INFECTION AND IMMUNITY, July 2005, p. 4281–4287
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 7
The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed
during Natural Mucosal Gonococcal Infection
Sarika Agarwal,1Carol A. King,1Ellen K. Klein,1David E. Soper,2Peter A. Rice,1,3
Lee M. Wetzler,1,3and Caroline A. Genco1,3*
Department of Medicine, Section of Infectious Diseases,1and Department of Microbiology,3Boston University School
of Medicine, Boston, Massachusetts 02118, and Medical University of South Carolina,
Charleston, South Carolina 294252
Received 29 October 2004/Returned for modification 4 January 2005/Accepted 2 March 2005
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.
Neisseria gonorrhoeae, the causative agent of gonorrhea, is
one of the most common causes of sexually transmitted infec-
tions in the world, with over 62 million new cases estimated by
the World Health Organization in 1999 alone (see http:
Control of gonorrhea has been complicated by the develop-
ment of resistance to antimicrobial agents. Manifestations of
gonococcal disease include urethritis and epididymitis in men
and urethritis, cervicitis, salpingitis, and endometritis in
women. If left untreated, gonococcal infection in women may
lead to the development of pelvic inflammatory disease, which
can result in infertility or ectopic pregnancy. Recent data also
suggest that gonorrhea upregulates production of human im-
munodeficiency virus (HIV) in seminal plasma of men coin-
fected with both agents and is accompanied by increased trans-
mission of HIV to female sex partners (9).
Bacteria are limited in their capacity to multiply in vivo by
their hosts’ “iron-withholding” defense mechanism (32, 41).
Bacteria require iron (0.3 to 1.8 ?M) for optimal growth (6),
but as bacteria colonize and then proliferate in the host, they
utilize elaborate mechanisms to acquire iron from the host.
The best-characterized mechanism is to scavenge iron; this
involves the synthesis of siderophores, which bind iron with
high affinity. Pathogenic Neisseria, however, does not produce
siderophores, but instead has evolved outer membrane recep-
tors that bind directly to host iron sources, such as transferrin,
lactoferrin, and hemoglobin. All gonococcal isolates can utilize
iron from transferrin and hemoglobin (29), but only 50 to 70%
of strains can internalize iron bound to lactoferrin (28). The
transferrin receptor consists of a highly conserved integral
outer membrane receptor, TbpA (10), and a variable surface-
exposed lipoprotein, TbpB (2, 11). Together, these proteins
bind human transferrin, specifically facilitating the removal of
iron by Neisseria in an energy-dependent manner (12). Once
iron is removed from transferrin, it is bound by periplasmic
ferric binding protein (FbpA), which ferries it to a cytoplasmic
membrane acceptor (FbpB), where it is internalized by an
energy-dependent process (8). In the human male urethral
challenge model of gonococcal infection, expression of a func-
tional transferrin uptake system (but not necessarily the lacto-
ferrin system) is essential for gonococcal colonization after
urethral installation of the challenge inoculum, thereby em-
phasizing the importance of this system in human infection
The expression of genes that encode gonococcal transferrin-
binding proteins is controlled at the transcriptional level by the
iron-dependent regulatory protein Fur (ferric uptake regula-
* Corresponding author. Mailing address: Department of Medicine,
Section of Infectious Diseases, Boston University School of Medicine,
650 Albany Street, Room 637, Boston, MA 02118. Phone: (617) 414-
5305. Fax: (617) 414-5280. E-mail: firstname.lastname@example.org.
tory protein) (31). Fur functions as a general global regulator
and controls the expression of genes required for iron trans-
port and also controls genes that are required for virulence (20,
39). Fur forms a dimer with ferrous iron and binds to a con-
sensus sequence (Fur-box) that overlaps the promoters of iron-
regulated genes and results in inhibition of transcription. Al-
though Fur may also act as a positive regulator in controlling
gene expression (15–17, 25), the interactions between the op-
erator regions of the iron-activated genes have not been stud-
ied in detail. We have determined previously that the gono-
coccal Fur protein binds to the promoter regions of several
well-defined iron transport genes in Neisseria and to additional
genes involved in catabolic, secretory, and recombination path-
ways. These include tonB, fur, recN, secY, sodB, hemO, hmbR,
fumC, and the opa family of genes (39). Furthermore, we
recently demonstrated with DNA microarray technology, using
Neisseria meningitidis strain MC58, that ?10% of the entire
bacterial genome is regulated in response to growth with iron
(20). While these recent observations demonstrate that patho-
genic Neisseria may regulate the expression of specific genes
globally in response to in vitro iron, little is known about gene
expression in response to iron in vivo.
