductaseania AT annejerse lipidmodifiedazurinofneisseriagonorrhoeaeisnotsurfaceexposedanddoesnotinteractwiththenitritereductaseania AT aleksandraesikora lipidmodifiedazurinofneisseriagonorrhoeaeisnotsurfaceexposedanddoesnotinteractwiththenitritereductaseania AT aleksandraesikora lipidmodifiedazurinofneisseriagonorrhoeaeisnotsurfaceexposedanddoesnotinteractwiththenitritereductaseania |