ABSTRACT
Although Chlamydia trachomatis is a human genital tract pathogen, chlamydial organisms have frequently been detected in both vaginal and rectal swab samples of animals and humans. The plasmid-encoded pGP3, a genital tract virulence factor, is essential for Chlamydia muridarum to colonize the mouse gastrointestinal tract. However, intracolon inoculation to bypass the gastric barrier rescued the colonization ability of a pGP3-deficient C. muridarum mutant, suggesting that pGP3 is required for C. muridarum to reach but not to colonize the large intestine. The pGP3-deficient mutant was rapidly cleared in the stomach and was 100-fold more susceptible to gastric killing. In mice genetically deficient in gastrin, a key regulator for gastric acid production, or pharmacologically treated with a proton pump inhibitor, the ability of pGP3-deficient C. muridarum to colonize the gastrointestinal tract was rescued. The pGP3-dependent resistance was further recapitulated in vitro with treatments with HCl, pepsin, or sarkosyl. In the genital tract, deficiency in pGP3 significantly reduced C. muridarum survival in the mouse vagina and increased C. muridarum susceptibility to vaginal killing by ∼8 times. The pGP3-deficient C. muridarum was more susceptible to lactic acid killing, and the pGP3 deficiency also significantly increased C. trachomatis susceptibility to lactic acid. The above-described observations together suggest that Chlamydia may have acquired the plasmid-encoded pGP3 to overcome the gastric barrier during its adaptation to the gastrointestinal tract and the pGP3-dependent resistance may enable chlamydial evasion of the female lower genital tract barrier during sexual transmission.
INTRODUCTION
Chlamydia trachomatis is a sexually transmitted bacterial pathogen that causes pathologies in the upper genital tract (1). However, C. trachomatis is also frequently detected in the gastrointestinal (GI) tract (2–5). Although the medical significance of C. trachomatis in the human GI tract remains unclear, Chlamydia muridarum in the mouse GI tract has been proposed to have an impact on both C. muridarum infection and pathogenicity in the genital tract, depending on the order of exposure to C. muridarum (6, 7). When naive mice are exposed to C. muridarum via intragastric inoculation, C. muridarum organisms can colonize the GI tract for long periods of time without causing any significant pathology (8–12). More importantly, the GI tract colonization is also able to induce transmucosal immunity against subsequent C. muridarum challenge infection in both genital (6) and airway (13) tissues. These observations have led to the proposal that chlamydial nonpathogenic colonization in the GI tract may be explored for developing an oral chlamydial vaccine. In contrast, when naive mice are first exposed to C. muridarum in the genital tract, via intravaginal inoculation for example, genital C. muridarum is known to induce long-lasting hydrosalpinx and infertility (14–16), closely mimicking the tubal adhesion/infertility observed in women (17–19), which is why the murine model has been extensively used for studying the mechanisms of C. trachomatis pathogenesis and immunity (20–25). However, the precise mechanism by which genital C. muridarum induces long-lasting hydrosalpinx after the genital C. muridarum organisms are cleared remains unclear. Recent studies have shown that vaginal C. muridarum can not only ascend to the oviduct but also spread to the GI tract (26) via a hematogenous route (27) to establish long-lasting colonization in the GI tract. Interestingly, this genital-to-GI-tract spreading seems to correlate with C. muridarum induction of hydrosalpinx in the genital tract (28–30). These observations have led to the proposal of a two-hit model partially explaining C. muridarum pathogenicity in the upper genital tract (7). The genital C. muridarum ascension may cause the initial damage to the oviduct epithelia, while C. muridarum spreading to the GI tract may induce a Chlamydia-specific profibrotic response that is then recruited to the injured oviduct for promoting/maintaining tubal fibrosis as a second hit. Although the oral vaccine proposal needs further evaluation and the two-hit model is still to be tested, the potentially significant impacts of GI tract Chlamydia on the genital tract pathogenesis sufficiently justify in-depth investigations into the biology of chlamydial colonization in the GI tract.
Orally delivered C. muridarum is known to colonize the mouse GI tract for long periods of time (8–12). During the first 2 weeks after inoculation, C. muridarum organisms spread to the entire GI tract and also to extra-GI tract tissues (8, 9). However, the systemic spreading is transient and most organisms gradually home to the cecum/colon tissues by the fourth week (12, 26). Once in the cecum/colon, C. muridarum can establish stable colonization for hundreds of days. It is not clear how C. muridarum overcomes the gastric barrier and achieves long-lasting colonization in the colon. We have recently shown that both the cryptic plasmid and its encoded pGP3 are important for C. muridarum to colonize the GI tract (28, 30). However, the precise mechanisms by which pGP3 promotes C. muridarum colonization in the GI tract remain unknown.
In the current study, we have used the C. muridarum mouse model to investigate mechanisms of C. muridarum colonization in the GI tract by focusing on pGP3 (28, 30). Interestingly, by using an intracolon inoculation to bypass the gastric barrier, the ability of a pGP3-deficient C. muridarum mutant to colonize the GI tract for a long period of time was rescued, suggesting that pGP3 is required for C. muridarum to overcome barriers in the upper GI tract but not the colon. Careful comparison of the numbers of live organisms recovered from the stomach for wild-type and mutant C. muridarum organisms following intragastric inoculation revealed that pGP3 deficiency increased the mutant’s susceptibility to gastric killing by >100-fold. The gastric restriction of the pGP3-deficient mutant was dependent on gastric acid but not on host immune responses. The pGP3-dependent resistance to gastric acid was recapitulated in vitro. These observations together suggest that C. muridarum may have acquired the plasmid-encoded pGP3 to overcome the gastric barrier during its adaptation to the GI tract. Interestingly, the same pGP3-dependent resistance to the gastric barrier may enable C. muridarum to selectively evade the vaginal barrier during genital tract transmission, since deficiency in pGP3 significantly reduced C. muridarum survival in the vagina. Thus, we have revealed a novel mechanism by which the plasmid-encoded pGP3 promotes C. muridarum colonization in both the GI and genital tracts.