In this study, we have directly assessed the expression of the
iron- and Fur-regulated genes fbpA, tbpA, tbpB, and fur in
urethral samples obtained from male subjects with uncompli-
cated gonococcal infections. Levels of antibody directed to a
subset of the proteins encoded by these genes were also mea-
sured to assess the immunogenic capacities of these iron- and
Fur-regulated gene products when they are expressed in vivo.
MATERIALS AND METHODS
Study population. Male subjects 18 years of age and older with uncomplicated
gonorrhea were enrolled from the Public Health Clinics at Boston Medical
Center (BMC), Boston, Mass., and the Medical University of South Carolina
(MUSC), Charleston, S.C. Men were excluded if they had been treated with
antibiotics in the past month or were HIV infected. Informed consent was
obtained and a current and past sexual history recorded. Routine laboratory
examination of urethral swab specimens, including enumeration of polymorpho-
nuclear leukocytes and nucleic acid amplification testing for Chlamydia tracho-
matis, was performed. Separate urethral swabs were obtained for this study from
men who were diagnosed with gonococcal infections as evidenced by Gram’s
stains of urethral exudate that showed gram-negative intracellular diplococci or
who exhibited positive tests for neisserial H8 antigen by use of immunochro-
matographic detection assays (27). The diagnoses were confirmed by the growth
of N. gonorrhoeae on Thayer-Martin media or by positive hybridization tests
(Gen-Probe, San Diego, CA) or transcription-mediated amplification assays
(Gen-Probe, San Diego, CA) performed on the urethral specimens. The separate
urethral swabs to be used for this study were placed in 1 ml TRIZOL reagent
(Invitrogen, Carlsbad, CA) for subsequent RNA isolation and stored at ?80°C.
Specimens from MUSC were shipped on dry ice by overnight delivery to Boston
Medical Center, and specimens from both sites were processed within 2 days. All
55 samples were analyzed for fbpA and tbpA mRNA transcripts. Forty-seven
samples were tested for fur transcripts and 16 for tbpB transcripts. At MUSC,
sera were also collected to measure levels of immunoglobulin G (IgG) antibody
against gonococcal TbpA and TbpB antigens and gonococcal porin isoforms IA
(PIA) and IB (PIB), with the latter two used as control antigens. Control sera
were also obtained from five uninfected volunteers with no history of gonococcal
infection or contact with gonococcal antigens.
In vitro growth of N. gonorrhoeae strain F62 and RNA isolation. To determine
the minimal concentration of RNA required to detect specific gonococcal
mRNA transcripts by reverse transcriptase (RT)-PCR, in vitro, we grew N.
gonorrhoeae strain F62 and isolated RNA from organisms grown under iron-
depleted and iron-sufficient conditions. Strain F62 was grown in chemically
defined medium (CDM) supplemented with 4.2% NaHCO3and in CDM plus an
iron chelator, 25 ?M Desferal (CDM/25 ?M Desferal) (Ciba-Geigy), for 3 h
aerobically at 37°C. Organisms grown under iron-restricted conditions were then
washed, resuspended, divided, and inoculated into fresh CDM/12.5 ?M Desferal
(iron-depleted liquid cultures) or CDM/100 ?M ferric nitrate (iron-sufficient
liquid cultures), each beginning with an absorbance at 660 nm (A660) of 0.06.
Growth was monitored and samples collected hourly for a total of 5 h (30).
