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. 2024 Jul 11;92(7):e0006324.
doi: 10.1128/iai.00063-24. Epub 2024 Jun 20.

Recognition of Chlamydia trachomatis by Toll-like receptor 9 is altered during persistence

Aissata Diallo  1   2 , Grace Overman  1   2 , Prakash Sah  1   2 , George W Liechti  1
Affiliations

Recognition of Chlamydia trachomatis by Toll-like receptor 9 is altered during persistence

Aissata Diallo et al. Infect Immun. .

Abstract

Toll-like receptor 9 (TLR9) is an innate immune receptor that localizes to endosomes in antigen presenting cells and recognizes single stranded unmethylated CpG sites on bacterial genomic DNA (gDNA). Previous bioinformatic studies have demonstrated that the genome of the human pathogen Chlamydia trachomatis contains TLR9 stimulatory motifs, and correlative studies have implied a link between human TLR9 (hTLR9) genotype variants and susceptibility to infection. Here, we present our evaluation of the stimulatory potential of C. trachomatis gDNA and its recognition by hTLR9- and murine TLR9 (mTLR9)-expressing cells. Utilizing reporter cell lines, we demonstrate that purified gDNA from C. trachomatis can stimulate hTLR9 signaling, albeit at lower levels than gDNA prepared from other Gram-negative bacteria. Interestingly, we found that while C. trachomatis is capable of signaling through hTLR9 and mTLR9 during live infections in HEK293 reporter cell lines, signaling only occurs at later developmental time points. Chlamydia-specific induction of hTLR9 is blocked when protein synthesis is inhibited prior to the RB-to-EB conversion, exacerbated by the inhibition of lipooligosaccharide biosynthesis, and is significantly altered during the induction of aberrance/persistence. Our observations support the hypothesis that chlamydial gDNA is released during the conversion between the pathogen's replicative and infectious forms and during treatment with antibiotics targeting peptidoglycan assembly. Given that C. trachomatis inclusions do not co-localize with TLR9-containing vacuoles in the pro-monocytic cell line U937, our findings also hint that chlamydial gDNA is capable of egress from the inclusion, and traffics to TLR9-containing vacuoles via an as yet unknown pathway.

Keywords: Chlamydia; TLR9; innate immunity; pathoadaptation; persistence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Genomic DNA from C. trachomatis induces hTLR9 signaling in vitro. A human TLR9 (hTLR9) HEK 293 reporter system was used to evaluate the stimulatory potential of sonicated genomic DNA isolated from E. coli (strain MG1655), C. trachomatis (strain L2 434/Bu), and Chlamydia muridarum (strain Nigg). Data presented are the mean of three independent, biological replicates and error bars represent standard error of the mean. Groups were compared via one-way ANOVA with multiple comparisons. ****; P < 0.0001, ***; P < 0.001. All comparisons to the media control not shown were not significant.
Fig 2
Fig 2
hTLR9-containing vacuoles do not co-localize with C. trachomatis inclusions in U937 cells. (A) U937 (pro-monocytic, human myeloid leukemia derived) and (B) HeLa cells were infected via rocking incubation with C. trachomatis at a MOI of ~5. At 40 hpi, cells were fixed in 10% PFA, blocked with 3% BSA, and labeled with polyclonal antibodies to the pathogen’s major outer membrane protein (MOMP) and TLR9. Images presented are Maximum Intensity Projections from zStacks acquired from a Zeiss PS.1 ELYRA imaging system and are representative of over 20 fields of view observed over two separate labeling experiments. Scale bar ~2 µm.
Fig 3
Fig 3
Pre-exposure to stimulatory TLR9 ligands does not impact the development of C. trachomatis in U-937 cells. (A) The average number of inclusions observed per field of view (from 20 fields) in U937 cells that were left untreated or pretreated with the TLR9 agonist ODN 2006 for 18 hours prior to infection with C. trachomatis. Cell monolayers were fixed and labeled at 24 hpi. (B) Inclusion size measurements in U937 cells that were untreated or pretreated with ODN 2006 prior to infection. Data from two separate experiments were pooled for the analysis, and groups were compared via unpaired t test with Welch’s correction. *; P < 0.05. (C) Inclusion forming unit (IFU) counts from C. trachomatis-infected U937 cells that were untreated or pretreated with ODN 2006 prior to infection. Data presented are the mean of three separate experiments (biological replicates) and groups were compared via unpaired t test with Welch’s correction. ns; not significant. (D) Representative images of larger (> 5 µm) inclusions containing multiple RB-sized bacteria found in C. trachomatis-infected U937 cells. All cells were fixed and imaged 24 hpi. Scale bar, ~2 µm.
Fig 4
Fig 4
C. trachomatis-induced hTLR9 signaling occurs late in the pathogen’s developmental cycle in non-phagocytic cells. (A) SEAP activity was measured from the supernatants of hTLR9- and Null 1-HEK 293 reporter cells infected with C. trachomatis serovar L2 (strain Bu/434) at 24 and 40 hpi. (B) A comparison of the hTLR9-dependent SEAP activity present in supernatants obtained from reporter cells infected with either C. trachomatis or C. muridarum for 48 hours. (C) hTLR9 signaling assessed at 48 hpi for live and heat-killed C. trachomatis EBs. For all panels, columns represent the mean value calculated for data acquired from three separate experiments (biological replicates) and error bars represent standard error of the mean. Groups were compared via two-way and one-way ANOVA with multiple comparisons, respectively. ****; P < 0.0001, ***; P < 0.001, ns; not significant.
Fig 5
Fig 5
C. trachomatis and C. muridarum signal through mTLR9 during live infections of HEK293 reporter cells. SEAP activity was measured from the supernatants of mTLR9-HEK 293 reporter cells infected with C. trachomatis and C. muridarum at 24 (A) and 48 hpi (B). For all panels, columns represent the mean value calculated for data acquired from three biological replicates and error bars represent standard error of the mean. Groups were compared via one-way ANOVA with multiple comparisons. ****; P < 0.0001, ***; P < 0.001, **; P < 0.01, *; P < 0.05, ns; not significant.
Fig 6
Fig 6
Chlamydia-specific TLR9 signaling in HEK293 cells occurs as a result of the RB-to-EB conversion. (A) C. trachomatis-infected hTLR9-HEK293 reporter cells were treated with chloramphenicol (25 ug/ml; Cm25) at the time points indicated and SEAP activity was measured at 44hpi. (B) The effects of the LOS inhibitor LPC-011 on C. trachomatis-induced hTLR9 signaling. All columns represent mean values calculated for data acquired from three biological replicates and error bars represent standard error of the mean. Groups were compared via two-way ANOVA with multiple comparisons. ****; P < 0.0001, ns; not significant.
Fig 7
Fig 7
Persistence alters chlamydia-induced hTLR9 signaling. (A) C. trachomatis genome copies were measured in untreated cells as well as in cells infected in the presence of ampicillin, the iron chelator 2,2′-Dipyridyl (Dpd), and the LpxC inhibitor (LPC-011) over the span of 44 hours. Measurements were taken at 1, 24, and 44 hpi. (B–D) The effects of (B) ampicillin; Amp, (C) 2,2′-Dipyridyl; Dpd, and (D) tryptophan-depletion via interferon gamma (IFNγ) were assessed on hTLR9-stimulatory activity of ODN 2006 and infection by C. trachomatis. All columns represent mean values calculated for data acquired from three biological replicates and error bars represent standard error of the mean. groups were compared via two-way ANOVA with multiple comparisons. ****; P < 0.0001, ***; P < 0.001, **; P < 0.01, *; P < 0.05, ns; not significant.
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