RESULTS
The plasmid-encoded pGP3 is not required for C. muridarum to colonize the mouse cecum/colon.We have previously shown that the plasmid-encoded genital tract virulence factor pGP3 is essential for C. muridarum colonization in the GI tract (30). To further define the role of pGP3 in promoting chlamydial colonization in the different segments of the GI tract, we compared the numbers of live organisms recovered from rectal swabs following intragastric versus intracolonic inoculations (Fig. 1). Following intragastric inoculation, no live organisms were recovered from mice inoculated with the pGP3-deficient C. muridarum mutant, although wild-type C. muridarum colonized the GI tract throughout the 2-month period. However, when chlamydial organisms were inoculated directly into the colon, the ability of the pGP3-deficient mutant to colonize the GI tract was rescued. Mice intracolonically inoculated with the mutant continued to shed live organisms in rectal swab samples taken throughout the 2-month period. The intracolonically inoculated mutant organisms were directly detected in the cecum and colon tissues (Fig. 1B), demonstrating that the pGP3-deficient mutant is able to colonize the large intestine.
The pGP3-deficient C. muridarum mutant is able to colonize the gastrointestinal tract following intracolon but not intragastric inoculation. (A) Groups of C57BL/6J mice were inoculated with 1 × 105 inclusion-forming units (IFUs) of wild-type C. muridarum organisms (strain CM-pGFP) (c and d) or mutant C. muridarum organisms deficient in pGP3 due to a premature stop codon in the pGP3 coding region (strain CM-pGP3S) (a and b) either intragastrically (a and c) or intracolonically (b and d). At different time points as shown along the x axis, mice were monitored for live chlamydial organism recovery from rectal swab samples, and the results are expressed as log10 IFUs per swab, shown along the y axis. Each group had 8 to 10 mice, and the data were obtained from 2 or 3 independent experiments. Note that although the pGP3-deficient mutant failed to colonize the gastrointestinal tract after intragastric inoculation, intracolonic inoculation rescued its colonization. (B) Parallel groups of mice with intracolonic inoculation with either the pGP3-deficient mutant (e to g) or wild-type C. muridarum (h to j) were sacrificed on days 7, 14, and 28 after inoculation, as shown on top of each panel, to monitor live organism recoveries from different gastrointestinal tissue segments from the stomach and duodenum (Duod.) to the rectum, and extragastrointestinal tissues, such as the genital tract and spleen, as indicated along the x axis. The numbers of live organisms are expressed as log10 IFUs per tissue segment, shown along the y axis. Each group had 3 to 5 mice, and the data were obtained from 2 independent experiments. Note that intracolonically inoculated mutant organisms were restricted to the cecum/colon throughout the experiment, while the wild-type organisms transiently spread to the small intestine and stomach before homing to the cecum/colon. Error bars show standard deviations.
pGP3 is essential for C. muridarum to survive in the stomach.To identify the tissue site in which the pGP3-deficient C. muridarum mutant is restricted following intragastric inoculation, we carefully monitored the tissue distribution of live organisms (Fig. 2). The wild-type organisms were recovered from all GI tract tissues from the stomach to the rectum during the first 2 weeks and gradually homed to the cecum/colon and rectum by day 28, which is a typical distribution pattern of C. muridarum in the mouse GI tract (6, 12). However, no live organisms were recovered from any GI tract tissues in mice intragastrically inoculated with the same amount of pGP3-deficient mutant organisms throughout the experiment. Only when the inoculum of the mutant was increased 100-fold to 1 × 107 inclusion-forming units (IFUs) were live mutant organisms detected, but at minimal levels and only in a few tissues, including the duodenum on day 7, duodenum/jejunum on day 14, and jejunum on day 21. By day 28, there were no longer any live mutant organisms in the GI tract. At no time were live mutants detected in the stomach, while live wild-type organisms were detected in the stomach during the first 2 weeks after inoculation. These results suggest that the stomach is the site most detrimental to the mutant. Thus, we further compared the survival of the mutant and the wild type in the stomach soon after intragastric inoculation (Fig. 2B). At 24 h after inoculation, live mutants were recovered only when the inoculation dose reached 1 × 107 IFUs. No more live mutants were detected when the dose was reduced to 1 × 106 or 1 × 105 IFUs. This observation is consistent with the above-described result that live organisms were only detected in the small intestine on days 7 to 21 after inoculation when the inoculation dose was at 1 × 107 IFUs. In contrast, high levels of live wild-type C. muridarum organisms were recovered when the inoculation dose was at 1 × 105 IFUs. These results together suggest that the pGP3-deficient mutant is at least 100 times more susceptible to gastric inactivation than the wild type. Furthermore, by day 3, the mutant had significantly reduced recovery in the stomach, which is consistent with the observation that no live organisms were detected in the stomach by day 7 in mice inoculated with 1 × 107 IFUs of the mutant (Fig. 2A). In contrast, live organisms were recovered at similar levels on both days 1 and 3 in mice inoculated with 1 × 105 IFUs of wild-type C. muridarum. In addition, on day 3, live organisms were still recovered in the stomach of 33% of mice intragastrically inoculated with 1,000 IFUs of wild-type C. muridarum (compared to 0% of mice with 1 million IFUs of the pGP3-deficient mutant). These results further validated that the pGP3-deficient mutant is significantly more susceptible to gastric acid killing than the wild type, suggesting that pGP3 is essential for maintaining chlamydial resistance to gastric acid. The failure of the pGP3-deficient C. muridarum mutant to survive the stomach acid largely explains its lack of colonization in the GI tract following intragastric inoculation.
