Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA

Abstract

Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of N. gonorrhoeae necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of N. gonorrhoeae anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC 50 value of C7-3 and C7-3m2 against anaerobically grown N. gonorrhoeae was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development.

Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants

“…Compared with WHO F, PorB was present at similar levels only in WHO G and N and was downregulated in 12 strains. PilQ (96) was ubiquitously expressed in 10 strains, whereas expression of Opa proteins was widely variable, as expected (104,105). The TbpA level was similar in 8 strains; however, we did not detect TbpB (106).…”

Genetic Similarity of Gonococcal Homologs to Meningococcal Outer Membrane Proteins of Serogroup B Vaccine

“…Among common OMPs identified in our study, PorB, PilQ, Omp85 (BamA), OpcA, LbpA, NspA, MtrE, MetQ, TbpB, and AniA have been experimentally tested as potential gonorrhea vaccine antigens due to their immunogenic and antigonococcal properties in vitro and/or in a mouse model (9,12,13,41). Previous findings describe the significance of MetQ (60) and AniA (44)(45)(46) as targets for antigonococcal vaccines and therapeutic intervention. Of those previously tested as gonorrhea vaccine candidates, PilQ, Omp85 (BamA), NspA, MtrE, MetQ, and LbpA have been identified within OMVs of the NmB strain NZ98/254 (24,30,32,34) and had Ն93% amino acid sequence similarity to N. gonorrhoeae, suggesting their potential contribution to mechanisms of cross-protection.…”

“…Other major OMV proteins, such as FetA, PilQ, Omp85 (BamA), RmpM, and MtrE of NZ98/298 consistently showed Ն93% mean amino acid sequence similarity to and within the N. gonorrhoeae isolates. The NZ98/298 nitrite reductase AniA, essential for N. gonorrhoeae anaerobic respiration and biofilm formation (44)(45)(46), showed 84% and 99% sequence similarity to and within N. gonorrhoeae isolates, respectively ( Table 2).…”

Quantitative proteomics of the 2016 WHONeisseria gonorrhoeaereference strains surveys vaccine candidates and antimicrobial resistance determinants

“…AniA was present at different levels in 7 strains, ubiquitous in five, and up-regulated in two isolates. Immunoblotting experiments with anti-AniA antisera corroborated these findings (105). The cellular pool of NspA (113) varied in ten isolates, while lactoferrin binding protein LbpA (114) was variable in five strains and was ubiquitous in WHO L and G (Table 2).…”

“…We first evaluated expression profiles of extensively studied gonorrhea vaccine candidates including MtrE (93–95), PorB (96, 97), PilQ (98), TbpA (99, 100), Opa (101, 102), and AniA (19, 103–105). MtrE and PorB were up-and down-regulated, respectively, in 12 isolates (Table 2).…”