Inhibition of Neisseria gonorrhoeae epithelial cell interactions by vaginal Lactobacillus species

Abstract

High levels of Lactobacillus, the dominant genus of the healthy human vaginal microbiota, have been epidemiologically linked to a reduced risk of infection following exposure to the sexually transmitted pathogen Neisseria gonorrhoeae. In this work, a cell culture model of gonococcal infection was adapted to examine the effects of lactobacilli on gonococcal interactions with endometrial epithelial cells in vitro. Precolonization of epithelial cells with Lactobacillus jensenii, Lactobacillus gasseri ATCC 33323, or L. gasseri ATCC 9857 reduced gonococcal adherence by nearly 50%. Lactobacilli also inhibited gonococcal invasion of epithelial cells by more than 60%, which was independent of the effect on adherence. Furthermore, lactobacilli were able to displace adherent gonococci from epithelial cells, suggesting that these organisms have potential as a postexposure prophylactic. Thus, vaginal lactobacilli have the ability to inhibit gonococci at two key steps of an infection, which might have a significant effect in determining whether the gonococcus will be able to successfully establish an infection following exposure in vivo.

FIG. 1.

Growth of N. gonorrhoeae in the presence or absence of L. jensenii. N. gonorrhoeae (10⁶ CFU/ml inoculum) was grown in the presence (□) or absence (♦) of L. jensenii (10⁷ CFU/ml inoculum). Serial dilutions were plated on selective medium at 2-h intervals to quantify CFU. Data are the averages of three independent experiments. Error bars indicate standard deviations.

FIG. 2.

Adherence of N. gonorrhoeae to Hec-1-B cells precolonized with lactobacilli. Epithelial cells were incubated with lactobacilli for 3 h. Nonadherent lactobacilli were removed prior to the addition of gonococci, and the Hec-1-B cells were incubated for an additional 3 h. Relative adherence is the percentage of adherent gonococci in wells containing lactobacilli divided by the percentage of adherent gonococci in the control. The data are averages of three independent experiments performed in duplicate. Error bars indicate standard deviations. *, statistically significant.

FIG. 3.

Adherence of N. gonorrhoeae to Hec-1-B cells in the presence of adherent and nonadherent lactobacilli. Relative adherence is the percentage of adherent gonococci in wells containing lactobacilli divided by the percentage of adherent gonococci in the control. The data are the averages of three independent experiments performed in triplicate. Ratios of gonococci to lactobacilli were 1:0, 10⁶ gonococci to no lactobacilli; 1:1, 10⁶ gonococci to 10⁶ lactobacilli; 1:10, 10⁶ gonococci to 10⁷ lactobacilli. At 3 h p.i. the number of CFU of N. gonorrhoeae doubled, and the total number of CFU of gonococci in the control was not significantly different from that of the Lactobacillus-treated samples (P value of 0.324 for 1:1 ratio and of 0.536 for 1:10 ratio). Error bars indicate standard deviations. Student's t test P values were 0.11 for 1:1 and 0.004 for 1:10. *, statistically significant.

FIG. 4.

Displacement of N. gonorrhoeae adherent to Hec-1-B cells by lactobacilli. N. gonorrhoeae adherent to Hec-1-B cells after 3 h were then infected with L. gasseri ATCC 33323 or L. jensenii at an MOI of 100 for 1 h. Adherence data are presented as the percentages of adhered gonococci in the well divided by that of the control and are the averages of four or more experiments performed in triplicate. Error bars indicate standard deviations. Student's t test P values were 0.019 (L. gasseri ATCC 33323) and 0.174 (L. jensenii). *, statistically significant.

FIG. 5.

Gram stains from an adherence assay performed as for the experiment shown in Fig. 2 with Hec-1-B cells precolonized with 10⁷ CFU of L. jensenii. (A) Gonococci and lactobacilli adherent to epithelial cells. (B) Gonococci adherent to epithelial cells in the absence of lactobacilli. (C) Arrangement of gonococci and lactobacilli in the supernatant. In all panels the white arrow points to lactobacilli, and the black arrow points to gonococci (Olympus model CHBS microscope; ×1,000 magnification).

FIG. 6.

Adherence of gonococci in medium preconditioned with L. jensenii and Hec-1-B cells. Percent adherence is the ratio of adherent gonococci to the total number of gonococci for each condition. Number of lactobacilli used to condition the medium: fresh medium; no lactobacilli and fresh DMEM-5% FCS-GC SII; preconditioned medium at an MOI of 10, 10⁶ CFU lactobacilli to 10⁵ Hec-1-B cells; preconditioned medium at an MOI of 100, 10⁷ CFU lactobacilli to 10⁵ Hec-1-B cells. Data are the averages of four independent experiments performed in triplicate. Error bars indicate the standard deviations. P values are 0.631 (MOI of 10) and 0.884 (MOI of 100).