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J Infect Dis. Author manuscript; available in PMC Sep 22, 2009.
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Thioredoxin Reductase Is Essential for Protection of Neisseria gonorrhoeae against Killing by Nitric Oxide and for Bacterial Growth during Interaction with Cervical Epithelial Cells


In Neisseria gonorrhoeae, the MerR family transcription factor NmlR activates 3 operons in response to disulfide stress. In the present study, we show that trxB, a monocistronic operon under the control of NmlR, encodes a functional thioredoxin reductase. It is shown that neisserial TrxB has biochemical properties similar to those of its homologue from Escherichia coli. Analysis of a trxB mutant of N. gonorrhoeae showed that it was more sensitive to disulfide stress and to stress induced by organic hydroperoxides, superoxide, and nitric oxide than wild-type gonococcus. TrxB was found to be essential for the microaerobic induction of aniA and norB, the genes encoding nitrite reductase and nitric oxide reductase, respectively. The importance of TrxB during natural infection was demonstrated by the fact that the survival of gonococci within human cervical epithelial cells, as well as biofilm formation on these cells, was greatly reduced for a trxB mutant compared with a wild-type strain.

Neisseria gonorrhoeae is an obligate human pathogen that primarily colonizes the genitourinary tract, resulting in the sexually transmitted infectious disease gonorrhea. N. gonorrhoeae is associated with inflamed mucosa of the urethra in males [13] but often results in asymptomatic infection of the cervix in females [36]. A key component of the innate immune response during N. gonorrhoeae infection is the production of reactive oxygen and nitrogen species. These compounds are also unavoidable by-products of bacterial metabolic processes and, in addition, are produced by commensal microorganisms occupying the same environmental niche as the gonococcus. Some of the strategies used by N. gonorrhoeae to combat oxidative and nitrosative stress have been characterized (reviewed in [7]). We have previously described a transcriptional regulator, NmlR, in N. gonorrhoeae that responds to disulfide stress [8]. NmlR is a transcription factor belonging to the MerR family of regulators and binds to 2 sites on the N. gonorrhoeae chromosome. The first site exhibits dyad symmetry between the divergent and overlapping promoters of nmlR (NGO0602) and adhC (NGO0601), and a second binding site has a similar arrangement between trxB (NGO0580) and the divergent gene copA (NGO0579) (figure 1). NmlR was shown to act as both a repressor and an activator of adhC, trxB, and copA, with activation occurring in the presence of the thiol-specific oxidant diamide. Analysis of AdhC in Escherichia coli and many eukaryotic systems has suggested that it plays a role in the defense against reactive nitrogen species through metabolism of S-nitrosoglutathione [911]. However, we have found that adhC of N. gonorrhoeae is a pseudo-gene resulting from a frameshift mutation that leads to a premature stop codon [12]. trxB is annotated in the N. gonorrhoeae genome sequence database (http://stdgen.northwestern.edu) as encoding a putative thioredoxin reductase that is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoproteins (which also includes glutathione reductase, mercuric reductase, and dihydrolipoamide dehydrogenase) [13]. Thioredoxin reductase catalyzes the NADPH-dependent reduction of thioredoxin [14]. Thioredoxin reductase and thioredoxin can act as an electron donor for ribo-nucleotide reductase [15] but play a wider role in cellular redox processes, including defense against oxidative stress [16]. The present study aimed to characterize the functions of TrxB in N. gonorrhoeae infection and to investigate its role in biofilm formation and in the interaction between gonococci and cervical epithelial cells.

Figure 1
Arrangement of the trxB (NGO0580) and copA (NGO0579) genes on the Neisseria gonorrhoeae chromosome. The inset shows the nucleic acid sequence of the overlapping and divergent promoter regions of the 2 genes, with the −10 and −35 RNA polymerase ...


