We have confirmed that a subset of gonococcal iron- and Fur-regulated genes are expressed in men with uncomplicated gonococcal infections. Furthermore, we have demonstrated that these subjects exhibit antibodies to TbpA and TbpB proteins. In the majority of subjects with gonococcal infections, we detected fbpA
, and fur
transcripts. The Neisseria
Fur appears to act as a global regulator with the ability to act both as a repressor and as an activator of gene transcription. While several studies have recently demonstrated fur
expression during in vitro growth (14
), our study is the first to describe the expression of the fur
transcript during natural gonococcal infection (in 79% of infected samples).
Our studies also demonstrated that a high proportion of male subjects with uncomplicated gonococcal infections exhibited levels of IgG antibody against TbpA and TbpB antigens that were significantly higher than levels measured in uninfected controls. The majority of sera from infected subjects in our study also contained anti-PIA and anti-PIB IgG antibody levels that were elevated relative to the levels in control sera (Table ). Previous studies have shown measurable levels of IgG antibody to gonococcal porins in infected subjects (7
). Despite elevated levels of TbpA antibodies measured in gonorrhea-infected men, bactericidal function against TbpA is highly dependent on activity directed against native or conformational epitopes (1
). Several studies have also suggested that TbpB should be considered as a candidate for a possible vaccine against N. meningitidis
). TbpB antibodies can be measured in convalescent-phase sera from patients with meningococcal disease (18
); they are protective in a mouse model of infection, and they are also bactericidal in laboratory animals (24
). However, TbpB is highly variable in different strains of N. gonorrhoeae
and, taken together with lower tbpB
transcript amounts produced in subject samples, may explain why we observed lower titers of IgG antibody against TbpB antigen than against TbpA. The gene-specific primers that we used for RT-PCR may have lacked the homology necessary to recognize all the separate tbpB
genes. Interestingly, we have found that tbpB
, when examined by microarray analysis (unpublished data), is expressed at levels higher than those found with RT-PCR, such as we have reported here. In the microarray analysis, we used a 50-bp oligonucleotide conserved across all the known tbpB
genes to represent the tbpB
gene, compared to a 350-bp internal tbpB
fragment that was used here in RT-PCR analysis, containing both conserved and unique (variable) tbpB
Recently, Price et al. (35
) reported IgG anti-TbpA and -TbpB antibody levels similar to those we report here for gonorrhea-infected male subjects but indicated that these were not different from the levels in uninfected controls (35
). This may be explained by differences in the sources of control sera used to measure antibody specificity. In our study, control sera were obtained from normal volunteers with no previous history of neisserial disease and no contact with gonococcal antigens. In comparison, control sera used by Price et al. (35
) were heavily weighted to include subjects from a sexually transmitted disease clinic who were culture negative for N. gonorrhoeae
at the time blood was drawn for antibody determinations and who had no known prior history of gonococcal infection, reflecting antibody levels ~10 times higher than those seen in our controls and those found by others (19
In our study, a trend between tbpA gene expression and antibody levels in sera was observed only in subjects with initial gonococcal infections, suggesting that the increases in antibody levels over a low baseline (e.g., control sera) may come about from single gonococcal infections. Those with previous gonococcal infection(s) exhibited antibody levels, but this bore no relationship to tbpA gene expression at the time of the current infection and suggests the possibility of carryover of IgG antibodies from previous infection.
Cross-reactivity between gonococcal and meningococcal Tbp's cannot be ruled out. However, the following three observations reported here indicate that much of the IgG antibody against TbpA/TbpB was the result of past and present gonococcal infection. (i) Antibody levels in serum taken from male subjects with gonorrhea are displayed at significantly higher levels (7.7-fold higher for anti-TbpA and 4.6-fold higher for anti-TbpB) than those from normal sera obtained from individuals with no history of gonorrhea and no contact with gonococcal antigens. (ii) In N. gonorrhoeae-infected male subjects who also had prior histories of gonococcal infection, anti-TbpA and anti-TbpB levels were 8.3-fold higher than in the normal controls. These subjects did not show correlations of their antibody levels with normalized expressions of tbpA (expression ratios), indicating possible carryover of antibody from previous gonococcal infection. (iii) In first-time gonococcal infection, a correlation was found between anti-TbpA levels and normalized expression of tbpA (expression ratio) (Fig. ).
In conclusion, we have shown that iron-regulated and Fur-regulated fbpA, tbpA, tbpB, and fur genes are expressed in vivo and that men with gonorrhea express measurable antibodies in their sera directed against certain of these gene products (TbpA and TbpB). We have also demonstrated that the iron- and Fur-regulated genes are differentially expressed in mucosal samples. Levels of antibody to TbpAB are present in male subjects with uncomplicated gonorrhea; in the case of TbpA, antibody levels correlate with the expression of the tbpA gene.