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. 2021 Oct 15;89(11):e00219-21. doi: 10.1128/IAI.00219-21

The Role of 17β-Estrogen in Escherichia coli Adhesion on Human Vaginal Epithelial Cells via FAK Phosphorylation

Xia Liu a,b,#, Ting Luan a,#, Wanqing Zhou c,#, Lina Yan a, Hua Qian b, Pengyuan Mao a, Lisha Jiang a,d, Jingyan Liu a, Can Rui a, Xinyan Wang a, Ping Li a,✉, Xin Zeng a,✉
Editor: Denise Monacke
PMCID: PMC8519280  PMID: 34424749

ABSTRACT

Estrogen, the predominant sex hormone, has been found to be related to the occurrence of vaginal infectious diseases. However, its role in the occurrence and development of bacterial vaginitis caused by Escherichia coli is still unclear. The objective of this study was to investigate the role of 17β-estrogen in E. coli adhesion on human vaginal epithelial cells. The vaginal epithelial cell line VK2/E6E7 was used to study the molecular events induced by estrogen between E. coli and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation with cell adhesion. The phosphorylation status of FAK and estrogen receptor α (ERα) upon estrogen challenge was assessed by Western blotting. Specific inhibitors for ERα were used to validate the involvement of ERα-FAK signaling cascade. The results showed that, following stimulation with 1,000 nM estrogen for 48 h, transient activation of ERα and FAK was observed, as was an increased average number of E. coli cells adhering to vaginal epithelial cells. In addition, estrogen-induced activation of ERα and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10 μM concentration and acted for 1 h, and a decrease in the number of adherent E. coli cells was observed simultaneously. However, this inhibitory effect diminished as the concentration of estrogen increased. In conclusion, FAK and ERα signaling cascades were associated with the increasing E. coli adherence to vaginal epithelial cells, which was promoted by a certain concentration of estrogen.

KEYWORDS: 17β-estrogen, Escherichia coli, vaginal epithelial cells, adhesion, focal adhesion kinase

INTRODUCTION

The vagina is a complex microecological system. There are dozens of microorganisms in the vagina of healthy women, in which the bacterial content of vaginal secretions is about 105 to 106 CFU/ml, while the vaginal bacteria in the infected state can be as much as 109 to 1011 CFU/ml (1). Women of reproductive age are prone to vaginal infections that not only bring heavy embarrassment to patients but also causes pelvic inflammatory diseases (2), infertility (3), ectopic pregnancy (4), abortion (5), premature labor (6), and neonatal infectious disease (7). Although vaginal infectious diseases, including aerobic vaginitis, trichomonas vaginitis, vulvovaginal candidiasis, and bacterial vaginosis, have been studied for more than 15 years (8), their underlying mechanisms are still poorly understood. Therefore, it is very important to explore their pathogenesis.

Bacterial vaginosis (BV), characterized by a reduction of vaginal lactobacilli, is the primary disease of vaginal microbiota imbalance in reproductive-age women. It is difficult to treat BV, as it is caused by diversified microorganisms (9). Through the analysis of pathogenic bacteria in BV patients, it has been found that Escherichia coli is one of the common pathogenic bacteria, in addition to Gardnerella, Prevotella, and Enterococcus faecalis (10, 11). E. coli is a conditional pathogen that can colonize the vaginal microbiome, usually without symptoms. However, E. coli can also be a highly virulent and frequently deadly pathogen when organism immunity declines or under stress conditions (12).

As a small natural steroid hormone in mammals, estrogen has many types, such as 17β-estradiol (E2), estrone (E1), and estriol (E3). Among them, E2, whose serum concentration fluctuates between 48 and 1,835 pmol/liter, has a broad and main physiological role in premenopausal women (13). It not only facilitates and maintains female reproductive organs and secondary sexual characteristics but also has extensive functions in the endocrine, cardiovascular, and metabolic systems and bone growth and skin (14). In addition, many kinds of female diseases have been confirmed to be associated with estrogen. However, the effect of estrogen on E. coli infection is currently inconclusive. Yang et al. found that estrogen inhibits the overgrowth of E. coli in the rat intestine under simulated microgravity (15). However, Gümüş et al. demonstrated that the expression of virulence genes (cnf 1, sfa-foc, and usp) of E. coli was significantly upregulated by the presence of estrogen (16). Sobel et al. also found that estrogen may contribute to genitourinary tract infection in premenopausal females by enhancing the adhesion and colonization of E. coli to epithelial cells (17). We currently know little about the role of estrogen in the process of E. coli adherence and how it invades the vaginal epithelial cells.