Bacterial strains and growth conditions

N. gonorrhoeae strain 1291 was routinely cultured on brain-heart infusion (BHI) agar (Oxoid) supplemented with 10% (vol/vol) Levinthal base and 1% (vol/vol) IsoVitaleX (Beckton Dickinson). GC agar (Oxoid) was used for disc diffusion sensitivity assays. Growth on solid medium was performed at 37°C in 5% CO2, and liquid cultures were incubated at 37°C. Aerobic/microaerobic liquid cultures were established by altering the culture-volume-to-flask-volume ratio in a manner similar to that described by Cooper et al. [17]—that is, shaking 5-mL (aerobic) or 30-mL (microaerobic) cultures in 50-mL Erlenmeyer flasks at 200 rpm (aerobic) or 100 rpm (microaerobic). E. coli was cultured on Luria-Bertani (LB) medium at 37°C. E. coli strain K1380 (trxB15::kan) and its parent strain, W3110, were obtained from the E. coli Genetic Stock Center, Yale University (New Haven, Connecticut). Ampicillin and kanamycin were used at a concentration of 100 μg/mL.

Genetic techniques

A N. gonorrhoeae trxB strain was constructed through insertion of the kanamycin-resistance cassette from pUC4kan into the trxB gene on the N. gonorrhoeae chromosome 1291 (figure 1). This cassette does not have a promoter that is functional in neisseriae and gives resistance only when cloned in the same orientation as the gene to be inactivated [18]. As a consequence, it does not exert a polar effect on the expression of downstream genes. Mutant strains were confirmed by polymerase chain reaction (PCR) using primers that bind external to the site of recombination and to the kanamycin-resistance cassette (trxB-F/R-check and Kan-F/R) (table 1). Expression of N. gonorrhoeae trxB in E. coli was achieved by amplifying trxB by PCR using primers trxB-pPROEX-F/R (table 1) and cloning this product into the pPROEX vector (Invitrogen) downstream of its lac operator/promoter by use of NcoI/HindIII, creating the plasmid pPROEX::NgtrxB. E. coli strains were transformed with this plasmid by electroporation. Overexpression of NgtrxB was achieved by adding 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) to exponentially growing cells and incubating for a further 5 h at room temperature.

Table 1
Primers used for polymerase chain reaction (PCR) and RT-PCR.

Thioredoxin reductase activity assays

Thioredoxin reductase activity was measured using the method described by Luthman and Holmgren [19]. Exponentially growing cells were harvested from liquid culture by centrifugation at 4500 g for 10 min and washed with 0.1 mol/L potassium phosphate (pH 7.5). N. gonorrhoeae cell-free extracts were prepared by subjecting cell suspensions to 5 freeze-thaw cycles followed by centrifugation at 18,000 g for 10 min. E. coli cells were disrupted by sonication before centrifugation. The protein concentration of cell-free extracts was determined spectrophotometrically using the following formula: mg/mL protein = 1.55 × A280 − 0.76 × A260, where A280 and A260 are absorbance read at 260 and 280 nm [20]. Assay mixtures contained cell-free extract, 0.1 mol/L potassium phosphate (pH 7.5), 1 mmol/L EDTA, 80 μmol/L insulin, 3 μmol/L E. coli thioredoxin (Sigma), and 0.2 mmol/L NADPH (Sigma) in a final volume of 1 mL. The reaction rate was determined at 25°C by following the oxidation of NADPH at 340 nm. Activity was expressed as micromoles of NADPH oxidized per minute per milligram of protein, using an extinction coefficient of 6220 L mol−1cm−1.

Bacterial sensitivity assays

Disc diffusion sensitivity assays were conducted by placing paper discs (Oxoid) containing 10 μL of the appropriate stress reagent onto a lawn of ~1 × 106 N. gonorrhoeae cells on GC agar plates. Plates were incubated overnight (37°C in 5% CO2), and the zone of clearing around the discs was measured. Killing assays were performed by making a suspension of ~1 × 106 cells in 200 μL of PBS, adding 10 μL of the appropriate stress reagent, and incubating at 37°C. Stress reagents were used at a final concentration of 60 mmol/L diamide, 25 mmol/L paraquat, 0.25 mmol/L menadione, 0.1% (vol/vol) cumene hydroperoxide, 1% (vol/vol) hydrogen peroxide, and 3 mmol/L 2-(N,N-diethylamino)-diazenolate 2-oxide (DEA-NONOate). Serial dilutions of the suspensions were made at the indicated time points and plated onto BHI agar plates. Plates were incubated overnight (37°C in 5% CO2), and the number of viable colonies was recorded. All reagents were purchased from Sigma.

