Porphyromonas gingivalis-mediated Epithelial Cell Entry of HIV-1

Abstract

HIV-1 relies on the host's cell machinery to establish a successful infection. Surface receptors, such as CD4, CCR5, and CXCR4 of T cells and macrophages, are essential for membrane fusion of HIV-1, an initiate step in viral entry. However, it is not well defined how HIV-1 infects CD4-negative mucosal epithelial cells. Here we show that there is a specific interaction between HIV-1 and an invasive oral bacterium, Porphyromonas gingivalis. We found that HIV-1 was trapped on the bacterial surface, which led to internalization of HIV-1 virions as the bacteria invaded CD4-negative epithelial cells. Both bacterial and viral DNA was detected in HeLa and TERT-2 cells exposed to the HIV-1-P. gingivalis complexes 2 hr after the initial infection but not in cells exposed to HIV-1 alone. Moreover, epithelial cell entry of HIV-1 was positively correlated with invasive activity of the P. gingivalis strains tested, even when the binding affinities of HIV-1 to these strains were similar. Finally, it was demonstrated that the viral DNA was integrated into the genome of the host epithelial cells. These results reveal a receptor-independent HIV-1 entry into epithelial cells, which may be relevant in HIV transmission in other mucosal epithelia where complex microbial communities can be found.

Keywords: CCR5; CXCR4; HeLA cells; P. gingivalis 33277; TERT-2 cells; bacterial invasion.

Figure 1.

Interaction of HIV-1 and Porphyromonas gingivalis and Streptococcus gordonii. (A) P. gingivalis strains 33277, FAE, W83, as well as S. gordonii G9B and E. coli DH5α, were incubated with NL4.3 or YU2 (p24, 250 ng) for 30 min and the bacteria/HIV-1 complexes collected by centrifugation. Quantitation of HIV-1 bound to bacterial surfaces was conducted by a p24 assay. Asterisk (*) indicates a statistically significant difference in p24 levels for comparison of p24 reading of phosphate-buffered saline (PBS) with that of bacteria incubated with HIV-1 (t test; p < .05). (B) P. gingivalis 33277 cells were exposed to different doses of NL4.3 for 30 min. (C) Bacterial cells were mixed with green fluorescent protein (GFP)–tagged HIV-1 NL4.3 for 30 min, and free virions were removed by washing with PBS. P. gingivalis cells were identified by a rabbit anti-FimA polyclonal antibody, and S. gordonii G9B was identified by a rabbit anti-SpB polyclonal antibody. Both were visualized by TRITC-conjugated AffiniPure goat anti-rabbit IgG antibody (red).

Figure 2.

Porphyromonas gingivalis promotes HIV-1 entry into HeLa cells. (A) Comparison of invasive ability of oral bacteria into Hela cells 3 hr after the initial infection. (B) Comparison of the role of various bacteria in HIV-1_NL4.3 entry of HeLa cells and (C) in HIV-1_YU2 entry 3 hr after the initial infection. (D) The corresponding of bacterial 16S rRNA and HIV-1_NL4.3 gag DNA detected during a 24-hr period after the initial infection. Each bar represents the relative fold increase in P. gingivalis 16S rRNA or HIV-1_NL4.3 DNA detected in HeLa cells compared to its detection levels in noninfected cells (as 1 unit). Error bars represent SD (n = 3). An asterisk indicates a statistically significant difference in host cell levels with and without infection (t test; p < .05).

Figure 3.

Visualization of intracellular HIV-1_NL4.3 and Porphyromonas gingivalis 33277 in HeLa cells with a confocal microscopy. HeLa cells were grown in a glass bottom dish (MatTek, Boston) for 16 hr and infected with P. gingivalis–NL4.3 complexes for 30 min. Intracellular P. gingivalis cells and HIV-1_NL4.3 were visualized under a confocal microscope (Nikon A1R). (A) Visualization of green fluorescent protein–tagged NL4.3 (green). (B) P. gingivalis cells were visualized by Alexa Fluor 546–conjugated anti-rabbit IgG secondary antibody (red). (C) Infected cells presented by differential interference contrast images. The dashed line indicates the boundary around an infected HeLa cell containing colocalization of both P. gingivalis and NL4.3.

Figure 4.

Integration of HIV-1 into the genome of HeLa cells. HeLa cells were infected with cell-free NL4-3 or Porphyromonas gingivalis 33277–associated NL4-3 for 1 hr. The infected HeLa cells were incubated for another 2 hr after the unbound bacteria and virions were removed. Quantitation of integrated provirus was determined via an Alu-long terminal repeat–based real-time nested polymerase chain reaction assay. Each bar represents a mean of copies of integrated provirus per 10⁶ cells ± SD. The results were obtained from 3 independent experiments. An asterisk indicates a statistically significant difference in provirus copies in the cells infected with cell-free NL4.3 versus cells infected with P. gingivalis–NL4.3 complexes (t test; p < .05).

Figure 5.

Expression of CXCR4 and CCR5 coreceptors on the surfaces of HeLa and TERT-2 cells. Following exposure to Porphyromonas gingivalis 33277, HIV-1, or P. gingivalis–HIV-1 complexes, the expressions of coreceptors were determined via an ELISA. (A) Anti-hCXCR4 and (B) anti-hCCR5 were applied to wells coated with HeLa or TERT-2 cells. The cells were then incubated with horseradish peroxidase–conjugated antibodies against mouse IgG. Each bar represents the relative expression of HIV-1 coreceptor on the surface of epithelial cells infected with the bacteria and/or HIV-1 compared to that in the cells noninfected (as 1 unit) (n = 3). Student’s t test was used to determine statistical significance of the differences in the expression profiles.