Vaginal epithelial cells act as the first line of defense against E. coli in the female vagina. Once the vaginal microecosystem is out of balance, E. coli grows quickly and attacks host cells and then adheres to vaginal epithelial cells (18). It is known that all infections begin by adhesion of pathogens, and adhesion ability of a bacterium is closely related to its pathogenicity (19). Cell adhesion molecules are in varied forms, such as integrins, selectins, cadherins, immunoglobulins, CD15, and MLA (20). It has been found that focal adhesion kinase (FAK) is also a special adhesion molecule that mediates adhesion between cells or between cells and extracellular matrix (21). Xue et al. found that the adhesion ability of E. coli-infected epithelial cells is prevented and synchronized by FAK inactivation (22). Estrogens may be involved in the regulation of several cytoskeletal and membrane remodeling components as the focal adhesion complexes (23). Under the control of actin organization, estrogens could accelerate cell movement through their regulation of cell morphology and reciprocal action with extracellular matrix (24). There are three types of estrogen receptors (ERs), namely, ERα, ERβ, and GPR30 (GPER-1). Among them, classical estrogen signaling in the female reproductive tract is activated through ERα (25). ERα, which can be activated by phosphorylation of multiple amino acid residues, is predominately phosphorylated on serine 118 (Ser-118) in response to estrogen binding (26). The phosphorylation of ERα can affect its transcriptional function and recycling as well as the ability to bind to DNA and ligands (27). In this regard, it has been reported that estrogens could induce the phosphorylation of FAK through ERα as well as their subsequent translocation within the membrane sites where focal adhesion complexes are assembled (28).

Therefore, we suspected that estrogen activates the ERα pathway to induce the phosphorylation of FAK and ultimately affect vaginal infection of E. coli. Consequently, we set out to explore the effects of E2 treatment at different concentrations and different time points on the adhesion of E. coli to vaginal epithelial cells and its effects on phosphorylation of ERα and FAK.

RESULTS

Coculture time for E. coli to adhere to vaginal epithelial cells.

We aimed to explore the optimal coculture time for E. coli adhesion to vaginal epithelial cells. During this period, E. coli had not caused vaginal epithelial cell floating and dying. The adhesion of E. coli to vaginal epithelial cells was observed after the interaction of both sides for 0.5 h, 1 h, 2 h, and 3 h, and then the lysates of cells were collected to determine the amount of total protein of ERα and FAK as well as phospho-ERα and phospho-FAK through Western blotting. The results showed that the number of E. coli cells adhering to each cell raised gradually with the increase of interaction time (Fig. 1A). Further, 100 cells were randomly selected under the oil immersion lens to calculate the number of adherent E. coli cells, and the relevant results showed that the average number of E. coli cells was 9.06 when the interaction time was 0.5 h, 13.15 when it was 1 h, 16.59 when it was 2 h, and 21.2 when it was 3 h (Fig. 1B). It was clear that significant difference (P < 0.05) existed between the 0.5-h group and 1-h group, 2-h group, and 3-h group. Western blot results showed that total protein expression of ERα and FAK did not have obvious change, while their extent of phosphorylation gradually increased with time (Fig. 1Ca). Corresponding semiquantitative analysis of Western blot results are shown in Fig. 1Cb.

FIG 1.

FIG 1

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Coculture time for E. coli to adhere to vaginal epithelial cells. E. coli adhered to vaginal epithelial cells after 0.5 h (Aa), 1 h (Ab), 2 h (Ac), and 3 h (Ad) was observed under an oil immersion lens. (B) Data were expressed as the average number of E. coli cells adhering to each cell for different times. (Ca) The expression of total protein of ERα and FAK, as well as phospho-ERα (p-ERα) and phospho-FAK (p-FAK), was assayed using specific antibodies and Western blotting as described in Materials and Methods. (Cb) The Western blotting results were quantified by Image J software. Data are mean values ± standard deviations from three duplicate experiments. Analysis by Student's t test: **, P < 0.01 compared with the 1-h groups; ***, P < 0.001 compared with the 2-h groups; ***, P < 0.001 compared with the 3-h groups.

The concentration of E. coli adhering to vaginal epithelial cells.