Quantitative real-time PCR

Primers were designed for the target gene as well as the 16S rDNA gene (table 1). RNA was extracted from cells by means of the RNeasy Mini Kit (Qiagen), and cDNA was produced using Omniscript reverse transcriptase (Qiagen). Reactions were performed using SYBR Green dye (Applied Biosystems) and the ABI 7000 real-time PCR machine. Validation of the efficiency of each PCR was performed with varying concentrations of primer (100 to 400 nmol/L) and template. The cycle threshold value was plotted against the log concentration; an efficiency value could be calculated, as could the abundance of each of the transcripts relative to the 16S rDNA quantity [21].

Biofilm formation assays

N. gonorrhoeae biofilm was grown in continuous-flow chambers over glass as described by Seib et al. [22]. Biofilm was grown over cervical cells as follows. Primary cervical cells were obtained from cervical biopsies performed at the University of Iowa Hospitals and Clinics and were immortalized by the method developed by Klingelhutz et al. [23]. The resulting cell line was designated TCX (transformed cervical cells). TCX cells were cultured in 100-mm tissue culture plates in serum-free keratinocyte growth medium (K-SFM) supplemented with 12.5 mg of bovine pituitary extract, 0.08 μg of epidermal growth factor, and 1% (final concentration) penicillin/streptomycin (Gibco) at 37°C in 5% CO2. Once confluent, the cells were seeded on collagen-coated coverslips, grown at 37°C in 5% CO2 until confluent (2 days), and stained with Cell Tracker Orange (Invitrogen) just before infection. N. gonorrhoeae were grown in continuous-flow chambers (50 by 22 by 5 mm) adapted for tissue culture in JE medium (2 parts serum-free hybridoma medium, 1 part McCoy’s 5A medium, and 1 part defined K-SFM [Gibco]; JE medium is a serum-free medium that does not contain any supplements that might harbor complement proteins or other factors that would be toxic to the gonococcus [J. E. Edwards, unpublished data]) diluted to 20% in PBS and supplemented with 1% IsoVitaleX, 100 μmol/L sodium nitrite, 0.5 g/L sodium bicarbonate, and 5 μg/μL chloramphenicol (to maintain the pGFP vector). Flow chambers were inoculated at an MOI of 100, using cell suspensions prepared in biofilm medium (10% GC broth diluted in PBS with 1% IsoVitaleX, 100 μmol/L sodium nitrite, and 5 μg/μL chloramphenicol). Flow chambers were incubated under static conditions at 37°C in 5% CO2 for 1 h after infection, to allow gonococcal attachment to TCX cells. Chambers were then incubated for 48 h at 37°C at a flow rate of 180 μL/min before biofilm formation was assessed via confocal microscopy as described by Seib et al. [22].

Cervical cell invasion and survival assays

Primary human ectocervical epithelial (pex) cells were procured and maintained as described elsewhere [24]. Cervical biopsy samples used to grow primary cell cultures were obtained through the Cooperative Human Tissue Network located at The Research Institute at Nationwide Children’s Hospital and The Ohio State University (Columbus). The ability of N. gonorrhoeae strains to associate with, to invade, or to survive within pex cells was determined using the modified gentamicin survival assay described by Seib et al. [22]. Pex cell monolayers were challenged with gonococci for 90 min (association), treated with 100 μg/mL gentamicin (invasion), and then reincubated into antibiotic-free medium for an additional 1 or 3 h (survival), before plating of the pex cell lysate and enumeration of colony-forming units.


Thioredoxin reductase encoded by N. gonorrhoeae trxB

Thioredoxin reductases are flavoproteins that typically use the reducing power of NADPH to catalyse the reduction of thioredoxin [25]. To determine whether N. gonorrhoeae trxB encodes a typical thioredoxin reductase, the gene was disrupted through insertion of a kanamycin-resistance cassette (figure 1), and enzyme activity assays were conducted using E. coli thioredoxin as a substrate. N. gonorrhoeae 1291trxB was found to exhibit much lower thioredoxin reductase activity than 1291, the isogenic wild-type (WT) strain (figure 2A).

Figure 2
A, Thioredoxin reductase activity of Neisseria gonorrhoeae 1291 and 1291trxB. B, Thioredoxin reductase activity of Escherichia coli BL21(DE3) containing the pPROEX plasmid with or without the N. gonorrhoeae trxB gene (NgtrxB) cloned downstream of its ...