RT-PCR of N. gonorrhoeae strain F62 RNA. Total RNA was isolated from N.
gonorrhoeae strain F62 using the RNeasy kit (QIAGEN, Valencia, CA). Samples
were treated with DNase I (Invitrogen) before performing an RT-PCR using the
SuperScript one-step RT-PCR with the Platinum Taq kit (Invitrogen, Carlsbad,
CA). To the DNA-free RNA samples (200 ng), we added 25 ?l of 2? reaction
mix, 100 ng of each primer (Table 1), 1 ?l RT/Taq mix, and diethyl pyrocarbon-
ate (DEPC)-treated water to final volumes of 50 ?l. Samples were heated to 50°C
for 30 min and subsequently predenaturated at 94°C for 2 min. PCR amplifica-
tions were then carried out using the following parameters: denaturation at 94°C
for 30 s, annealing at 56°C for 45 s, and elongation at 72°C for 45 s, for 25 cycles.
For each sample, a control was also included to ensure the absence of DNA
contamination by performing a PCR lacking reverse transcriptase enzyme with
the isolated RNA sample using the rmp gene-specific primers for amplification.
RNA isolation and RT-PCR of clinical samples. Total RNA was isolated from
TRIZOL-preserved urethral swab specimens according to the manufacturer’s
instructions. Briefly, the sample in TRIZOL was repeatedly pipetted to disrupt
cells. The samples were incubated for 5 min at room temperature to permit
complete dissociation of nucleoprotein complexes, 0.25-ml portions of chloro-
form were added, and the samples were centrifuged at 12,000 ? g for 15 min at
4°C. Five milligrams of RNase-free glycogen and 0.5 ml of isopropyl alcohol were
introduced to precipitate nucleic acids for 15 min at room temperature, and the
pellets were washed with 75% ethanol (in DEPC-treated water). Pellets were
resuspended in DEPC-treated water, and DNase I (Invitrogen) treatment was
performed according to the manufacturer’s instructions. The total volume of
isolated RNA was divided equally for each amplification reaction, and all RT-
PCRs for a single specimen were performed simultaneously using parameters the
same as those described above for RNA from gonococcal strain F-62, except that
the number of amplification cycles was increased to 35. Amplification was per-
formed using gene-specific primers of gonococcal genes known to be regulated
by iron and Fur (fbpA, tbpA, tbpB, and fur) and by the constitutively expressed
rmp gene (Table 1). For each sample, a control sample was also included to
ensure the absence of DNA contamination of RNA prepared from specimen
samples by performing a PCR, lacking reverse transcriptase enzyme, with the
isolated RNA sample using rmp gene-specific primers for amplification.
Semiquantitative densitometry analysis of amplified cDNA bands. Amplified
cDNA fragments isolated by the RT-PCR methods indicated above were run on
a 1% agarose gel in 1? TAE (Tris-acetate-EDTA) buffer with 0.5 ?g/ml
ethidium bromide and then visualized under UV light (38). The density of each
DNA band on the 1% agarose gel was measured using Bio-Rad QUANTITY
TABLE 1. Primers used for RT-PCR in this study
aPrimers are written in the 5? to 3? direction.
4282AGARWAL ET AL.INFECT. IMMUN.
ONE 4.1.1 quantitation software. Background measurements were subtracted,
and a relative number was assigned to each band intensity (20).
Antigen production. TbpA protein was purified from F62 that was grown in
iron-deficient media using an affinity isolation procedure described earlier (5).
Gonococcal porins were purified by previously published purification procedures
using detergent extraction and column chromatography (4). Recombinant gono-
coccal outer membrane protein TbpB (36) was purified from Escherichia coli
(DH5?) expressing the maltose-binding fusion protein that contained TbpB by
affinity purification using an amylose resin column (36).
ELISA and antibody quantification. Levels of anti-TbpA- and anti-TbpB-
specific IgG antibody were measured in the sera of subjects infected with N.