To explore the suitable concentration of E. coli adhesion to vaginal epithelial cells, we examined the adhesion of E. coli at increasing concentrations to vaginal epithelial cells. The results showed that the number of E. coli cells adhering to each cell gradually increased, with the concentration in the range of 2.0 × 107 to 2.0 × 109 CFU/ml. When the concentration reached 2.0 × 1010 CFU/ml, the number of adhering E. coli cells decreased obviously, and the cells lost normal appearance and shrunk to globular size rather than extending around and eventually died (Fig. 2A). The results of the bacterial counting experiments showed that the average number of adherent E. coli cells at four increasing concentrations was 9.05, 14.2, 19.84, and 8.83 (Fig. 2B). Significant differences existed between the 2.0 × 107 CFU/ml group and 2.0 × 108 CFU/ml group as well as between the 2.0 × 107 CFU/ml group and 2.0 × 109 CFU/ml group (P < 0.05). However, there was no difference between the 2.0 × 1010 CFU/ml group and 2.0 × 107 CFU/ml group.

FIG 2.

FIG 2

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Concentration of E. coli infecting vaginal epithelial cells could maximize adhesion of E. coli to the cells. E. coli adhering to vaginal epithelial cells at concentrations of 2.0 × 107 CFU/ml (Aa), 2.0 × 108 CFU/ml (Ab), 2.0 × 109 CFU/ml (Ac), and 2.0 × 1010 CFU/ml (Ad), respectively, was observed under the oil immersion lens. (B) Data were expressed as the number of E. coli cells adhering to each cell on average at different concentrations. The data represent the number of cells (mean values ± standard deviations) from 3 duplicate experiments. Student's t test analysis: **, P < 0.01 compared with the 2.0 × 108 group; ***, P < 0.001 compared with the 2.0 × 109 group; #, P > 0.05 compared with the 2.0 × 1010 group.

The effect of E2 on adhesion of E. coli to vaginal epithelial cells.

VK2/E6E7 cells were pretreated with gradient concentrations of E2 (1 to 1,000 nM) for 24 h, 48 h, and 72 h, respectively. E. coli at a concentration of 2.0 × 109 CFU/ml was cocultured with these cells for 3 h. The results indicated that the number of E. coli cells adhered to cells gradually increased along with the raised concentration of E2 (Fig. 3A). The results of the bacterial counting experiments showed that after 24 h of E2 pretreatment, the average number of E. coli cells adhering to each cell in the blank group (KM alone with no E2), solvent group (KM with 0.01% alcohol), and experimental groups at four gradient concentrations were 2.7, 3.57, 3.54, 4.03, 6.64, and 13.7, respectively. The average number was separately 8.03, 9.12, 10.37, 11.42, 13.05, and 13.04 after 48 h, and the average number was 5.31, 6.56, 7.44, 8.86, 9.48, and 10.53 after 72 h, respectively (Fig. 3B). The results showed that the number of E. coli cells adhering to cells rose with the increase of E2 concentration. When E2 concentration reached 1,000 nM, the number of adhered E. coli cells reached the highest point, whereas when the interaction time of estrogen reached 48 h, the number of adhering E. coli cells was at its peak, and the adhering number decreased slightly at the interaction time of 72 h compared to the interaction time of 48 h. In addition, we collected the lysates of cells treated with increasing concentrations of E2 to determine the amount of total protein of ERα and FAK as well as the phospho-ERα and phospho-FAK. Western blot results showed that total protein expression of ERα and FAK did not have obvious changes after 48 h of E2 treatment, while their extent of phosphorylation gradually increased with elevated concentration of E2. Only the phosphorylation level of ERα was not significantly different between the 100 nM E2 group and the 1,000 nM E2 group (Fig. 3Ca). The semiquantitative analysis of corresponding Western blot results is shown in Fig. 3Cb.

FIG 3.

FIG 3

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Effects of different concentrations of E2 on adhesion of E. coli to vaginal epithelial cells after different times. E. coli adhered to vaginal epithelial cells in blank (Aa) and vehicle (Ab) and at E2 concentrations of 1 nM (Ac), 10 nM (Ad), 100 nM (Ae), and 1,000 nM (Af) was observed under the oil immersion lens. (B) Data were expressed as the average number of E. coli cells adhering to each cell after treatment of various concentrations of E2 for different times. (Ca) The expression of total protein of ERα and FAK as well as the phosphor-ERα (p-ERα) and phosphor-FAK (p-FAK) was assayed using specific antibodies and Western blotting as described in Materials and Methods. (Cb) The Western blotting results were quantified by Image J software. Data were mean values ± standard deviations from three duplicate experiments. Analysis by Student's t test: (24 h) *, P < 0.05 compared with vehicle group, 1 nM E2 group, and 10 nM E2 group; ***, P < 0.001 compared with 100 nM E2 group and 1,000 nM E2 group; (48, 72 h) *, P < 0.05 compared with vehicle group; **, P < 0.01 compared with 1 nM E2 group and 10 nM E2 group; ***, P < 0.001 compared with 100 nM E2 group and 1,000 nM E2 group.