To further investigate the properties of TrxB, the N. gonorrhoeae gene trxB (pPROEX::NgtrxB) was expressed in E. coli, and the thioredoxin reductase activity of cell-free extracts that had been prepared with or without IPTG induction of NgtrxB was then determined. Induction of NgtrxB expression in E. coli was found to correlate with a large increase in thioredoxin reductase activity that was clearly discernible from background E. coli thioredoxin reductase activity (figure 2B).

Inactivation of thioredoxin reductase in E. coli has been shown to result in hyperresistance to hydrogen peroxide (H2O2) challenge because of the increased expression of katG (catalase/hydroperoxidase I) in this strain [26]. The ability of NgtrxB to complement an E. coli thioredoxin reductase mutant strain was investigated. E. coli K1380 (trxB15::kan) and its parent strain, W3110, were transformed with the pPROEX::NgtrxB construct described above, and cell suspensions were challenged with 0, 2, and 4 mmol/L H2O2 for 60 min. E. coli K1380 (trxB::kan) was found to be significantly less sensitive to hydrogen peroxide challenge than W3110 (WT) (figure 2C), confirming the results of Takemoto et al. [26]. However, the K1380 (trxB::kan) transformant carrying the pPROEX::NgtrxB plasmid exhibited similar H2O2 sensitivity to W3110 (WT), whereas transformation of W3110 (WT) with this plasmid had no effect on its ability to survive H2O2 challenge (figure 2C). Taken together, these data confirm that the gonococcal TrxB is a thioredoxin reductase with biological properties that are similar to those of its E. coli homologue.

Requirement of N. gonorrhoeae TrxB for resistance to oxidative and disulfide stress

We previously demonstrated that an nmlR mutant is more sensitive to killing by reagents that cause disulfide stress, such as diamide, and by oxidants, such as cumene hydroperoxide [8]. To determine whether the phenotype of the trxB mutant was similar to that of the nmlR mutant, we used disc killing assays to investigate the sensitivity of N. gonorrhoeae 1291 (WT) and 1291trxB to diamide, H2O2, and cumene hydroperoxide as well as to the superoxide generators paraquat and menadione. N. gonorrhoeae 1291trxB was found to exhibit significantly higher sensitivity to diamide, paraquat, menadione, and cumene hydroperoxide than the WT strain (figure 3A). However, the reverse was true for H2O2, with the trxB strain exhibiting significantly higher resistance to this compound (figure 3A), consistent with the phenotype of an E. coli trxB mutant strain [26].

Figure 3
A, Sensitivity of Neisseria gonorrhoeae 1291 and 1291trxB to the thiol oxidizing agent diamide (DIA; 60 mmol/L), the superoxide donors paraquat (PQ; 25 mmol/L) and menadione (MD; 0.25 mmol/L), and the peroxides/oxidizing agents cumene hydroperoxide (CHP; ...

Requirement of N. gonorrhoeae TrxB for defense against nitric oxide (NO)

NO is synthesized by the human host and is an important component of the innate immune response. It is also an intermediate during anaerobic respiration in N. gonorrhoeae during the conversion of nitrite to nitrous oxide. Under microaerophilic conditions, N. gonorrhoeae expresses a copper-containing nitrite reductase (AniA) [27] and a NO reductase (NorB) [28]. Given the apparent importance of N. gonorrhoeae TrxB in the defense against a variety of reactive oxygen species (figure 3A), we investigated whether it was also required for defense against reactive nitrogen species. Cell suspensions of N. gonorrhoeae 1291 (WT) and 1291trxB were challenged with the NO generator DEA-NONOate for 60 min. N. gonorrhoeae 1291trxB was found to exhibit increased sensitivity to NO compared with the WT strain (figure 3B), suggesting that TrxB contributes to the ability of gonococci to detoxify reactive nitrogen species.