gonorrhoeae and in the sera of uninfected volunteers by use of quantitative
enzyme-linked immunosorbent assay (ELISA) (40, 42). Antibodies directed
against PIA and PIB were measured as positive controls, because previous
studies have shown the presence of PIA- and PIB-specific antibodies in all sera
from subjects with local (7, 23, 26, 33, 34) or disseminated (21, 37) gonococcal
disease. The Mann-Whitney U test was used to compare IgG antibody levels in
sera from subjects with control levels. We also compared levels of IgG antibody
against TbpA and TbpB in subject sera with those in control sera by determining
the number of subject sera that displayed antibody levels greater than 2 standard
errors of the mean (SEMs) (geometric mean) above the geometric mean(s) of
control sera. Fisher’s exact test was used to assess the difference between levels
of IgG antibody levels against TbpA and TbpB in subjects and those of control
sera. A possible correlation between tbpA gene expression and TbpA isoantibody
levels was assessed with Pearson’s linear correlation (InStat; GraphPad, San
Sensitivity of RNA isolation and detection of gonococcal
transcripts. Total RNA isolated from cultures of N. gonor-
rhoeae strain F62 in iron-depleted and -sufficient conditions
was analyzed for differential gene expression of the iron- and
Fur-regulated fbpA, tbpA, tbpB, and fur genes. The rmp gene
was also amplified at different time points in iron-depleted and
-sufficient conditions, and no variability of expression was ob-
served between the growth conditions, indicating that the rmp
gene was constitutively expressed in each of the growth condi-
tions. We confirmed that in iron-depleted conditions, the ex-
pression of the iron-regulated genes was increased compared
to the expression of the constitutively expressed rmp gene (Fig.
1). Total RNA isolated from N. gonorrhoeae strain F62 grown
in iron-sufficient conditions was also utilized to determine the
sensitivity of RT-PCR under the experimental conditions used
in this study. We amplified an rmp PCR product with as little
as 1 ng of total RNA obtained from cultures grown in iron-
sufficient conditions (data not shown).
Detection of gonococcal transcripts in urethral specimens
from male subjects. Using the methodology described above,
we next analyzed gonococcal gene expression in specimens
from male subjects with uncomplicated gonococcal infections.
The total amount of RNA isolated from urethral specimens
(host plus organism) typically ranged from 50 ng to 600 ng.
Differential net gene expression of specific iron- and Fur-reg-
ulated genes from urethral specimens was assessed following
RT-PCR and semiquantitative densitometry analysis of each
amplified product. Each gene examined in this manner was
assigned a relative densitometry value with Bio-Rad QUAN-
TITY ONE 4.1.1 quantitation software, and a ratio of the
relative densitometry values of the fbpA, tbpA, tbpB, and fur
gene transcripts was calculated against the rmp value for each
specimen. A ratio (deemed the expression ratio) of ?1.0 was
arbitrarily taken to represent a decrease in gene expression
versus that of rmp mRNA, and an expression ratio of ?1.0 was
taken to represent increased gene expression compared to rmp
gene expression. Over 90% of gonorrhea-positive specimens
expressed the rmp gene by RT-PCR. Of the 55 rmp-positive
specimens, 51 (93%) expressed the fbpA gene (Table 2). The
genes encoding the transferrin-binding proteins TbpA and
TbpB were expressed, respectively, in 48 (87%) specimens
positive for rmp transcripts and in 14 out of 16 (86%) rmp-
positive specimens that were also tested for the tbpB transcript.
We also detected a fur transcript in 37 of 47 (79%) specimens
positive for rmp transcripts that were also tested for the fur
transcript (Table 2). None of the RNA specimens prepared
from urethral swab specimens were contaminated with DNA,
as determined by PCRs lacking reverse transcriptase enzyme
with rmp-specific primers (data not shown).
Levels of the amplified transcripts for the iron-regulated
genes, relative to that of the rmp gene (the expression ratio),
varied greatly from subject to subject (Fig. 2 and 3). Expression
ratios ranged from 0.03 to 17.4 for fbpA transcripts; from 0.01
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
TABLE 2. Expression found by RT-PCR of iron- and Fur-
regulated genes: fbpA, tbpA, fur, and tbpB transcripts and rmp
transcripts amplified from total RNA isolated from urethral
specimens from gonorrhea-infected males
% of specimens
(mean ? SEM)b
0.64 (0.23 ? 0.012)
0.66 (0.15 ? 0.013)
0.50 (0.07 ? 0.013)
0.22 (0.06 ? 0.014)
aWhen both genes were examined in the same specimen.
bMedians and geometric means (mean ? SEM) of the expression ratios
defined as the relative densitometry measurements of the amplified transcripts of
fbpA, tbpA, fur, and tbpB genes compared to the level of the constitutively
expressed rmp gene measured in the same specimens. The expression ratio of the
band intensity of the transcript of interest divided by the band intensity of the
rmp transcript in 1% agarose gels was measured using Bio-Rad quantitation
software (QUANTITY ONE 4.1.1).