E. coli adhesion was inhibited by ER antagonist.

VK2/E6E7 cells were pretreated with 1,000 nM E2 solution for 48 h with 10 μM ICI182780 for 0 h, 1 h, 12 h, and 24 h. E. coli at a concentration of 2.0 × 109 CFU/ml was cocultured with the cells for 3 h. Subsequently, we noticed the adhesion of E. coli to vaginal epithelial cells. Additionally, we collected the lysates of cells treated with E2 and ICI182780 to determine the amount of total protein of ERα and FAK as well as the phospho-ERα and phospho-FAK. The results indicated that the number of E. coli cells adhering to cells decreased first and then increased with the prolongation of pretreatment time of ICI182780. When the pretreatment time reached 1 h, the adhered number was significantly reduced. However, this inhibitory effect was gradually relieved with the proceeding of ICI182780 action. In addition, no obvious difference was observed in adhering number between the ICI182780 12-h group and 0-h group (Fig. 4A). The results of the bacterial counting experiments showed that the average number of adherent E. coli was 10.63 after treatment with E2 alone and 8.54, 5.75, 9.42, and 12.28 after ICI182780 treatment for different times (Fig. 4B). Western blot results showed that total protein expression of ERα and FAK did not have obvious changes after the treatment of E2 and ICI182780. After E2 pretreatment, nevertheless, the level of phospho-ERα in cells treated with ICI182780 for 1 h was initially inhibited, and this response was more pronounced after treatment of ICI182780 for 12 and 24 h. The level of phospho-FAK in cells treated with ICI182780 for 1 and 12 h was obviously inhibited, and this inhibition was greatly abolished after treatment of ICI182780 for 24 h (Fig. 4Ca). The semiquantitative analysis of corresponding Western blot results is shown in Fig. 4Cb.

FIG 4.

FIG 4

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Duration of E. coli adhesion experiments with ICI182780. E. coli adhered to vaginal epithelial cells after 1,000 nM E2 action (Aa) and 10 μM ICI182780 for 0 h (Ab), 1 h (Ac), 12 h (Ad), and 24 h (Ae) was observed under the oil immersion lens. (B) Data were expressed as the average number of E. coli cells adhering to each cell after action of E2 and ICI182780. (Ca) The expression of total protein of ERα and FAK as well as the phospho-ERα (p-ERα) and phospho-FAK (p-FAK) was assayed using specific antibodies and Western blotting as described in Materials and Methods. (Cb) The Western blotting results were quantified by Image J software. Data are mean values ± standard deviations from three duplicate experiments. Analysis by Student's t test: *, P < 0.05 compared with 1,000 nM E2 plus ICI 0-h group; ***, P < 0.001 compared with 1,000 nM E2 plus ICI 1-h group; #, P > 0.05 compared with 1,000 nM E2 plus ICI 12-h group; *, P < 0.05 compared with 1,000 nM E2 plus ICI 24-h group.

VK2/E6E7 cells were pretreated with a series concentrations of E2 (10 to 1,000 nM) for 48 h and 10 μM ICI182780 for 1 h. After this, E. coli at a concentration of 2.0 × 107 CFU/ml was cocultured with the cells for 3 h. Subsequently, we observed the adhesion of E. coli to vaginal epithelial cells. Additionally, we collected the lysates of cells treated with E2 and ICI182780 to determine the amount of total protein of ERα and FAK as well as the phosphor-ERα and phospho-FAK. The results showed that the number of E. coli cells adhering to cells increased significantly after the pretreatment with 1,000 nM E2 compared with the control group but decreased after treatment with 10 μM ICI182780. When the concentration of E2 was 10 nM, the number of E. coli cells was more obviously reduced. However, when the concentration of E2 was 1,000 nM, the number of adhered E. coli cells increased more significantly than in the 10 nM E2 group and 100 nM E2 group (Fig. 5A). The results of the bacterial counting experiments showed that the average number of adherent E. coli cells was 12.95 in the solvent group (KM with 0.01% alcohol). The average adhesion number was 31.76 after treatment with 1,000 nM E2 alone and 17.48, 21.66, and 24 after treatment with 10 μM ICI182780 and E2 with a gradient concentration (10 to 1,000 nM) (Fig. 5B). Western blot results showed that there was no significant change in the expression of these four proteins after E2 or ICI182780 treatment alone. Nevertheless, the expression of phospho-ERα and phospho-FAK in cells treated with 10 μM ICI182780 and 10 nM E2 decreased distinctly, and their extent of phosphorylation gradually increased with concentration of E2 (Fig. 5Ca). The semiquantitative analysis of corresponding Western blot results is shown in Fig. 5Cb.