The pGP3-deficient C. muridarum mutant is rapidly cleared in the stomach following intragastric inoculation. (A) Groups of C57BL/6J mice were intragastrically inoculated with pGP3-deficient C. muridarum (mutant) organisms at a dose of 1 × 105 (a to d) or 1 × 107 (e to h) IFUs or wild-type C. muridarum organisms at 1 × 105 IFUs (i to l). At different time points as shown on top of the figure, mice were sacrificed to recover live chlamydial organisms in different segments of the gastrointestinal tract, as listed along the x axis. The numbers of live organisms recovered are expressed as log10 IFUs per tissue segment, as shown along the y axis. Data in each panel were acquired from 3 to 5 mice in two independent experiments. P < 0.01 (Wilcoxon), e versus i, f versus j, g versus k, or h versus l at either the individual tissue segment or the whole gastrointestinal tract level. Note that no live pGP3-deficient C. muridarum mutant organisms were recovered from any gastrointestinal tissues of mice inoculated with 1 × 105 IFUs and only a minimal number of live organisms was recovered from the duodenum or jejunum but not the stomach of mice inoculated with 1 × 107 IFUs. (B) In a parallel experiment, pGP3-deficient mutant (right) and wild-type (left) C. muridarum organisms were compared for survival in the stomach on days 1 (m) and 3 (n) following intragastric inoculation with different doses as listed along the x axis. The numbers of live organisms recovered from stomach were expressed as log10 IFUs as shown along the y axis, while the percentages of mice found positive for live organisms are indicated in the corresponding panels. Each data point came from 3 to 5 mice. Note that on day 1, 1 × 105 IFUs of the wild type were sufficient for infecting the stomach of 100% of mice, while 1 × 107 IFUs of the mutant were required. By day 3, the mutant organisms were rapidly reduced, while the wild type maintained infectivity in the stomach. Error bars show standard deviations.
pGP3 is required for C. muridarum resistance to gastric acid in the stomach.To determine the host factors responsible for killing the pGP3-deficient mutant in the stomach, we compared chlamydial colonization in the GI tract of mice deficient in different host factors, including gastrin (an essential regulator of gastric acid production [31]), MyD88 (an adaptor required for many innate immunity receptor signaling pathways [32]), Rag1 (recombination activation gene 1, required for the development of adaptive immunity receptors), interferon-gamma (IFN-γ) receptor (IFNgR), and interleukin 22 (IL-22). Following intragastric inoculation, although wild-type C. muridarum successfully colonized the GI tract of all mice regardless of the genetic deficiencies, the C. muridarum mutant was only able to colonize the GI tract of mice deficient in gastrin (Fig. 3). By day 14 after inoculation, significant numbers of live mutant organisms were recovered from the rectal swabs of all gastrin-deficient mice. Since overcoming gastric killing is necessary for the intragastrically inoculated chlamydial organisms to be detected live in rectal swabs and mice deficient in gastrin can no longer produce gastric acid, the above-described observations suggest that pGP3 is important for chlamydial evasion of gastric acid. This conclusion is consistent with the observation that C. muridarum organisms remained viable in the stomach of gastrin-deficient mice for at least 28 days regardless of the chlamydial pGP3 status, while chlamydial organisms were completely cleared from the stomach by this time in gastrin-competent mice (Fig. 4A). Furthermore, the gastrin deficiency-dependent survival of the pGP3-deficient mutant in the stomach allowed the mutant to achieve long-lasting colonization in the GI tract (Fig. 4B). Consistent with this, oral treatment of wild-type mice with a proton pump inhibitor to raise stomach pH also allowed the pGP3-deficient C. muridarum mutant organisms to achieve long-term colonization in the GI tract (Fig. 5). Thus, we have demonstrated that pGP3 may promote C. muridarum colonization in the GI tract by enabling chlamydial resistance to gastric acid.
Deficiency in gastrin but not immunity rescued the ability of the pGP3-deficient C. muridarum mutant to colonize the gastrointestinal tract following an intragastric inoculation. Groups of mice deficient (knockout [KO] mice) in gastrin (an essential regulator of gastric acid production) (a), MyD88 (a key adaptor molecule required for the innate immunity receptor signaling pathway) (b), recombination activation gene 1 (an essential enzyme required for forming adaptive immunity receptors) (c), IFN-γ receptor (a receptor required for inhibiting intracellular chlamydial growth in innate and adaptive immunity) (d), or IL-22 (a key cytokine for regulating gut function) (e) or without deficiency (wild-type C57BL/6J mice) (f) were intragastrically inoculated with 1 × 105 IFUs of pGP3-deficient C. muridarum (mutant, left) or wild-type C. muridarum (right) organisms. At different time points as shown along the x axis (from day 3 to day 28 after inoculation), mice were monitored for live chlamydial organisms in rectal swab samples, and the results are expressed as log10 IFUs per swab as shown along the y axis. There were 5 mice in each group. P < 0.01 (Wilcoxon), a versus any of b to f among mice inoculated with pGP3-deficient C. muridarum mutant. Note that the mutant organisms colonized the gastrointestinal tract only in mice deficient in gastrin and not any other mice. Error bars show standard deviations.
The pGP3-deficient C. muridarum mutant is able to both colonize the stomach and descend to the colon in mice deficient in gastrin. (A) Groups of mice with (a and c) or without (b and d) deficiency in gastrin were intragastrically inoculated with 2 × 105 IFUs of the pGP3-deficient C. muridarum mutant (a and b) or C. muridarum wild type (c and d) organisms. On day 28 after inoculation, mice were sacrificed to monitor the numbers of live C. muridarum organisms recovered from different tissue segments of the gastrointestinal tract from the stomach and duodenum (Duod.) to the rectum, as well as extragastrointestinal organs, such as the genital tract and spleen, as indicated along the x axis. The live organism recoveries are expressed as log10 IFUs per tissue segment as shown along the y axis. Data came from 3 mice per group. Note that both the mutant and wild-type C. muridarum organisms remained viable in the stomach tissues of the gastrin-deficient mice on day 28 after the initial inoculation. (B) Groups of mice were inoculated as described for panel A. At different time points as shown along the x axis, mice were monitored for live chlamydial organisms in rectal swabs as shown along the y axis in log10 IFUs per swab. The data came from 5 to 10 mice per group. Note that gastrin deficiency rescued the ability of the mutant to colonize the gastrointestinal tract for 63 days. Error bars show standard deviations.