VOL. 73, 2005Fur-REGULATED GENES EXPRESSED IN GONOCOCCAL ISOLATES4283
to 20.4 and from 0.01 to 20.1 for tbpA and fur transcripts,
respectively; and from 0.01 to 1.09 for the tbpB transcript (Fig.
2). Overall, 73% of the specimens exhibited expression ratios
for fbpA, tbpA, and fur transcripts of ?1.0, and 27% had ex-
pression ratios of ?1.0. Two specimens demonstrated no dif-
ferences in fbpA or tbpA gene expression compared to rmp
gene expression (expression ratio, 1.0). Although expression
ratios for fbpA, tbpA, and fur genes fell above (28%, 28%, and
24%) and below (72%, 72%, and 76%) 1.0, among the 14
specimens positive for tbpB transcripts, expression ratios were
?1.0 in 13.
Antibody responses to iron-regulated proteins in male sub-
jects. We measured IgG antibody responses to gonococcal
transferrin-binding proteins TbpA and TbpB in sera from male
subjects with gonorrhea by use of quantitative ELISA to assess
whether IgG antibody responses correlated with upregulation
of the genes encoding these proteins. IgG antibodies against
TbpA and TbpB were measured because they exhibit comple-
ment-dependent bactericidal activity in Neisseria-infected
mice, which may be protective in neisserial infection (24).
Levels of IgG antibody against TbpA ranged widely, from 10 to
6,970 ng/ml (median, 630 ng/ml; geometric mean ? SEM, 379
? 1.4 ng/ml). Levels of IgG against TbpB also ranged widely
(20 to 6,700 ng/ml) (median, 134 ng/ml; geometric mean ?
SEM, 164 ? 1.5 ng/ml) (Fig. 4). Measured levels of IgG anti-
body against TbpA and TbpB from gonorrhea-infected sub-
jects were significantly higher than the corresponding levels (49
? 1 and 36 ? 1.3 ng/ml) measured in control sera (P ? 0.003
and P ? 0.02, respectively). Ninety-one percent of subjects had
TbpA IgG antibody levels that exceeded the geometric mean
(? 2 SEM) level of the control sera (P ? 0.004), compared to
73% for TbpB (P ? 0.04). As expected, levels of IgG against
PIA and PIB antigens were also higher than levels in control
sera (Table 3). These results indicate that subjects exhibit
above-normal levels of IgG antibody to these iron-regulated
protein antigens during natural gonococcal infection. A corre-
lation of tbpA gene expression to antibody levels against TbpA
in sera from infected male specimens was determined by cal-
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.
4284 AGARWAL ET AL.INFECT. IMMUN.
culating the correlation coefficient using the InStat program
(version 3.06; GraphPad Software, San Diego, CA). The ex-
pression ratios of tbpA to rmp versus IgG antibody levels in
10/22 subjects infected with N. gonorrhoeae were plotted (Fig.
5). These 10 subjects reported first-time gonococcal infections;
the remaining 12 had had gonorrhea before, and some exhib-
ited elevated antibody levels in the absence of tbpA gene ex-
pression. The correlation coefficient (r) in the 10 subjects was
0.65 (P ? 0.04) (Fig. 5).
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 pro-
teins. In the majority of subjects with gonococcal infections, we
detected fbpA, tbpA, tbpB, 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 expres-
sion during in vitro growth (14, 39), 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 exhib-
ited levels of IgG antibody against TbpA and TbpB antigens
that were significantly higher than levels measured in unin-
fected 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 3). Previous studies have shown measurable levels of
IgG antibody to gonococcal porins in infected subjects (7, 23,
26, 40). Despite elevated levels of TbpA antibodies measured
in gonorrhea-infected men, bactericidal function against TbpA
is highly dependent on activity directed against native or con-
formational epitopes (1). Several studies have also suggested
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
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 gonococ-
cal infection (n ? 10) are represented as closed boxes. Infected sub-
jects with prior histories of gonococcal infection (n ? 12) are repre-
sented as open circles. The regression line and the r value were
determined for infected subjects with no known prior history of gono-
coccal 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 cor-
relation (InStat; GraphPad).