FIG 5.

FIG 5

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Adhesion of E. coli to vaginal epithelial cells was inhibited by ICI182780. E. coli adhered to cells in vehicle (Aa), 1,000 nM E2 (Ab), 10 μM ICI182780 and 10 nM E2 (Ac), 10 μM ICI182780 and 100 nM E2 (Ad), and 10 μM ICI182780 and 1,000 nM E2 (Ae) was observed under the oil immersion lens. (B) Data were expressed as the average number of E. coli cells adhering to each cell after action of E2 and ICI182780. (Ca) The expression of total protein of ERα and FAK, as well as phospho-ERα (p-ERα) and phospho-FAK (p-FAK), were assayed using specific antibodies and Western blotting as described in Materials and Methods. (Cb) The Western blotting results were quantified by Image J software. Data are mean values ± standard deviations from three duplicate experiments. Analysis by Student's t test: ***, P < 0.001 compared with 1,000 nM E2 group; *, P < 0.05 compared with 10 μM ICI182780 plus 10 nM E2 group; **, P < 0.01 compared with 10 μM ICI182780 plus 1,000 nM E2 group.

DISCUSSION

With the progress of the Human Microbiome Project (HMP), more than 20 microorganisms have been found in the vaginal microenvironment. Many of them are potentially pathogenic, but they are interrelated and interact with each other and adhere to and colonize the vaginal mucosal epithelium to maintain the vaginal microecological balance (11, 29, 30). E. coli is a common conditional pathogen in human digestive and urogenital tracts, with an infection rate of 23% among symptomatic women with aerobic vaginitis (31). Vaginal colonization and infection by E. coli in pregnant women can lead to serious consequences. Reuschel et al. showed that inflammatory immune responses induced by lipopolysaccharides (LPS) of Gram-negative bacteria, especially E. coli and E. aerogenes, play an important role in the pathogenesis of preterm labor and delivery and in neonatal disorders (32). Watt et al. found that E. coli strains responsible for amniotitis during pregnancy and at birth and for early bacteremia and meningitis in neonates, and intestinal strains, have colonized the vaginal microbiota (33). In addition, several researchers have extensively studied the virulence factors and pathogenic mechanism of E. coli (34). It is generally known that early molecular events in the interaction of bacteria and cells include adhesion, colonization, and invasion. Adhesion is a universal biological phenomenon in the relationship between microorganisms and their hosts as well as the first step in infection (35). The adhesion process of E. coli to mammalian cells remains elusive and requires the integration and coordination of complex biochemical and biomechanical signals. The formation of new adhesions involves a variety of signaling mediated by integrins and other adhesion receptors.

Some studies have demonstrated that estrogen is involved in the regulation of vaginal epithelial keratin and mucin expression, which promotes protective keratinization of vaginal epithelial cells (36). Estrogen can also reduce production of inflammation cytokines and inhibit neutrophil infiltration in vaginal epithelium when subjected to certain stimuli (37). FAK is a cytosolic protein tyrosine kinase that binds to integrin and growth factor receptor to promote cell adhesion, migration, and invasion (38). Until now, accumulating evidence has shown that the FAK-paxillin signaling pathway is involved in the migration and invasion of tumor cells through various molecular mechanisms (39). Increased expression of FAK showed good correlation with increased invasion and migration of several metastasis tumors, including colon tumors, breast tumors, ovarian tumors, and melanomas (40). Recently, some groups proved that PCTK3/CDK18 could regulate cell migration and adhesion by negatively modulating the FAK/Rho signaling pathway (41). Xue et al. found that FAK signaling cascades are involved in the early interaction between the intestinal epithelial cells and E. coli (22). Therefore, our study focused on the effect of estrogen on E. coli adhesion to vaginal epithelial cells and FAK signaling pathway.