Antiacid treatment is sufficient for rescuing the ability of pGP3-deficient C. muridarum to colonize the gastrointestinal tract. Groups of C57BL/6J mice (n = 5) with (a and c) or without (b and d) antiacid treatment with proton pump inhibitor (omeprazole) were intragastrically inoculated with pGP3-deficient mutant C. muridarum organisms at a dose of 1 × 105 (a and b) or 1 × 107 (c and d) IFUs. At different time points up to day 63 as shown along the x axis, mice were monitored for numbers of live chlamydial organisms in rectal swab samples as shown along the y axis in log10 IFUs per swab. Data were from 3 to 5 mice per group. Note that the antiacid treatment rescued the ability of pGP3-deficient C. muridarum to colonize the gastrointestinal tract for a long period of time. Error bars show standard deviations.
The plasmid-encoded pGP3 promotes C. muridarum resistance to treatment with HCl, pepsin, or sarkosyl.The above-described mouse experiments have demonstrated that pGP3 promotes the ability of C. muridarum to colonize the GI tract by enabling its resistance to gastric acid. To evaluate whether the pGP3-dependent resistance to gastric acid relies on pGP3 that is secreted from chlamydial organisms or associated with the organism’s outer membrane (33, 34), we next compared purified C. muridarum elementary bodies (EBs) with or without pGP3 deficiency for their resistance to HCl treatment in an in vitro assay (Fig. 6A). Although the saline-HCl solution at low pHs effectively killed both wild-type and pGP3-deficient mutant organisms, the mutant was significantly more susceptible to HCl inactivation between pH 3.5 and 4.5 when the incubation was carried out for 10 min. This differential susceptibility was maintained when the incubation was maintained for up to 2 h. These observations agreed with the in vivo finding that pGP3 can promote C. muridarum resistance to gastric acid. Since pepsin is a major protease in the stomach, we further evaluated whether pGP3-deficient C. muridarum was also more susceptible to pepsin treatment (Fig. 6B). Starting at the concentration of 2.5 mM, pepsin preferentially killed pGP3-deficient mutant organisms when incubated for 2 h, and the differential pepsin effect was detected after incubation for only 30 min when pepsin was used at 5 mM. To directly test whether lack of the outer membrane-associated pGP3 makes the outer membrane more susceptible to sarkosyl permeabilization, we carried out an experiment using serial extraction with sarkosyl (Fig. 7). Limited sarkosyl extraction is known to produce gradual permeabilization of the outer membrane, leading to sequential release of chlamydial cytosolic and periplasmic proteins, such as heat shock protein 60 (HSP60) (34). The gradual loss of HSP60 from the purified EBs was tracked by monitoring the remaining HSP60 that was still associated with EBs. We found that pGP3-deficient mutant EBs released HSP60 much faster than the wild type did, indicating that the mutant was more vulnerable to sarkosyl-mediated permeabilization. This observation suggests that pGP3 is essential for maintaining C. muridarum outer membrane integrity.
The pGP3-deficient C. muridarum mutant organisms are more susceptible to HCl and protease killing. (A) Aliquots of 1 × 107 IFUs of wild-type C. muridarum (CM-pGFP) or pGP3-deficient C. muridarum (CM-pGP3S) organisms were each incubated either at 37°C for 10 min in 100 μl saline with pH adjusted using HCl to various values as listed along the x axis (a) or in a pH 4.5 saline-HCl solution for various times (min) as indicated along the x axis (b). The live organisms remaining were recovered by rapid dilution in SPG buffer and inoculated onto HeLa cell monolayers for titration. The numbers of live organisms recovered are expressed as log10 IFUs per sample as shown along the y axis. Data came from 4 to 5 independent experiments. *, P < 0.05; **, P < 0.01 (Wilcoxon). Note that the pGP3-deficient mutant C. muridarum organisms were significantly more susceptible to inactivation by HCl. (B) Pepsin solutions prepared in saline at different concentrations as indicated along the x axis (c) were each reacted with an aliquot of 1 × 107 IFUs of wild-type or pGP3-deficient mutant C. muridarum organisms for 120 min (c) or in 5 mM pepsin solution for various periods of time as indicated along the x axis (d). The live organisms remaining in each treatment sample were recovered by rapid dilution in SPG buffer and inoculated onto HeLa cell monolayers for titration. The numbers of live organisms recovered are expressed as log10 IFUs per sample as shown along the y axis. Data came from 4 to 5 independent experiments. *, P < 0.05; **, P < 0.01 (Wilcoxon). Note that the pGP3-deficient mutant C. muridarum organisms were significantly more susceptible to inactivation by pepsin. Error bars show standard deviations.
The pGP3-deficient C. muridarum mutant organisms are more susceptible to sarkosyl permeabilization. Aliquots of 5 × 108 IFUs of wild-type or pGP3-deficient mutant C. muridarum organisms were each suspended in 50 μl of 2% sarkosyl–PBS solution for 10 min on ice, followed by centrifuging (13,000 rpm in a microcentrifuge) at 4°C for 30 min to pellet chlamydial ghosts. After carefully removing the supernatant, the remaining pellet was defined as the extraction 1 pellet. To extract the same pellet for a second time, a parallel extraction 1 pellet was reextracted in 50 μl of 2% sarkosyl solution as described above to produce an extraction 2 pellet. Some parallel pellets were extracted a total of three or four times as indicated on top of the figure. To each pellet, 50 μl of 2× SDS sample buffer was added and 10 μl was loaded to each lane. After electrophoresis and transfer, the membranes were blotted with mouse (M) anti-chlamydial HSP60 MAb (clone BC7.1) (a), anti-chlamydial major outer membrane protein (MOMP) MAb (clone MC22) (b), or a mouse anti-denatured pGP3 polyclonal antibody (PAb) (c). Molecular sizes (kDa) are shown to the left. Note that the pGP3-deficient mutant displayed more rapid loss of HSP60, while the patterns of MOMP bands were similar between the wild-type and mutant organisms.
