MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies

Abstract

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea.

Keywords: ABC transporters; MetQ; Neisseria gonorrhoeae; adhesins; gonorrhea; methionine; vaccine candidate.

FIG 1

Schematic of the gonococcal locus encoding MetQ. (A) Structure of the NGAG_00167-NGAG_00172 locus from N. gonorrhoeae strain 1291 containing the metNIQ operon (light gray) and flanking genes (white). Arrows represent open reading frames, with the genome locus tag being shown below the arrow and the known/probable function being shown above. The locations of conserved motifs and domains in metNIQ, as described in the text, are shown by dark gray boxes. OM, outer membrane. (B) Alignment of the sequence of the putative MetQ (NGAG_00171 gene locus) of Neisseria gonorrhoeae (Ng) with the GNA1946 sequence of Neisseria meningitidis (Nm), the MetQ sequence of Escherichia coli (Ec), and the Tp32 sequence (Tp0821 gene locus) of Treponema pallidum (Tp). The percent identity and the percent similarity of each protein to the MetQ of N. gonorrhoeae are indicated. The signal peptides of N. gonorrhoeae and N. meningitidis are shown, as are the amino acids involved in binding to methionine in the T. pallidum structure (*, identical in N. gonorrhoeae; #, not conserved in N. gonorrhoeae).

FIG 2

Cell surface localization of MetQ. (A) Western blot of the N. gonorrhoeae 1291 wild type (WT), metQ knockout (ΔmetQ), and complemented (C-metQ) strains using polyclonal anti-MetQ antibodies. The samples analyzed included whole-cell lysates, OMP fractions, and OMVs. (B) Coomassie-stained SDS-polyacrylamide gel and Western blot of recombinant His-tagged MetQ (rec. MetQ) probed with polyclonal antibodies raised against either heat-inactivated whole cells, OMPs, or OMVs of the N. gonorrhoeae 1291 wild type. (C) Flow cytometry of whole cells of the N. gonorrhoeae wild type, ΔmetQ, and C-metQ strains, with the expression of MetQ on the cell surface being determined by the detection of binding of polyclonal anti-MetQ antibody and secondary Alexa Fluor 488 anti-mouse immunoglobulin antibody. (D) Whole-cell ELISAs of untreated and trypsin-treated (+trypsin) N. gonorrhoeae wild type, ΔmetQ, and C-metQ strains using polyclonal anti-MetQ antibodies. The results for the negative control (−ve), containing secondary antibody only, are also shown. The graph shows the average absorbance at 450 nm from three independent replicates ±1 standard deviation. By Student's t test, P was <0.00001 for the ΔmetQ strain, the wild-type strain treated with trypsin, or the C-metQ strain treated with trypsin versus the wild type; P was <0.002 for the ΔmetQ strain versus the wild type or C-metQ strain; and P was >0.4 for the ΔmetQ strain, the wild-type strain treated with trypsin, or the C-metQ strain treated with trypsin versus the negative control. (E) Western blot analysis of whole-cell lysates of the N. gonorrhoeae wild type treated with trypsin for 0, 15, 30, or 60 min and probed with antibodies to MetQ or the periplasmic protein NGAG_01228 (meningococcal GNA1030/NUbp homologue).

FIG 3

l-Methionine binding to MetQ. (A) (i and ii) SPR sensograms (i) and line-of-best-fit curve (ii) of l-methionine binding to immobilized recombinant MetQ protein. The line of best fit was generated in Biacore T100 Evaluation software, with χ² values for all experiments falling under 0.1 of the maximum response units (R_max). Single-cycle kinetics were used to generate the K_D of the interactions between MetQ and the l-methionine substrate. (iii) SPR sensograms showing no interaction between the negative control (alanine) and immobilized MetQ. (B) ITC of the interaction of the N. gonorrhoeae 1291 wild-type (i), metQ knockout (ΔmetQ) (ii), and complemented (C-metQ) (iii) strains with l-methionine. The graphs show heat changes upon injection of l-methionine, which is dependent on the presence of MetQ.

FIG 4

Role of MetQ in adherence to cervical epithelial cells. Adherence and invasion of ME180 (A) and tCX (B) human cervical epithelial cells by the N. gonorrhoeae 1291 wild-type (WT), metQ knockout (ΔmetQ), and complemented (C-metQ) strains. Data represent the average percent adherence or invasion for triplicate samples as a percentage of that for the inoculum and are shown relative to the results obtained with the wild-type strain (the results for the wild type, set at 100%, are 2.9 × 10⁵ adherent CFU and 1.1 × 10⁴ invasive CFU for ME180 cells and 5.5 × 10⁶ adherent CFU and 1.3 × 10⁵ invasive CFU for tCX cells). Error bars represent ±1 standard deviation. ***, P ≤ 0.001 for the ΔmetQ strain relative to the wild type, using a two-tailed Student's t test. Experiments were performed on at least three occasions, and representative results are shown.

FIG 5

Role of MetQ in survival in monocytes, macrophages, and human serum. Survival of the N. gonorrhoeae 1291 wild-type (WT), metQ knockout (ΔmetQ), and complemented (C-metQ) strains with primary monocytes (A), activated macrophages (B), and normal human serum (NHS) (C). Data represent the average percent survival for triplicate samples as a percentage of the inoculum size and are shown relative to the results obtained with the wild-type strain (the results for the wild type, set at 100%, are 2.4 × 10⁴ CFU for monocytes, 1.2 × 10⁵ CFU for macrophages, 7.0 × 10⁴ CFU for 40% normal human serum, and 1.9 × 10⁴ CFU for 80% normal human serum). Error bars represent ±1 standard deviation. **, P ≤ 0.01 for the ΔmetQ strain relative to the wild type, using a two-tailed Student's t test; ***, P ≤ 0.001 for the ΔmetQ strain relative to the wild type, using a two-tailed Student's t test. Experiments were performed on at least three occasions, and representative results are shown.

FIG 6

Functional activity of anti-MetQ antibodies. (A) Serum bactericidal activity. The survival of the wild-type (WT) strain in the presence of a 1/10 dilution of preimmune serum (PI) or 2-fold dilutions of heat-inactivated anti-MetQ mouse polyclonal serum with 10% normal human serum (preabsorbed with N. gonorrhoeae) as a source of complement is shown. (B) Blocking of adherence to cervical epithelial cells. The adherence of the wild-type strain in the presence of a 1/20 dilution of preimmune serum or 2-fold dilutions of heat-inactivated anti-MetQ mouse polyclonal sera is shown. Data represent the average survival (A) or adherence (B) for triplicate samples as a percentage of the inoculum size and the adherence of the inoculum and are shown relative to the result obtained with the untreated wild-type strain (the results for the untreated wild type, set at 100%, are 1.8 × 10⁵ CFU for serum bactericidal activity and 9.1 × 10⁴ CFU for adherence). Error bars represent ±1 standard deviation. In both panels A and B, there was a statistically significant difference between groups, as determined by one-way ANOVA [F(9, 20) = 46.58 and P = 1.8 × 10⁻¹¹ and F(6, 21) = 32.5 and P = 1.4 × 10⁻⁹, respectively]. **, P ≤ 0.01 relative to the untreated wild type, using a two-tailed Student's t test; ***, P ≤ 0.001, relative to the untreated wild type, using a two-tailed Student's t test. Experiments were performed on at least three occasions, and representative results are shown.