In this study, our findings suggested that with a certain level of E2, when vaginal epithelial cells had interacted with E. coli, the key element of adhesion-FAK was activated through phosphorylation, and the vaginal epithelial cells turned more permissive to E. coli adherence, with the number of E. coli cells adhering to vaginal epithelial cells increased markedly. From this, we infer that one of the early molecular events caused by estrogen-promoting adhesion of E. coli to vaginal epithelial cells is rapid and sustained activation of the FAK cascade. This is similar to the report from Rigiracciolo et al. (42), who found that the FAK signaling pathway is rapidly and temporarily activated in triple-negative breast cancer cells by estrogen. Based on our observations, E2 activates FAK and the E2/ERα/FAK cascade is involved in the enhancement of E. coli adherence to epithelial cells, which is supported by the following evidence: (i) phosphor-FAK, the activated form of FAK, is detected on vaginal epithelial cells stimulated with a suitable concentration of E. coli and estrogen; (ii) an improved level of phosphorylation of FAK is accompanied by an increase in the number of E. coli cells adhered to vaginal epithelial cells; and (iii) specific ER antagonists can suppress the FAK activation in vaginal epithelial cells and reduce the number of adherent E. coli cells. However, the mechanism of this phenomenon remains unclear.

In vitro adherence of E. coli to isolated human genitourinary tract epithelial cells from women has been reported to change with the stages of the menstrual cycle of the cell donors (43). Sugarman et al. found that hormonal changes may influence bacterial adherence by altering epithelial cell number and morphology, modifying receptor site synthesis on epithelial cells, changing the surrounding pH or mucus composition, etc. (44). Therefore, the variable E2/ERα/FAK cascade-induced alteration in the cell membranes or in the cell metabolism that may lead to the enhancing of E. coli adherence will be the focus of our future research. Cheng et al. showed that Grb2 is a new FAK activator, and the activating action involves paxillin (45). Daly et al. also found that in hormone-responsive breast cancer cells, Grb2-associated binder 2 (Gab2) expression was induced by E2 via ER in a manner sensitive to anti-estrogen ICI182780 (46). Therefore, it is worth investigating the role of paxillin and Grb2 in the observed phenotypic increased E. coli adherence and E2/ERα/FAK cascade. In addition, bacteria employ adhesins on the cell surface to attach to the host. Dozens of the adhesins are identified as intracellular chaperones or enzymes in glycolysis or other central metabolic pathways (47). Thus, the effect of estrogen on the expression of intracellular chaperones and enzymes may also affect the results of this experiment, and we need to further explore the effect of estrogen on the metabolism of E. coli.

Since FAK regulates multiple biologic activities in normal cells, such as cell proliferation, differentiation, cell cycle traversal, and survival, we are currently performing additional correlative studies to delineate and characterize the expression and the specific roles of FAK in the interaction between vaginal epithelial cells and E. coli. FAK is not defined as an oncogene, but an increase in FAK expression has been reported in tumors of various tissue origins (48). A number of basic, clinical, and epidemiological studies have confirmed that inflammation is a clear risk factor for cancer, especially by infectious agents (49). However, estrogen can promote the adhesion of E. coli to the vagina and the activation of FAK in vaginal epithelial cells. Whether this process could cause precancerous lesions or even malignant tumors in the female genital tract is also the focus of our future research.

In conclusion, the present study reported that the sequential activation of FAK, in response to E2, and the E2/ERα/FAK cascade is involved in the enhancement of E. coli adherence to vaginal epithelial cells. The observation illustrated that adhesion is the first step in the infection of vaginal epithelial cells by E. coli, and this process may be closely related to the estrogen-FAK signaling pathway. BV caused by E. coli and its drug resistance are common in the clinic, and the bottleneck of screening new antibacterial drugs makes the choice of drugs for bacterial vaginitis difficult. We can try to explore new antibacterial substances that inhibit vaginal E. coli infection by blocking the E2-ERα-FAK signaling pathway. The results of our research could provide new clues for clinical treatment and drug development of bacterial colonization or infection caused by E. coli.

MATERIALS AND METHODS

Materials.