The plasmid-encoded pGP3 promotes chlamydial survival in the mouse vagina.Since Chlamydia is considered a genital tract pathogen, we next tested whether the pGP3-dependent resistance to the gastric barrier impacts C. muridarum survival in the genital tract. We first compared the survival of the wild-type and pGP3-deficient mutant C. muridarum organisms in the mouse vagina (Fig. 8). Live organisms were recovered from both vagina and cervix tissues 24 h after mice were intravaginally inoculated with 1,000 IFUs of wild-type but not pGP3-deficient mutant C. muridarum organisms. Live mutant organisms were only detectable when the inoculation dose was increased to 10,000 IFUs. Furthermore, the titer of the mutant live organisms recovered was significantly lower than that of the wild type inoculated at 1,000 IFUs and the mutant live organisms were restricted to the vagina only and did not spread to the cervix. Only when the mutant inoculation was increased to 1 × 105 IFUs were live organisms recovered from both vagina and cervix, demonstrating that the pGP3-deficient C. muridarum was significantly more susceptible to vaginal killing than the wild type. The 50% infection dose (ID50) of the mutant in the vagina was ∼8 times higher than that of the wild type, which further quantitatively demonstrates that pGP3 plays an essential role in C. muridarum survival in the vagina. Since normal vaginal pH is acidic due to the production of lactic acid, we further tested whether the pGP3-deficient mutant was also more susceptible to lactic acid killing (Fig. 9). Similar to what was observed in the HCl killing assay, a saline-lactic acid solution at pHs ranging from 3.5 to 4.5 preferentially killed the mutant when incubated for 10 min. The differential killing of the mutant was maintained when the incubation was maintained for up to 4 h at a pH of 5.5. Thus, the outer membrane-associated pGP3 can also promote chlamydial resistance to lactic acid, which may provide an explanation for the pGP3-dependent survival in the vagina. To test whether C. trachomatis may also use a pGP3-dependent mechanism to overcome vaginal lactic acid, we compared the susceptibilities of a wild-type C. trachomatis L2-pGFP clone and that of a pGP3-deficient mutant L2 clone, L2-pGP3S, to lactic acid killing (Fig. 10). We found that the pGP3-deficient mutant L2 organisms were significantly more susceptible to lactic acid killing than the wild-type organisms. This observation is consistent with a previous finding that vaginal microbiota lactobacillus species may enhance vaginal resistance to C. trachomatis infection by secreting lactic acid (35).
The pGP3-deficient C. muridarum is highly susceptible to mouse vaginal killing. (A) Groups of C57BL/6J mice were intravaginally inoculated with C. muridarum organisms without (a to d, wild type) or with (e to h, mutant) deficiency in pGP3 at various doses from 1 × 102 to 1 × 105 IFUs as indicated in the panels. Twenty-four hours after inoculation, mice were sacrificed to recover live chlamydial organisms in different segments of the genital tract, including the vagina (VG), cervix (CV), left uterine horn (LU), right uterine horn (RU), left oviduct/ovary (LO), and right oviduct/ovary (RO) as listed along the x axis. The numbers of live organisms recovered are expressed as log10 IFUs per tissue segment as shown along the y axis. Data in each panel were acquired from 3 to 6 mice from two independent experiments. **, P < 0.05 (Wilcoxon, vaginal IFUs between the mutant and wild-type organisms). Note that the numbers of live organisms recovered from vaginas inoculated with 1 × 105 or 1 × 104 IFUs of mutant C. muridarum organisms were significantly lower than those from vaginas inoculated with 1 × 104 or 1 × 103 IFUs of wild-type C. muridarum organisms. (B) ID50s in the form of log10 IFUs were calculated using the IC50 calculator from Aat Bioquest (https://www.aatbio.com/tools/ic50-calculator/) based on the rates of IFU positivity in mouse vagina or cervix tissue homogenates. Note that the ID50s in the vagina and cervix of mice inoculated with the mutant were 27- and 37-fold higher, respectively, than those from mice inoculated with the wild-type C. muridarum. (C) Illustration of how a mouse genital tract was separated into different tissue segments from the vagina (VG) to the oviducts/ovaries on both sides (LO and RO) as listed along the y axis of panel B.
The pGP3-deficient C. muridarum mutant is more susceptible to lactic acid killing. Aliquots of 1 × 106 IFUs of wild-type (CM-pGFP, filled bars) or pGP3-deficient (CM-pGP3S, open bars) C. muridarum organisms were each incubated in 100 μl saline containing 110 mM lactic acid with pH adjusted to various values using NaOH as listed along the x axis at 37°C for 10 min (a) or in saline-lactic acid solution (pH 5.5) for various times as indicated along the x axis (b). The live organisms remaining were recovered by rapid dilution in SPG buffer and inoculated onto HeLa cell monolayers for titration. The numbers of live organisms recovered are expressed as log10 IFUs per sample as shown along the y axis. Data came from 4 to 5 independent experiments. *, P < 0.05; **, P < 0.01 (Wilcoxon). Note that the pGP3-deficient mutant C. muridarum organisms were significantly more susceptible to inactivation by lactic acid. Error bars show standard deviations.
The pGP3-deficient C. trachomatis mutant is also more susceptible to lactic acid killing. Aliquots of 1 × 106 IFUs of wild-type (L2-pGFP, filled bars) or pGP3-deficient mutant (L2-pGP3S, open bars) C. trachomatis L2 organisms were each incubated in 100 μl saline containing 110 mM lactic acid with pH adjusted to various values using NaOH as listed along the x axis at 37°C for 10 min (a) or in saline-lactic acid solution (pH 5.5) for various times as indicated along the x axis (b). The live organisms remaining were recovered by rapid dilution in SPG buffer and inoculated onto HeLa cell monolayers for titration. The numbers of live organisms recovered are expressed as log10 IFUs per sample as shown along the y axis. Data came from 5 or 6 independent experiments. *, P < 0.05 (Wilcoxon). Note that the pGP3-deficient mutant C. trachomatis organisms were significantly more susceptible to inactivation by lactic acid. Error bars show standard deviations.