TABLE 3. Percentages of samples displaying levels of IgG antibody
against the indicated antigens from male subjects with
uncomplicated gonococcal infection greater than 2 SEM above the
geometric means of levels measured in control sera
% of samples
(no. of positive
aComparison of antibody levels in subjects and controls by Fisher’s exact test.
VOL. 73, 2005 Fur-REGULATED GENES EXPRESSED IN GONOCOCCAL ISOLATES4285
that TbpB should be considered as a candidate for a possible
vaccine against N. meningitidis infection (1–3, 19). TbpB anti-
bodies can be measured in convalescent-phase sera from pa-
tients with meningococcal disease (18, 19, 22); they are pro-
tective 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 sub-
ject 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 re-
ported here. In the microarray analysis, we used a 50-bp oli-
gonucleotide 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 sequences.
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 transmit-
ted 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, re-
flecting 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 gono-
coccal 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 anti-
bodies from previous infection.
Cross-reactivity between gonococcal and meningococcal
Tbp’s cannot be ruled out. However, the following three ob-
servations reported here indicate that much of the IgG anti-
body 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 sub-
jects 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 (ex-
pression 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. 5).
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 muco-
sal 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.
This study was supported by grants AI48611 (C.A.G.), AI40944
(L.M.W.), and U19AI38515 (P.A.R.).
We thank Andrea Dandridge, Cresene Sanglap, Stephanie Crane,
Rosalyn Liu, Faye Huang, and Linda Richard from Boston Medical
Center Public Health Clinic and Faye LeBoeuf, Beth Collins-Sharp,
Lisa Steed, Emily Betsille, and Sandy Hirschmann from the Medical
University of South Carolina, Charleston, for their invaluable help in
collecting subject samples.
1. Ala’Aldeen, D. A., P. Stevenson, E. Griffiths, A. R. Gorringe, L. I. Irons, A.
Robinson, S. Hyde, and S. P. Borriello. 1994. Immune responses in humans
and animals to meningococcal transferrin-binding proteins: implications for
vaccine design. Infect. Immun. 62:2984–2990.
2. Anderson, J. E., P. F. Sparling, and C. N. Cornelissen. 1994. Gonococcal
transferrin-binding protein 2 facilitates but is not essential for transferrin
utilization. J. Bacteriol. 176:3162–3170.
3. Archibald, F. S., and I. W. DeVoe. 1980. Iron acquisition by Neisseria men-
ingitidis in vitro. Infect. Immun. 27:322–334.
4. Blake, M. S., and E. C. Gotschlich. 1982. Purification and partial character-
ization of the major outer membrane protein of Neisseria gonorrhoeae. Infect.
5. Bonnah, R. A., R. Yu, and A. B. Schryvers. 1995. Biochemical analysis of
lactoferrin receptors in the Neisseriaceae: identification of a second bacterial
lactoferrin receptor protein. Microb. Pathog. 19:285–297.
6. Braun, V., and H. Killman. 1999. Bacterial solutions to the iron-supply
problem. Trends Biochem. Sci. 24:104–109.
7. Brooks, G. F., and C. J. Lammel. 1989. Humoral immune response to
gonococcal infections. Clin. Microbiol. Rev. 2(Suppl):S5–S10.
8. Chen, C. Y., S. A. Berish, S. A. Morse, and T. A. Mietzner. 1993. The ferric
iron-binding protein of pathogenic Neisseria spp. functions as a periplasmic
transport protein in iron acquisition from human transferrin. Mol. Microbiol.
