HIV-associated penile anaerobes disrupt epithelial barrier integrity

Abstract

Specific anaerobic taxa within the penile microbiome-the Bacteria Associated with Seroconversion, Inflammation and Immune Cells (BASIC) species-enhance HIV-1 susceptibility, in part by recruiting susceptible cells to the inner foreskin. However, their effect on epithelial barrier integrity has not been described. Using foreskin tissues and penile swabs from 116 males undergoing voluntary medical male circumcision, we assessed the relationship between BASIC species and foreskin epithelial thickness, junction protein expression, and cellular proliferation. The absolute abundance of BASIC species was associated with reduced tissue expression of the epithelial junction proteins claudin-1 and E-cadherin, and with elevated soluble E-cadherin in penile secretions, suggesting proteolytic cleavage. These effects were not seen in participants with a high abundance of control taxa without high levels of BASIC species. The BASIC species Prevotella bivia, but not Peptostreptococcus anaerobius or Dialister micraerophilus, was shown to directly degrade recombinant human E-cadherin and to increase the release of soluble E-cadherin from foreskin epithelial cells in vitro. In vivo BASIC species absolute abundance was also linked to a thicker nucleated epithelium and increased keratinocyte proliferation, with no change in stratum corneum thickness. Therefore, BASIC species may enhance penile HIV susceptibility by directly disrupting epithelial integrity, in addition to previously described target cell recruitment.

Fig 1. Representative immunofluorescence images of staining and quantification of epithelial junction proteins.

Inner and outer foreskin tissue, showing a representative full tissue section (A), and staining (magenta) for junction proteins E-cadherin (B, C), claudin-1 (D, E), and desmoglein-1 (F, G). The stratum corneum was visualized by staining for filaggrin (yellow) and cell nuclei are shown in cyan (DAPI). A neuriteness filter was applied to create the net structure (H), and a threshold was applied to generate a binary net (I) for quantification. Image scale bars are 1000 µm (A) and 100 µm (B–G). Brightness and contrast have been enhanced from the original images for visualization purposes.

Fig 2. BASIC species are associated with altered expression of epithelial junction proteins in the inner foreskin.

Inner and outer foreskin tissue samples were divided into 3 groups; No BASIC (n = 25), High Control (n = 21), and High BASIC (n = 21). Total bacterial density (A, 16S rRNA qPCR) and BASIC species density (B, 16S rRNA sequencing and qPCR) was determined from penile swabs. Relative expression of epithelial junction proteins E-cadherin (C, F), claudin-1 (D, G), and Desmoglein-1 (E, H) in foreskin tissues was determined by quantitative immunofluorescent microscopy on tissues from both the inner (C, D, E) and outer (F, G, H) aspects of the foreskin. Statistical comparisons were made using the Wilcoxon rank-sum test, lines shown for median in each group.

Fig 3. Prevotella bivia can cleave E-cadherin.

Soluble E-cadherin from all participants was quantified in penile swabs and negatively correlated with tissue E-cadherin (immunofluorescence) (A, n = 116, Spearman’s correlation). Soluble E-cadherin was compared between No BASIC (n = 25), High BASIC (n = 21), and High Control (n = 21) groups (B). In vitro, BASIC species were grown in monocultures or together then added as live bacteria (C) or concentrated conditioned media (D) to primary human foreskin epithelial cells; soluble E-cadherin release was quantified by ELISA. Data represent means of three independent experiments for (C) and individual technical replicates for (D). Live bacteria (E) or concentrated conditioned media (F) were also added directly to recombinant human E-cadherin to assess cleavage. L = ladder, E = E-cadherin alone, CT = Corynebacterium tuberculostearicum, PB = Prevotella bivia, PA = Peptostreptococcus anaerobius, DM = Dialister Micraerophilus. Lines represent means in all figures. Student’s t-test (B), or ANOVA with Tukey’s test was used to compare groups (C, D), both α = 0.05.

Fig 4. BASIC species are associated with thicker nucleated cell layers, but not stratum corneum thickness.

The edges of the stratum corneum (A) and nucleated epithelium (B) were manually traced from immunofluorescence images and the mean distance between traced lines was quantified for the stratum corneum (C–D) and nucleated epithelial layers (E–F). Mean thicknesses were compared between men in the No BASIC (n = 25) group and men in the High BASIC (n = 21) and High Control (n = 21) groups (Student’s t-test, lines represent means). Representative inner foreskin images from High BASIC and No BASIC groups shown in G and H, respectively (scale bars 100 µm). Representative images were chosen from samples near the median thickness in each group. Brightness and contrast have been enhanced from the original images for visualization purposes.

Fig 5. BASIC species are associated with increased keratinocyte proliferation.

Ki-67 expression was quantified by immunofluorescence in a subset of participants with High BASIC (n = 10) or No BASIC (n = 10) species (representative images in A and B). Mean percentage of Ki-67 positive cells was compared between groups (C, Student’s t-test, lines represent means). Percent Ki-67 + cells correlated with thickness of the nucleated cell layers of the epithelium (D, Spearman’s correlation). Brightness and contrast have been enhanced from the original images for visualization purposes.