DISCUSSION
In the current study, we have presented experimental evidence to support the hypothesis that C. muridarum may have acquired the plasmid-encoded pGP3 to overcome the gastric barrier during its adaptation to the GI tract, which may enable C. muridarum to evade the vaginal barrier during genital tract transmission. First, intracolon inoculation rescued the ability of pGP3-deficient C. muridarum to colonize the GI tract (Fig. 1), suggesting that once C. muridarum organisms reach the colon, pGP3 is no longer required. Thus, the pGP3-dependent colonization in the GI tract (30) may be due to pGP3’s role in promoting the ability of C. muridarum to overcome the upper GI tract barriers. Second, pGP3-deficient C. muridarum organisms are rapidly cleared in the stomach, within 24 h after direct inoculation into the stomach (Fig. 2). Deficiency in pGP3 increased the susceptibility of C. muridarum to gastric killing by more than 100-fold, since the number of live organisms recovered from stomach homogenates of mice inoculated with 1 × 107 IFUs of pGP3-deficient C. muridarum was still significantly smaller than that from mice inoculated with 1 × 105 IFUs of wild-type C. muridarum. By day 3, the difference was even greater. Third, the ability of pGP3-deficient C. muridarum to colonize the stomach was successfully rescued in mice deficient in gastrin but not immunity (Fig. 3 and 4), suggesting that gastric acid is the primary mechanism for inactivating the mutant in the stomach. This conclusion is further supported by the observations from the proton pump inhibition experiment (Fig. 5). Fourth, the pGP3-dependent resistance to gastric acid was recapitulated using in vitro assays (Fig. 6 and 7). The pGP3-deficient C. muridarum organisms were significantly more susceptible to HCl and pepsin killing, respectively, and their outer membranes were more vulnerable to detergent permeabilization. Finally, pGP3-deficient organisms had significantly reduced survival in the mouse vagina and were more susceptible to lactic acid killing (Fig. 8 and 10), suggesting that the same pGP3 function may enable Chlamydia to evade the lower genital tract barrier during genital transmission. Although mouse vaginal pH varies between 4.5 and 7.5 depending on the stage of the estral cycle (36), the human vaginal pH is maintained at acidic levels between 2.8 and 4.2 (37). Since pGP3 is highly conserved between C. muridarum and C. trachomatis, we hypothesize that the pGP3-dependent resistance to gastric acid may enable C. trachomatis resistance to vaginal barriers of women during sexual transmission.
The mouse stomach pH is 3 (fed) or 4 (fasted) (38), which is sufficient to rapidly clear pGP3-deficient mutant C. muridarum organisms. Apparently, pGP3-sufficient wild-type C. muridarum is able to evade this barrier. The next question is how pGP3 promotes C. muridarum resistance to gastric acid. The pGP3 protein is an immunodominant (39–41) and trimeric antigen (42) that is known to be both secreted out of EBs (33) and associated with the EB outer membrane (34). We have previously shown that secreted pGP3 may be able to promote chlamydial pathogenesis by neutralizing host innate effectors, such as the cathelicidin CRAMP in mice or LL-37 in humans (43). We now hypothesize that it is the outer membrane-associated pGP3 that enables C. muridarum to resist gastric acid, possibly by maintaining the integrity of the outer membrane complex, an idea which is supported by the following observations. First, the resistance to gastric killing was rapid and effective within 24 h after inoculation. C. muridarum may not have enough time to produce secreted pGP3. Thus, it is likely that the immediate role may rely on the preexisting pGP3 already associated with the outer membrane. Second, pGP3-dependent resistance to HCl and pepsin, as well as lactic acid, was detected with purified EBs in a cell-free system, in which there was no significant biosynthesis. These observations have provided direct evidence that it is the organism-associated pGP3 that resists the killing by acid or protease. Finally, when both wild-type and pGP3-deficient mutant C. muridarum organisms were subjected to a controlled sarkosyl permeabilization treatment, accelerated release of the cytosolic protein HSP60 was detected from the mutant EBs, suggesting that in the absence of pGP3, the outer membrane complex is more susceptible to detergent permeabilization. The pGP3-free outer membrane became more vulnerable in an acidic environment, which may explain why pGP3-deficient organisms had reduced survival in both the stomach and vagina.
Bacteria have acquired the ability to resist acid (44) via distinct mechanisms (45). Outer membrane components of Gram-negative bacteria can contribute to bacterial resistance to both acid (46) and antimicrobial peptides (47). The periplasmic chaperones, such as HdeA and HdeB, have been shown to play critical roles in bacterial tolerance of an acidic environment (48). Various stress response systems have been found to protect bacteria from acid stress (49). Besides a pH-independent general stress resistance system that contributes to acid survival, bacteria also express low-pH-inducible acid tolerance response systems, including an acid-inducible acid survival system under oxidative growth conditions and an acid survival system requiring either glutamate or arginine. The function of the arginine acid survival system is dependent on the induction of arginine decarboxylase (50). Interestingly, C. muridarum, along with three other Chlamydia species that infect animals, encodes and expresses functional arginine decarboxylase (51), although most C. trachomatis serovars (except for serovar E) have accumulated loss-of-function mutations in this gene (52). Chlamydia species that infect animals, including C. muridarum, may still need a functional arginine decarboxylase to maintain oral-fecal transmission, while C. trachomatis serovars have adapted to the genital tract, where acid killing is less robust, thus allowing loss-of-function mutations to accumulate in this gene. Since the pGP3-dependent resistance to acid was detectable using purified EBs in vitro and free EBs are unable to undergo biosynthesis, the pGP3-dependent resistance may rely on the outer membrane-associated pGP3. The questions are whether and how the outer membrane pGP3 either directly neutralizes protons or indirectly regulates chlamydial acid tolerance mechanisms. It is also possible that the expression of acid tolerance system components like arginine decarboxylase is positively regulated by pGP3 and the pGP3-deficient mutant expresses lower levels of acid tolerance machineries. The fact that the pGP3-dependent acid resistance measured in vitro was not as robust as that measured in the mouse stomach suggests that pGP3 may exert its antiacid function by upregulating chlamydial acid tolerance responses in the mouse stomach, in addition to its direct roles.
