Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA.

Benjamin I Baarda,Aleksandra E Sikora,Ryszard A Zielke,Ann E Jerse

doi:10.3389/fmicb.2018.02915

Abstract

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.

Highlights

Azurins are a class of small copper-containing proteins with a characteristic strong spectroscopic absorbance at 600 nm (Ainscough et al, 1987; Nobrega et al, 2016)
To evaluate lipid-modified azurin (Laz) as a potential gonorrhea vaccine candidate, we first examined the level of conservation between Laz homologs within all Neisseria species with sequences available on the PubMLST Neisseria database
To analyze Laz diversity among N. gonorrhoeae and other Neisseria species further, phylogenetic trees were constructed using the translated amino acid sequences from all 18 alleles found in N. gonorrhoeae (Figure 2B), as well as the 503 alleles found in all available Neisseria isolates (Supplementary Figure S1)

Summary

Introduction

Azurins are a class of small copper-containing proteins with a characteristic strong spectroscopic absorbance at 600 nm (Ainscough et al, 1987; Nobrega et al, 2016). Azurin demonstrates a strong redox potential (Sutherland and Wilkinson, 1963), which allows it to function as an electron carrier for periplasmic or membrane-bound components of the electron transport chain, especially between cytochrome c-551 and cytochrome oxidase (Holwerda et al, 1976; Farver and Pecht, 1991) This electron shuttling ability enables azurin to protect P. aeruginosa from oxidative stress in vitro (Vijgenboom et al, 1997). Azurin proteins are not universally distributed among bacteria, nor are they present in all pathogenic bacteria (Sutherland and Wilkinson, 1963) They are found in Neisseria species, including pathogenic N. meningitidis and N. gonorrhoeae, as well as commensal N. lactamica, N. flava, and N. perflava (Woods et al, 1989). Laz appears to be surface exposed in N. meningitidis, as illustrated by the sensitivity of a non-encapsulated strain to the bactericidal effect of anti-Laz antiserum (Deeudom et al, 2015)

Methods

Results

Conclusion