The gonococcal Fur-regulated tbpA and tbpB genes are expressed during natural mucosal gonococcal infection

Abstract

Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

FIG. 1.

RT-PCR analysis of N. gonorrhoeae strain F62. Differential iron-regulated gene expression was monitored in growing cultures of N. gonorrhoeae strain F62 under iron-depleted (−) and -sufficient (+) conditions. Total bacterial RNA was isolated from a culture sample collected at 2 h, and RT-PCR was performed as described in Materials and Methods.

FIG. 2.

Ratio of densitometry measurements of iron-regulated fbpA, tbpA, fur, and tbpB genes to the constitutively expressed rmp gene, termed the expression ratio. Each individual specimen is represented by a distinct symbol. The median value of the expression ratio for each gene is marked as a straight line.

FIG. 3.

Differential expression of iron-regulated fbpA, tbpA, and fur genes found by RT-PCR in three urethral specimens from males with gonococcal infections (#1, #2, #3). The expression ratio is indicated below each lane.

FIG. 4.

Levels of IgG antibody in sera from male subjects with uncomplicated gonococcal infection directed against TbpA purified from gonococcal strain F62 and recombinant TbpB antigens (see Materials and Methods). Levels are represented as (log₁₀) ng/ml; values in control sera for antibodies against specific antigens are depicted as TbpA-C and TbpB-C (anti-PIA and -PIB antibody levels in infected subjects are also compared to control levels). The data are represented as box-whisker plots, in which the lower and upper levels of the boxes represent the 25th and 75th percentiles, respectively, and the whiskers represent the ranges of data points; the median values are depicted as horizontal lines in the boxes.

FIG. 5.

Expression ratios of the tbpA gene to the rmp gene versus levels of anti-TbpA IgG displayed for 22 gonorrhea-infected male subjects. Each symbol represents the expression ratio for an individual subject. Infected subjects with no known history of previous gonococcal infection (n = 10) are represented as closed boxes. Infected subjects with prior histories of gonococcal infection (n = 12) are represented as open circles. The regression line and the r value were determined for infected subjects with no known prior history of gonococcal infection (r = 0.65; P = 0.04). There was no correlation in infected subjects with known prior histories of gonococcal infection (note that three subjects had antibody levels whose expression ratios were minimal). The r value was calculated using Pearson's linear correlation (InStat; GraphPad).