It is worth noting that deficiency in pGP3 reduced C. muridarum survival in the stomach by more than 100-fold, while the reduction in survival in the vagina was only ∼8-fold. This discrepancy is consistent with previous observations that loss-of-function mutations in genes carried in the C. muridarum plasmid or genome caused a more dramatic phenotype in the GI tract than in the genital tract (28–30, 53). The GI tract antimicrobial machineries, such as gastric acid and digestive hydrolases, are more robust than those in the genital tract. The robust antimicrobial mechanisms in the GI tract may impose stronger selection pressures for Chlamydia to evolve countermeasures. For example, the chlamydial acquisition of pGP3 may be selected for due to conferring the ability to overcome the gastric barrier during chlamydial adaptation to the GI tract. The same pGP3-dependent resistance to the gastric barrier may also help chlamydial colonization in the lower genital tract when Chlamydia is introduced into the genital tract. Even though pGP3 only promoted chlamydial survival in the mouse vagina by ∼8-fold, this makes a big difference in medical significance, allowing the pGP3-competent C. muridarum to induce pathology in the upper genital tract (54). The fact that the genital tract virulence factor pGP3 exhibits a more dramatic phenotype in the GI tract than in the genital tract (30) suggests that investigating C. muridarum interactions with the mouse GI tract may represent a productive approach for revealing chlamydial pathogenic mechanisms in the genital tract.
Although the observations in the mouse model have provided exciting information for guiding studies on human chlamydial infections, it is worth emphasizing that maintenance of fitness for or adaption to the mouse GI tract by C. muridarum does not necessarily mean C. trachomatis also maintains fitness for the human GI tract. This is because C. trachomatis is known to transmit between human genital tracts, while C. muridarum may be naturally transmitted between mice via an oral-fecal route. It is conceivable that the oral-fecal route may be more efficient than genital tract contact in spreading microbes among mice. Genome sequence analyses (55) have revealed that many Chlamydia species adapted to animal hosts still maintain three copies of complete toxin genes encoding homologues of the large toxin from the enteric bacterium Clostridium, while C. trachomatis has accumulated many loss-of-function mutations in the toxin genes (56), suggesting that C. trachomatis, although it may have initially adapted to colonize the GI tract, is moving away from the GI tract. Nevertheless, since non-lymphogranuloma venereum (LGV) C. trachomatis is still frequently detected in rectal swab samples (2–5) and the medical significance of non-LGV C. trachomatis in the GI tract remains unclear, it will be worth the effort in investigating the biology of C. trachomatis interactions with the human GI tract, although with the above-named caveat in mind. Investigating the natural history of C. trachomatis interaction with both the human genital and GI tract may provide useful information for advancing our understanding of chlamydial pathogenic mechanisms.
MATERIALS AND METHODS
Chlamydial organisms.All Chlamydia muridarum clones used in the current study were derived from strain Nigg3 (GenBank accession number CP009760.1). A plasmid-free clone, CMUT3G5 (GenBank accession number CP006974.1), that was initially derived from Nigg3 (57) was used for transformation with the plasmid pCM:GFP to create strain CM-pGFP (designated the wild type in the current study) or with pCM:GFP with a premature stop codon in the pgp3 gene to create strain CM-pGP3S (designated the mutant in the current study), as described previously (54, 58). The genome and plasmid sequences of CM-pGFP and CM-pGP3S are nearly identical, with the exception of the premature stop codon in pgp3 in CM-pGP3S. Since both are transformants, the plasmid copy numbers are also similar. The C. trachomatis L2 organisms used in the current study include the wild-type L2-pGFP clone and the pGP3-deficient mutant L2-pGP3S clone as described previously (59). All organisms were propagated in HeLa cells and purified as elementary bodies (EBs) as mentioned previously (26, 60). Aliquots of the purified EBs were stored in SPG buffer (220 mM sucrose, 12.5 mM phosphate, and 4 mM l-glutamic acid, pH 7.5) at −80°C until use.
Mouse infection.The mouse experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (61). The protocol was approved by the Committee on the Ethics of Laboratory Animal Experiments of the University of Texas Health Science Center at San Antonio.
Purified C. muridarum EBs were used to inoculate 5- to 7-week-old mice (Jackson Laboratories, Inc., Bar Harbor, ME) intragastrically, intracolonically, intravaginally, or intrabursally with different numbers of inclusion-forming units (IFUs) as indicated for individual experiments and as described previously (16, 26, 30, 54). C57BL/6J mice were from Jackson Laboratories (stock number 000664), while breeding pairs of gastrin-deficient mice were from the University of Michigan (31). The remaining knockout (KO) mice were all purchased from Jackson and were as follows: Rag1 KO (B6.129S7-Rag1tm1Mom/J; stock number 002216), MyD88 KO [B6.129P2(SJL)-Myd88tm1.1Defr/J; stock number 009088], IFNgR KO (B6.129S7-Ifngr1tm1Agt/J; stock number 003288), and IL-22cre [C57BL/6-Il22tm1.1(icre)Stck/J (with the first IL-22 exon replaced with a codon-optimized Cre recombinase); stock number 027524].
For intragastric inoculation, live C. muridarum EBs suspended in 200 μl of SPG buffer were delivered to each mouse using a straight ball end needle (item number N-PK 020; Braintree Scientific, Inc., Braintree, MA). In some experiments, mice were treated with the proton pump inhibitor omeprazole (product number O104; Sigma-Aldrich, St. Louis, MO) to raise stomach pH. Three days prior to and throughout the experiment, omeprazole was cointragastrically inoculated twice a day at 2 mg/200 μl SPG buffer per inoculation. Intracolonic inoculation was used for delivering live organisms in 2 μl of SPG buffer to the mouse colon using an inoculation tube (NSET, catalog number 60010; ParaTechs Corp., Lexington, KY). Five days prior to intravaginal inoculation, mice were subcutaneously injected with 2.5 mg of colloidal depot medroxyprogesterone (Depo-Provera; Pharmacia & Upjohn LLC, Kalamazoo, MI) suspended in sterile phosphate-buffered saline (PBS). For intravaginal inoculation, the EB inoculum in 10 μl SPG buffer was delivered into the mouse vagina using a 20-μl micropipette tip. After the inoculations, mice were monitored for vaginal and rectal live organism shedding or sacrificed to titrate live organisms in the corresponding organs/tissues or evaluate pathology in the upper genital tract.