Requirement of TrxB for expression of denitrification genes

Expression of aniA and norB is an essential element of the adaptation of the gonococcus to microaerophilic conditions, an environment that is likely to predominate during gonococcal infection of the human host. Induction of aniA is dependent on the transcriptional regulator FNR, which is active only under microaerophilic conditions, in which its [4Fe-4S] iron-sulfur cluster is stable [29]. FNR has recently been shown to also induce the nmlR locus in N. gonorrhoeae [30], indicating that expression of the NmlR regulon is also important in gonococcal adaptation to microaerophilic growth. To determine whether the expression of genes essential for microaerobic growth and NO detoxification is influenced by the thioredoxin system, quantitative RT-PCR was used to measure the expression of aniA (primers aniA-RT-F/R [table 1]) and norB (primers norB-RT-F/R [table 1]) in N. gonorrhoeae 1291 (WT) and 1291trxB grown under aerobic or microaerobic conditions. In the WT strain, both genes were found to be expressed at a much higher level in cells grown microaerobically (figure 4A and 4B). However, in the trxB strain, no difference in aniA or norB expression was evident between cells grown aerobically and those grown microaerobically (figure 4A and 4B). These results indicate that transcriptional activation of aniA and norB under microaerobic conditions is dependent on a functional thioredoxin reductase.

Figure 4
Real-time quantification of aniA (A) and norB (B) mRNA transcripts in Neisseria gonorrhoeae 1291 and 1291trxB grown aerobically or microaerobically. Error bars indicate ±1 SD from the mean. Experiments were conducted in triplicate and repeated ...

Defectiveness of the trxB mutant in biofilm formation on glass and transformed cervical cells

It has been suggested that the formation of a biofilm by N. gonorrhoeae may contribute to its ability to persist in an asymptomatic state in the female genital tract [31]. To determine whether TrxB is important during this mode of growth, a comparison of the ability of the N. gonorrhoeae 1291 (WT) and 1291trxB strains to form a biofilm was evaluated after 2 days of growth under continuous-flow conditions. COMSTAT [32], a mathematical script written for MATLAB (version 5.3; Mathworks) that quantifies 3-dimensional biofilm structures by evaluating confocal image stacks, was used to assess the biomass and the average and maximum thickness of biofilm photomicrographs taken for each flow chamber. COMSTAT analyses showed that the trxB mutant was deficient in biofilm formation compared with the WT strain, both over glass and over TCX cells (figure 5). Three-dimensional images of these biofilms indicated that the trxB mutant formed diffuse biofilms over glass with large gaps between biofilm clusters and that these gaps appeared to be larger when the biofilm was grown over TCX cells (figure 5). This differed from the WT strain, which formed compact biofilms with fewer and smaller gaps between biofilm clusters (figure 5).

Figure 5
Gonococcal biofilm formation over glass and transformed cervical cells. Panel A depicts 3-dimensional reconstructions of stacked z-series of 1291 (left) and 1291trxB (right) biofilms grown over glass, which were taken at ×200 magnification and ...

Requirement of TrxB for N. gonorrhoeae survival during infection of cervical epithelial cells

Gonococci are typically associated with cervical and urethral epithelium in women and men, respectively, and are able to invade, survive, and replicate within these cells [33]. To determine whether TrxB is important during this process, the ability of N. gonorrhoeae 1291 (WT) and 1291trxB to associate with, invade, and survive within pex cells was tested. There was a small but significant difference observed in the ability of 1291trxB to associate with and invade pex cells compared with the WT strain (figure 6). However, 1291trxB was severely attenuated in its ability to survive and replicate within pex cells compared with the WT strain. These data indicate that TrxB plays an important role in promoting the survival of gonococci during cervical infection.

Figure 6
Gonococcal association with and intracellular survival within primary human ectocervical epithelial (pex) cells. The bars depicted in each graph represent the mean percentage of total association, invasion, or survival calculated from 3 experiments performed ...


In a previous study, we demonstrated that the NmlR transcription factor of N. gonorrhoeae is required for the defense of the bacterium against oxidative and disulfide stress [8]. The present study targeted trxB, a gene that appears to be under the control of NmlR in N. gonorrhoeae. We have now confirmed that N. gonorrhoeae trxB encodes a thioredoxin reductase that has biochemical and biological properties similar to those of its E. coli homologue.

