Chlamydia Lipooligosaccharide Has Varied Direct and Indirect Roles in Evading both Innate and Adaptive Host Immune Responses

Abstract

Chlamydia bacteria are obligate intracellular pathogens which can cause a variety of disease in humans and other vertebrate animals. To successfully complete its life cycle, Chlamydia must evade both intracellular innate immune responses and adaptive cytotoxic T cell responses. Here, we report on the role of the chlamydial lipooligosaccharide (LOS) in evading the immune response. Chlamydia infection is known to block the induction of apoptosis. However, when LOS synthesis was inhibited during Chlamydia trachomatis infection, HeLa cells regained susceptibility to apoptosis induction following staurosporine treatment. Additionally, the delivery of purified LOS to the cytosol of cells increased the levels of the antiapoptotic protein survivin. An increase in survivin levels was also detected following C. trachomatis infection, which was reversed by blocking LOS synthesis. Interestingly, while intracellular delivery of lipopolysaccharide (LPS) derived from Escherichia coli was toxic to cells, LOS from C. trachomatis did not induce any appreciable cell death, suggesting that it does not activate pyroptosis. Chlamydial LOS was also a poor stimulator of maturation of bone marrow-derived dendritic cells compared to E. coli LPS. Previous work from our group indicated that LOS synthesis during infection was necessary to alter host cell antigen presentation. However, direct delivery of LOS to cells in the absence of infection did not alter antigenic peptide presentation. Taken together, these data suggest that chlamydial LOS, which is remarkably conserved across the genus Chlamydia, may act both directly and indirectly to allow the pathogen to evade the innate and adaptive immune responses of the host.

Keywords: Chlamydia; antigen processing; apoptosis; dendritic cells.

FIG 1

Chlamydial LOS synthesis is necessary to prevent host cell programmed cell death. (A) HeLa cells were treated with staurosporine to induce nonspecific programmed cell death and DNA nicking, analyzed via the TUNEL assay (top) and via Hoechst staining (bottom) to detect changes to DNA nuclear morphology (bar, 10 μm). (B) C. trachomatis-infected HeLa cells were treated with either LPC-011 or ampicillin for the duration of the infection. At approximately 24 h postinfection, staurosporine was added, and 3 h later cells were analyzed for changes in DNA nuclear morphology. (C and D) A blinded observer quantified cells with changes to DNA nuclear morphology across five randomly selected fields for each treatment. Treatment with LPC-011 of infected cells resulted in a statistically significant increase in cells with changes to nuclear morphology consistent with the induction of programmed cell death compared to untreated or ampicillin-treated cells (**, P < 0.005; ***, P < 0.001; n.s, not significant).

FIG 2

Intracellularly delivered chlamydial LOS is not toxic and does not induce programmed cell death. (A) HeLa cells were infected with C. trachomatis L2 and treated with LPC-011 for 24 h or left untreated. Cells were then fixed and analyzed for LOS by staining with antibodies against LOS (green) or the inclusion membrane protein IncA (red) and appropriate secondary antibodies (bar, 10 μm). (B) JY cells were transfected with PBS or purified chlamydial LOS from C. trachomatis L2, allowed to adhere to poly-l-lysine-coated coverslips, and cultured for 2 h prior to confocal microscopy analysis. Fixed and permeabilized cells were analyzed for LOS (green), DNA (blue), or the plasma membrane (red), and sequential z-stacks were collected (bar, 10 μm). Transfected cells were compared to mock-transfected cells. Individual planes were analyzed to visualize the interior of the cell. (C) JY cells were transfected with equivalent doses of either chlamydial LOS or LPS derived from E. coli (from two different commercial sources). Three hours later, cells were analyzed for PI uptake to measure cell death. (D) JY cells were transfected with various doses of E. coli LPS, and cell death was measured by PI uptake. (E and F) JY cells were transfected with either chlamydial LOS or one of two doses of E. coli LPS, cultured for 3 h, and analyzed by Western blotting for the loss of procaspase 3 (E) or PARP (F). IB, immunoblot.

FIG 3

Chlamydial LOS increases survivin levels. (A) JY cells were transfected with PBS or chlamydial LOS and cultured for 3 h prior to analysis by apoptosis protein array. The fold change in protein levels of LOS-transfected cells compared to PBS controls for each protein was calculated. The average Z-score from four independent experiments was determined for each protein, and changes in the scores for survivin, cSMAC/Diablo, and XIAP were noted to be statistically significantly different from zero (*, P < 0.05). (B) The increase in survivin levels following LOS transfection was confirmed by Western blot analysis (*, P < 0.05; n = 5). (C) Survivin levels were quantified by Western blotting following C. trachomatis infection with or without LPC-011 treatment. Infected untreated cells had an increased level of survivin compared to all other cells (*, P < 0.05; n = 5).

FIG 4

Chlamydial LOS is a poor stimulator of DC maturation. BMDC were derived from HLA-A2 transgenic mice on a C57BL/6 background by culturing bone marrow cells in GM-CSF for 10 days. Various doses of either E. coli LPS (black circles) or C. trachomatis L2 LOS (white circles) were added to cells, and cells were cultured overnight to induce DC maturation. Cells were stained for particular DC maturation markers, and the mean fluorescence intensity (MFI) for the population (for CD40, HLA-A2, OX40L, PD-L1, and I-A^b) or the percentage of cells positive for CD80 is indicated. Dotted lines represent background staining of immature BMDC. The results are representative of three independent experiments.

FIG 5

Intracellular LOS does not increase self-peptide antigen presentation. (A) JY/SCRAP-SVG cells were infected with C. trachomatis L2, treated with LPC-011, and cultured for 12 h prior to the addition of Shield-1. Cells were cultured for an additional 12 h and analyzed for HLA-A2-SVG peptide presentation by staining cells with the MAb RL15A and measuring the MFI using flow cytometry. (B) JY/SCRAP-SVG cells were transfected with different doses of LOS (or PBS controls) and cultured for 12 h in the presence of Shield-1 prior to staining with RL15A MAb to measure HLA-A2-SVG peptide levels (*, P < 0.05).