Polyclonal antibodies against β-actin, ERα, phospho-ERα, FAK, and phospho-FAK were purchased from Cell Signaling Technology (Danvers, MA), while goat anti-mouse and goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) were from Santa Cruz Biotechnology (CA, USA). 17β-estrogen (E2, Sigma, USA) was prepared as a stock solution at a concentration of 10−2 M with absolute ethanol and then diluted with keratinocyte medium (KM; ScienCell, USA) to the concentration required for experiments. ER antagonist (ICI182780; Fulvestrant; Tocris Bioscience, Britain), a specific antagonist of ER without estrogen-like effect, was prepared to stock solution at a concentration of 50 mM with absolute ethanol (Sigma, USA) and kept in a dark place at −20°C for further use. The solution was diluted with KM to the concentration required for experiments.

E. coli culture.

E. coli K-12 strain 5034 (ATCC 29425; USA) used in this research was provided by Microbiological Lab of Nanjing Drum Tower Hospital and grown on Sabouraud dextrose agar at 37°C. In order to prepare E. coli suspension, this strain was inoculated in Luria-Bertani (LB) medium (volume ratio of E. coli to LB medium was 1:50; Beijing Leagene, China), shaken at 220 rpm, 37°C, for 12 h, and finally cryopreserved in 15% glycerinum at −80°C. Simultaneously, LB medium was also adopted to adjust the E. coli suspension to the desired concentration. A hemocytometer (Isolab, Germany) was used for colony counting.

Cell lines and cell culture.

The VK2/E6E7 cell line (ATCC CRL-2616) was maintained in KM supplemented with keratinocyte growth supplement (KGS; Sigma, USA) and penicillin-streptomycin (Invitrogen, USA) solution in an atmosphere of 5% CO2 at 37°C. Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM–F-12; ATCC 30-2006), containing 10% fetal bovine serum (FBS; ATCC 30-2021), was used to neutralize the trypsin and cryopreserve cells. Furthermore, 4 × 104 cells in 12 ml medium were seeded onto 100-mm culture dishes and grown to 90% confluence before experiments, and every 2 days the medium was changed.

Time-dependent experiments of E. coli adhesion.

VK2/E6E7 cells in logarithmic phase were digested, blended, and counted. Subsequently, these cells were inoculated in a 60-mm culture dish (430166; Corning, USA) at a cell density of 5 × 105/ml and cultivated in a CO2 constant temperature incubator (Thermo Fisher Scientific, Waltham, MA) for 48 h to achieve 80% cell adherence. Two milliliters of E. coli suspension at a concentration of 2.0 × 109 CFU/ml was added into four 60-mm culture dishes and incubated in a CO2 constant temperature incubator for 0.5 h, 1 h, 2 h, and 3 h. To clear away E. coli that did not adhere to the cells, the culture medium was removed and the cells were washed three times with phosphate-buffered saline (PBS; Invitrogen, USA). Here, these cells were immobilized with −20°C pure methanol. Subsequently, 100 cells were randomly selected under the oil immersion lens to count E. coli cells adhering to each cell after gram staining. The experiments were repeated three times with the same techniques and methods.

Dose-dependent experiments of E. coli adhesion.

VK2/E6E7 cells were processed as described above. Two milliliters of E. coli suspension at four different concentrations of 2.0 × 107 CFU/ml, 2.0 × 108 CFU/ml, 2.0 × 109 CFU/ml, and 2.0 × 1010 CFU/ml was added in each of the four 60-mm culture dishes and then incubated in a CO2 constant temperature incubator at 37°C for 3 h. After that, we removed the culture medium and washed the cells three times with PBS and fixed cells with −20°C pure methanol. Next, 100 cells were randomly selected under the oil immersion lens after gram staining, and the average number of E. coli cells adhered to each cell was counted. The above-described experiments were repeated three times with the same techniques and methods.

E. coli adhesion experiments with gradient concentrations of E2 at different time points.

VK2/E6E7 cells were processed as described above; immediately after, the KM solution in each culture dish was replaced with the medium required for experiments as described below. Eighteen culture dishes were equally divided into three groups, and each group included a blank group (KM alone with no E2), a solvent group (KM with 0.01% alcohol), 1 nM E2 solution group, 10 nM E2 solution group, 100 nM E2 solution group, and 1,000 nM E2 solution group. The three experimental groups were cultured in a CO2 constant temperature incubator at 37°C for 24 h, 48 h, and 72 h, respectively. Two milliliters of E. coli suspension at a concentration of 2.0 × 109 CFU/ml was separately added into each of the above-described culture dishes and incubated in a CO2 constant temperature incubator at 37°C for 3 h afterwards. Thereafter we removed the unadhered E. coli, fixed the cells with methanol, and randomly selected 100 cells to count E. coli adhered to each cell after gram staining. The experiments were repeated three times with the same techniques and methods.