Titrating live chlamydial organisms recovered from swabs and tissue homogenates.To quantitate live chlamydial organisms in vaginal or rectal swab samples, each swab was soaked in 0.5 ml of SPG buffer and vortexed with glass beads, and the chlamydial organisms released into the supernatants were titrated on HeLa cell monolayers in duplicate. The infected cultures were processed for immunofluorescence assay as described previously (57, 62) and below. Inclusions were counted in five random fields per coverslip under a fluorescence microscope. For coverslips with less than one IFU per field, entire coverslips were counted. Coverslips showing obvious cytotoxicity of HeLa cells were excluded. The total number of IFUs per swab was calculated based on the mean number of IFUs per view, the ratio of the view area to that of the well, the dilution factor, and the inoculation volume. Where possible, a mean number of IFUs/swab was derived from the serially diluted and duplicate samples for any given swab. The total number of IFUs/swab was converted into log10, which was used to calculate the mean value and standard deviation across mice of the same group at each time point.
For quantitating live organisms from mouse organs and tissue segments, each organ or tissue segment was transferred to a tube containing 0.5 to 5 ml SPG buffer, depending the sizes of the organs. Each GI tract was cut into 7 segments/portions, including stomach, duodenum, jejunum, ileum, cecum, colon, and anorectum (rectum), while each genital tract was divided into 6 segments, including vagina/exocervix, endocervix, left uterine horn, right uterine horn, left oviduct/ovary, and right oviduct/ovary. The organs and tissue segments were homogenized in cold SPG buffer using a 2-ml tissue grinder (catalog number K885300-0002; Fisher Scientific, Pittsburgh, PA) or an automatic homogenizer (Omni tissue homogenizer, order number TH115; Omni International, Kennesaw, GA). The homogenates were briefly sonicated and spun at 3,000 rpm for 5 min to pellet remaining large debris. The supernatants were titrated for live C. muridarum organisms on HeLa cells as described above. The results were expressed as log10 IFUs per organ or tissue segment.
Immunofluorescence assay.For immunofluorescence labeling of C. muridarum organisms in HeLa cells, a rabbit antibody (R1604, raised with purified C. muridarum elementary bodies) was used as a primary antibody to label C. muridarum, which was visualized with a goat anti-rabbit IgG conjugated with Cy2 (green, catalog number 111-225-144; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The DNA dye Hoechst 3328 (blue; Sigma-Aldrich, St. Louis, MO) was used to visualize nuclei. The doubly labeled samples were used to count C. muridarum organisms under a fluorescence microscope (AX70; Olympus) equipped with a charge-coupled-device (CCD) camera (Hamamatsu).
In vitro killing assays.Aliquots of strains CM-pGFP (wild-type [pGP3-sufficient] C. muridarum), CM-pGP3S (pGP3-deficient C. muridarum), L2-pGFP (wild-type C. trachomatis), or L2-pGP3S (pGP3-deficient C. trachomatis) organisms at 1 × 106 or 1 × 107 IFUs were each incubated in 100 μl saline with the pH adjusted to desired values using HCl or lactic acid for different time periods as described for individual experiments. The remaining live organisms were recovered by rapid dilution of each reaction mixture in SPG buffer and inoculated onto HeLa cell monolayers for titration as described above. For in vitro treatment with pepsin (product number P7000-25g; Sigma-Aldrich), pepsin solutions prepared in saline at different concentrations as indicated for individual experiments were each reacted with an aliquot of wild-type or pGP3-deficient mutant C. muridarum organisms for different periods of time as indicated for individual experiments. The live organisms remaining in each treatment sample were recovered by rapid dilution in SPG buffer and inoculated onto HeLa cell monolayers for titration. The numbers of live organisms recovered were expressed as log10 IFUs per sample.
Sarkosyl extraction and Western blot assay.To compare the levels of EB outer membrane susceptibility to sarkosyl permeabilization between wild-type and pGP3-deficient mutant C. muridarum organisms, a sequential sarkosyl extraction experiment was carried out as described previously (34). Aliquots of 5 × 108 IFUs of C. muridarum organisms were each suspended in 50 μl of 2% sarkosyl (product number L9150-50g; Sigma-Aldrich) in PBS for 10 min on ice, followed by centrifuging at 13,000 rpm at 4°C for 30 min in an Eppendorf microcentrifuge (catalog number 24-282; Genesee Scientific Corporation, San Diego, CA) to pellet the remaining chlamydial ghosts. After carefully removing the supernatant, the remaining pellet was defined as the “extraction 1” pellet. To extract the same pellet for a second time, a parallel extraction 1 pellet was resuspended in 50 μl of 2% sarkosyl solution as described above to produce an “extraction 2” pellet. Similarly, some parallel pellets were repeatedly extracted a total of 3 or 4 times. To each pellet, 50 μl of 2× SDS sample buffer was added, and a 10-μl amount was loaded to each lane. After electrophoresis and transfer, the membranes were blotted with mouse anti-chlamydial HSP60 monoclonal antibody (MAb) (clone BC7.1) (40, 63), anti-chlamydial major outer membrane protein (MOMP) MAb (clone MC22) (34), or mouse anti-denatured pGP3 antibody (33, 42, 43). The primary antibody bindings were visualized using a standard enhanced chemiluminescence (ECL) assay as described previously (34).
Statistical analyses.Comparison of the numbers of live organisms (in IFUs) between different samples was done using the Wilcoxon rank sum test. The 50% infectious dose (ID50) was calculated using the IC50 (50% inhibitory concentration) calculator from Aat Bioquest (https://www.aatbio.com/tools/ic50-calculator/), based on the rates of IFU positivity in mouse samples.
ACKNOWLEDGMENT
This study is supported in part by grants from the U.S. NIH (G.Z.).
FOOTNOTES
- Received 25 November 2018.
- Returned for modification 14 January 2019.
- Accepted 6 March 2019.
- Accepted manuscript posted online 11 March 2019.
- Copyright © 2019 American Society for Microbiology.