9. Cohen, M. S., I. F. Hoffman, R. A. Royce, P. Kazembe, J. R. Dyer, C. C. Daly,
D. Zimba, P. L. Vernazza, M. Maida, S. A. Fiscus, J. J. Eron, Jr., et al. 1997.
Reduction of concentration of HIV-1 in semen after treatment of urethritis:
implications for prevention of sexual transmission of HIV-1. Lancet 349:
10. Cornelissen, C. N., J. E. Anderson, I. C. Boulton, and P. F. Sparling. 2000.
Antigenic and sequence diversity in gonococcal transferrin-binding protein
A. Infect. Immun. 68:4725–4735.
11. Cornelissen, C. N., J. E. Anderson, and P. F. Sparling. 1997. Characteriza-
tion of the diversity and the transferrin-binding domain of gonococcal trans-
ferrin-binding protein 2. Infect. Immun. 65:822–828.
12. Cornelissen, C. N., J. E. Anderson, and P. F. Sparling. 1997. Energy-depen-
dent changes in the gonococcal transferrin receptor. Mol. Microbiol. 26:25–
13. Cornelissen, C. N., M. Kelley, M. M. Hobbs, J. E. Anderson, J. G. Cannon,
M. S. Cohen, and P. F. Sparling. 1998. The transferrin receptor expressed by
gonococcal strain FA1090 is required for the experimental infection of hu-
man male volunteers. Mol. Microbiol. 27:611–616.
14. Delany, I., R. Rappuoli, and V. Scarlato. 2004. Fur functions as an activator
and as a repressor of putative virulence genes in Neisseria meningitidis. Mol.
15. Delany, I., G. Spohn, R. Rappuoli, and V. Scarlato. 2001. The Fur repressor
controls transcription of iron-activated and -repressed genes in Helicobacter
pylori. Mol. Microbiol. 42:1297–1309.
16. Dubrac, S., and D. Touati. 2000. Fur positive regulation of iron superoxide
dismutase in Escherichia coli: functional analysis of the sodB promoter. J.
17. Dubrac, S., and D. Touati. 2002. Fur-mediated transcriptional and post-
transcriptional regulation of FeSOD expression in Escherichia coli. Micro-
4286AGARWAL ET AL.INFECT. IMMUN.
18. Ferreiros, C. M., L. Ferron, and M. T. Criado. 1994. In vivo human immune
response to transferrin-binding protein 2 and other iron-regulated proteins
of Neisseria meningitidis. FEMS Immunol. Med. Microbiol. 8:63–68.
19. Gorringe, A. R., R. Borrow, A. J. Fox, and A. Robinson. 1995. Human
antibody response to meningococcal transferrin binding proteins: evidence
for vaccine potential. Vaccine 13:1207–1212.
20. Grifantini, R., S. Sebastian, E. Frigimelica, M. Draghi, E. Bartolini, A.
Muzzi, R. Rappuoli, G. Grandi, and C. A. Genco. 2003. Identification of
iron-activated and -repressed Fur-dependent genes by transcriptome analysis
of Neisseria meningitidis group B. Proc. Natl. Acad. Sci. USA 100:9542–9547.
21. Hadfield, S. G., and A. A. Glynn. 1982. Analysis of antibodies in local and
disseminated Neisseria gonorrhoeae infections by means of gel electrophore-
sis-derived ELISA. Immunology 47:283–288.
22. Johnson, A. S., A. R. Gorringe, A. J. Fox, R. Borrow, and A. Robinson. 1997.
Analysis of the human Ig isotype response to individual transferrin binding
proteins A and B from Neisseria meningitidis. FEMS Immunol. Med. Micro-
23. Lammel, C. J., R. L. Sweet, P. A. Rice, J. S. Knapp, G. K. Schoolnik, D. C.
Heilbron, and G. F. Brooks. 1985. Antibody-antigen specificity in the im-
mune response to infection with Neisseria gonorrhoeae. J. Infect. Dis. 152:
24. Lissolo, L., G. Maitre-Wilmotte, P. Dumas, M. Mignon, B. Danve, and M. J.
Quentin-Millet. 1995. Evaluation of transferrin-binding protein 2 within the
transferrin-binding protein complex as a potential antigen for future menin-
gococcal vaccines. Infect. Immun. 63:884–890.
25. Masse, E., and S. Gottesman. 2002. A small RNA regulates the expression
of genes involved in iron metabolism in Escherichia coli. Proc. Natl. Acad.
Sci. USA 99:4620–4625.