E. coli adhesion experiments with ER antagonist at different time points.

VK2/E6E7 cells were processed as described above. The KM solution in the five culture dishes was replaced by 100 nM E2 solution and then placed in the incubator for 24 h. After this step, 10 μM ICI182780 was added into four of these dishes, and the durations of ICI182780 treatment were 0 h, 1 h, 12 h, and 24 h. Next, 2 ml E. coli suspension at a concentration of 2.0 × 109 CFU/ml was added into each of the five culture dishes and incubated in a CO2 constant temperature incubator at 37°C for 3 h afterwards. Using the same methods as those described above, we removed E. coli that did not adhere to the cells, and the cells were immobilized with −20°C pure methanol. Subsequently, we randomly selected 100 cells under the oil immersion lens to count E. coli adhered to each cell after gram staining. The experiments were repeated three times with the same techniques and methods described above.

E. coli adhesion experiments inhibited by ER antagonist.

VK2/E6E7 cells were processed as described above. The KM solution in the first and second culture dishes were separately substituted by 0.01% alcohol solution and 100 nM E2 solution, respectively. However, the KM solution in the third, fourth, and fifth culture dishes were separately replaced by 10 nM, 100 nM, and 1,000 nM E2 solution. Next, 10 μM ICI182780 was added in these three culture dishes. After that, all of the above culture dishes were placed in the incubator for 24 h, and then each of the five culture dishes was added with 2 ml E. coli suspension at a concentration of 2.0 × 109 CFU/ml and incubated in a CO2 constant temperature incubator at 37°C for 3 h afterwards. Subsequently we cleared away the unadhered E. coli, fixed the cells with methanol, and randomly selected 100 cells to count E. coli adhered to each cell after gram staining. The experiments were repeated three times with the same techniques and methods.

Western blotting.

The cells in relevant culture dishes, which were stimulated in the above-described experiments, were washed with PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer at 4°C for 3 min. The cell suspension was centrifuged at 12,000 × g for 15 min at 4°C, after which the supernatants were collected. The BCA (bicinchoninic acid; Pierce) protein assay reagent was used to determine the protein concentration in the supernatant. The protein samples were added with 5× loading buffer and denatured by incubating at 95°C for 5 min; later, the samples were separated on 6% polyacrylamide gels (15 μg/lane) and transferred to polyvinylidene difluoride (PVDF) membrane. Afterwards, the blots were blocked with 1× TBST (0.1 mol/liter Tris, 0.9% NaCl, 0.1% Tween 20) overnight at 4°C containing 5% bovine serum albumin. Subsequently, the blots were incubated with a first antibody (1:2,000 dilution) for 2 h at room temperature and then washed with 1× TBST for 25 min (five times for 5 min each). Immediately after that, the blots were incubated with goat anti-rabbit or goat anti-mouse IgG (1:5,000 dilution) for 2 h at normal room temperature and washed with 1× TBST for 25 min (five times for 5 min each). Those blots were eventually developed with SuperSignal chemiluminescence reagent (Pierce) and exposed to X-ray with the aim of visualizing the proteins. The Western blot results were further analyzed by scanning the images with Gel Doc2000 (Bio-Rad, USA) and semiquantifying the gray bands with Image J software (Rawak Software Inc., Stuttgart, Germany).

Statistical analysis.

SPSS 16.0 was used in this study for statistical analysis. All data were expressed as means ± standard deviations. One-way analysis of variance was used to compare significant differences among multiple groups. A Student's t test was used to compare the differences between two groups, and a P value of <0.05 was considered significant.

ACKNOWLEDGMENTS

This research was financially supported by National Natural Science Foundation of China (82071602, 82073392, and 81671410), Six Talent Project in Jiangsu Province (WSY-119 and WSW-120), Postgraduate Research and Practice Innovation Project in Jiangsu Province JX10413752, Nanjing Medical Science and Technique Development Foundation QRX17072, and Science and Technology Development Fund of Nanjing Medical University 2017NJMUZD076.

Contributor Information

Ping Li, Email: njfyliping@163.com.

Xin Zeng, Email: august555482@126.com.

Denise Monack, Stanford University.

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