The hyperresistance of an E. coli trxB strain to H2O2 is attributed to constitutive up-regulation of katG (catalase/hydroperoxidase I), an element of the OxyR regulon [26]. Although the OxyR regulon of N. gonorrhoeae is smaller than that of E. coli (consisting of just 3 genes encoding catalase, glutathione reductase, and peroxiredoxin/glutaredoxin [22]), we found that this regulon is also constitutively activated in a N. gonorrhoeae trxB mutant strain (data not shown). These data are consistent with the H2O2 hyperresistance phenotype we observed for the N. gonorrhoeae trxB mutant. However, constitutive activation of the OxyR regulon in the gonococcal trxB mutant does not protect N. gonorrhoeae from challenge with superoxide, organic peroxide, or disulfide stress. This indicates that the response of N. gonorrhoeae to H2O2 via OxyR is distinct from the response to other oxidizing species, of which TrxB is a key component. The sensitivity of the trxB mutant to oxidative and disulfide stress may be linked to a failure to maintain a sufficiently reduced thioredoxin pool. This manifests itself in a reduced doubling time for the trxB mutant (3 h) compared with that for WT cells (1 h) under aerobic conditions (A.J.P., S.P.K., A.G.M., and M.P.J., unpublished data).

We also observed that gonococcal TrxB is important in the defense against NO. It is known that thiol groups react with NO-derived species to form S-nitrosothiols [34]. The human and E. coli thioredoxin systems are able to catalyze the denitrosation of protein S-nitrosothiols [35] and S-nitrosoglutathione [36]. Thus, TrxB and thioredoxin may play a direct role in the protection of N. gonorrhoeae against nitrosative stress. However, TrxB and thioredoxin may play an additional or alternative indirect role in the defense against NO, because the observation was also made that aniA (nitrite reductase) and norB (NO reductase) are not induced in vitro under microaerophilic conditions in the N. gonorrhoeae trxB strain. The failure of the trxB mutant to activate aniA expression suggests that the oxygen-sensing transcription factor FNR may have lower activity in this mutant. There is evidence that, in E. coli, the thioredoxin system promotes iron-sulfur cluster biogenesis by providing a reducing environment for the action of the iron-sulfur cluster biosynthetic proteins IscA and IscU [37]. Interestingly, NO has been shown to inactivate FNR under anaerobic conditions [38]. Given that FNR is not required for the induction of norB, the low expression of this gene in a trxB mutant requires a different explanation. In this regard, it is known that N. gonorrhoeae norB expression is activated by the ferric-uptake regulator (FUR) protein [39]. NO inhibition of FUR activity has also been demonstrated [40], and, furthermore, a proteomic analysis of E. coli found that the FUR protein interacts with thioredoxin [41]. Therefore, it is possible that TrxB and thioredoxin are required to maintain the activator form of this transcriptional regulator. This hypothesis is supported by our observation that expression of nuoD, which encodes a subunit of NADH dehydrogenase and possesses a FUR box in its operator/promoter, was reduced 3.8-fold in a trxB mutant (figure 7).

Figure 7
Expression of nuoD in Neisseria gonorrhoeae wild-type and trxB strains grown under microaerobic conditions.

Gonococci are able to invade, survive, and replicate within polymorphonuclear leukocytes [42, 43] as well as within epithelial cells of the urogenital tract [33]. We have demonstrated an important role for TrxB in the survival of gonococci within primary cervical epithelial cells. Oxidative/nitrosative killing mechanisms of these cells have not yet been fully explored. However, recent data have indicated that pex cells produce NO in response to gonococcal infection and, furthermore, that NO actually promotes the intracellular survival of these bacteria during ex vivo infection of these cells (J.L.E., unpublished data). In view of this, the importance of TrxB for survival within these cells may be reflective of its importance for NO tolerance.

TrxB is absolutely required for biofilm formation, both on glass and on cervical epithelial cells. It has been suggested that the majority of bacterial cells growing within a biofilm are oxygen limited [44]. This suggests that gonococci will need to respire using the AniA-NorB partial denitrification pathway to form a biofilm. The biofilm phenotype of N. gonorrhoeae trxB may reflect the lack of induction of these respiratory genes in this strain. That the trxB strain was more attenuated for biofilm formation on TCX cells than on glass may suggest that reactive oxygen and/or nitrogen species are produced by urogenital epithelial cells as an antibacterial mechanism.


National Health and Medical Research Council of Australia (program grant 284214 to M.P.J. and A.G.M.); The Research Institute at Nationwide Children’s Hospital (funding to J.L.E.); National Institutes of Health (grant AI045728 to M.A.A.).

We thank the Cooperative Human Tissue Network (Columbus, Ohio) for providing cervical tissue specimens.


Potential conflicts of interest: none reported.


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