26. McMillan, A., G. McNeillage, and H. Young. 1979. Antibodies to Neisseria
gonorrhoeae: a study of the urethral exudates of 232 men. J. Infect. Dis.
27. McQuillen, D. P., S. Gulati, S. Ram, A. K. Turner, D. B. Jani, T. C. Heeren,
and P. A. Rice. 1999. Complement processing and immunoglobulin binding
to Neisseria gonorrhoeae determined in vitro simulates in vivo effects. J. In-
fect. Dis. 179:124–135.
28. Mickelsen, P. A., E. Blackman, and P. F. Sparling. 1982. Ability of Neisseria
gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to ob-
tain iron from lactoferrin. Infect. Immun. 35:915–920.
29. Mickelsen, P. A., and P. F. Sparling. 1981. Ability of Neisseria gonorrhoeae,
Neisseria meningitidis, and commensal Neisseria species to obtain iron from
transferrin and iron compounds. Infect. Immun. 33:555–564.
30. Morse, S. A., and L. Bartenstein. 1980. Purine metabolism in Neisseria
gonorrhoeae: the requirement for hypoxanthine. Can. J. Microbiol. 26:13–20.
31. Morton, D. J., J. M. Musser, and T. L. Stull. 1993. Expression of the
Haemophilus influenzae transferrin receptor is repressible by hemin but not
elemental iron alone. Infect. Immun. 61:4033–4037.
32. Payne, S. M. 1993. Iron acquisition in microbial pathogenesis. Trends Mi-
33. Plummer, F. A., H. Chubb, J. N. Simonsen, M. Bosire, L. Slaney, I. Maclean,
J. O. Ndinya-Achola, P. Waiyaki, and R. C. Brunham. 1993. Antibody to
Rmp (outer membrane protein 3) increases susceptibility to gonococcal
infection. J. Clin. Investig. 91:339–343.
34. Plummer, F. A., H. Chubb, J. N. Simonsen, M. Bosire, L. Slaney, N. J.
Nagelkerke, I. Maclean, J. O. Ndinya-Achola, P. Waiyaki, and R. C. Brun-
ham. 1994. Antibodies to opacity proteins (Opa) correlate with a reduced
risk of gonococcal salpingitis. J. Clin. Investig. 93:1748–1755.
35. Price, G. A., M. M. Hobbs, and C. N. Cornelissen. 2004. Immunogenicity of
gonococcal transferrin binding proteins during natural infections. Infect.
36. Retzer, M. D., R. H. Yu, and A. B. Schryvers. 1999. Identification of se-
quences in human transferrin that bind to the bacterial receptor protein,
transferrin-binding protein B. Mol. Microbiol. 32:111–121.
37. Rice, P. A., H. E. Vayo, M. R. Tam, and M. S. Blake. 1986. Immunoglobulin
G antibodies directed against protein III block killing of serum-resistant
Neisseria gonorrhoeae by immune serum. J. Exp. Med. 164:1735–1748.
38. Sambrook, J., and D. Russel (ed.). 2001. Molecular cloning: a laboratory
manual, 3rd ed., vol. 1, p. 5.14–5.17. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.
39. Sebastian, S., S. Agarwal, J. R. Murphy, and C. A. Genco. 2002. The gono-
coccal Fur regulon: identification of additional genes involved in major
catabolic, recombination, and secretory pathways. J. Bacteriol. 184:3965–
40. Simpson S. D., Y. Ho, P. A. Rice, and L. M. Wetzler. 1999. T lymphocyte
response to Neisseria gonorrhoeae porin in individuals with mucosal gono-
coccal infections. J. Infect. Dis. 180:762–773.
41. Weinberg, E. D. 1993. The development of awareness of iron-withholding
defense. Perspect. Biol. Med. 36:215–221.
42. Wetzler, L. M., M. S. Blake, K. Barry, and E. C. Gotschlich. 1992. Gono-
coccal porin vaccine evaluation: comparison of Por proteosomes, liposomes,
and blebs isolated from rmp deletion mutants. J. Infect. Dis. 166:551–555.
Editor: V. J. DiRita
VOL. 73, 2005Fur-REGULATED GENES EXPRESSED IN GONOCOCCAL ISOLATES4287