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The phylum Chlamydiae contains the human pathogens Chlamydia trachomatis, C. psittaci, and C. pneumoniae. Chlamydiae are obligate intracellular, gram negative bacteria that undergo a biphasic developmental cycle with the bacteria in either an infectious and metabolically constrained state, termed the elementary body (EB), or a metabolically active and replicative state that is non-invasive, and termed a reticulate body (RB)1,2. The intracellular development of chlamydiae occurs within a parasitophorous vacuole that is referred to as an inclusion3. C. trachomatis contains multiple serological variants that demonstrate specific organotropism and associated disease. Serovars A–C are associated with endemic, blinding trachoma, serovars D–K are responsible for the common sexually transmitted urogenital infections, and serovars L1–L3 are the causative agents of lymphogranuloma venereum, a more invasive disease4,5,6.

C. trachomatis utilizes a type III secretion system to deliver effectors across the inclusion membrane and into the cytosol of the host cell7. A subset of these T3SS effector proteins, termed Incs, localize to the inclusion membrane via a bi-lobed transmembrane domain8,9. Incs are oriented in the inclusion membrane in a manner that exposes them to the cytosol of the host cell10,11, enabling the Incs to interact with cytosol-exposed proteins12. While Incs are typically distributed relatively evenly around the inclusion membrane, a subset of Incs are localized to discrete, punctate sites on the membrane, termed microdomains, that are also enriched in cholesterol and active host Src-family kinases13. Currently, the C. trachomatis effectors CT101 (MrcA), CT147, CT222, CT223 (IPAM), CT224, CT228, CT232 (IncB), CT233 (IncC), CT288, and CT850 have been identified as Incs enriched in inclusion microdomains9,13,14,15. Early findings demonstrated microdomains function as hubs for interactions with the cytoskeleton and promote the positioning of the inclusion at the microtubule organizing center13,16. Microdomains also influence extrusion-based dissemination, a process in which all or part of an intact chlamydial inclusion is exocytosed from the host cell for dissemination to distal anatomic sites. The phosphorylation of myosin light chain 2 (MLC2) at the microdomain is a critical regulator of this dissemination process14, which occurs via ionized calcium (Ca2+)-dependent pathways17.

Cytosolic Ca2+ acts as a second messenger for a number of pathways in eukaryotes influencing fertilization, embryonic axis formation, cell differentiation, cell proliferation, transcription factor activation, and cell fate decisions18,19. Cytosolic Ca2+ is tightly regulated to ensure these pathways are activated only when required. This homeostatic regulation of cytosolic Ca2+ concentration is achieved through Ca2+ pumps, exchangers, channels, buffering proteins, and sensors to control Ca2+ movement across the plasma membrane and the membranes of intracellular Ca2+ stores20. In excitable and non-excitable cells, store-operated Ca2+ entry (SOCE) is a major regulator of Ca2+ homeostasis21. The SOCE process is initiated by the depletion of endoplasmic reticulum (ER) Ca2+. The reduction in ER luminal Ca2+ is sensed by stromal interaction molecule 1 (STIM1) and subsequently initiates a STIM1 conformational change enabling STIM1 to interact with Orai1 to generate Ca2+ release-activated Ca2+ (CRAC) influx channels at the plasma membrane. The STIM1-CRAC channel interaction opens the channel resulting in an ingress of extracellular Ca2+ into the cytosol22. This influx of extracellular Ca2+ into the cytosol refills Ca2+ stores via the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA)23 and is a signal for select Ca2+-dependent pathways.

The C. trachomatis inclusion interacts with the host ER, a major intracellular Ca2+ store, through multiple interactions including the inclusion membrane protein IncD interaction with ceramide transfer protein (CERT)24, the direct interaction of IncV with VAP25, the interaction of STIM1 with the inclusion membrane by an unknown mechanism17,26, and the interaction of MrcA with the inositol triphosphate receptor (IP3R)17. STIM1 and IP3R localization to microdomains of the inclusion membrane17,26 demonstrate that regulators of Ca2+ homeostasis are recruited during chlamydial development. Therefore, we investigated if chlamydial development disrupts host cell Ca2+ homeostasis. Here we provide evidence that SOCE is impaired by the midpoint of the chlamydial developmental cycle and the SOCE-inducible NFAT/calcineurin signaling pathway is concurrently abrogated, which could have major implications on host cell physiology.

Results

Store-operated Ca2+ entry is impaired by mid-cycle of the C. trachomatis developmental cycle

To investigate the impact of chlamydial infections upon intracellular Ca2+ mobilization, the ratiometric Ca2+ indicator, Fura-2, AM was used to assess changes in host cell intracellular Ca2+ concentration ([Ca2+]i). HeLa cells either uninfected or infected with C. trachomatis L2 were loaded with Fura-2, AM at the desired time post infection. The binding of Ca2+ to Fura-2, AM induces a shift in its fluorescence excitation from 380 to 340 nm. Therefore, a 340 nm/380 nm fluorescence ratio of Fura-2, AM was used to determine relative changes in Ca2+ concentration. A Ca2+ re-addition assay27 was used to obtain ratiometric measurements in a resting state, during induced ER Ca2+ leakage, and throughout SOCE. Following a resting state baseline reading in a Ca2+-free Ringer’s solution, cells were incubated with Ca2+-free Ringer’s solution containing either thapsigargin (TG) or the vehicle control, DMSO. TG is an inhibitor of the SERCA Ca2+ pump responsible for mobilizing Ca2+ from the cytosol into the lumen of the ER or sarcoplasmic reticulum. TG thus causes an increase in cytosolic Ca2+ by impairing ER Ca2+ uptake while ER Ca2+ depletion occurs via passive ER Ca2+ leakage through ER translocon complexes28,29. When TG treated and DMSO control HeLa cells were moved to a Ca2+-containing Ringer’s solution, a distinctive increase in [Ca2+]i was detected in TG-treated, but not DMSO treated, cells indicating that Ca2+ depletion of the ER resulted in the activation of SOCE (Fig. 1a). A STIM1 siRNA knockdown was performed to verify this methodology (Extended Data Fig. 1).

Figure 1
figure 1

C. trachomatis impairs host cell store-operated calcium entry (SOCE) by a mid-cycle developmental time point. (a) Ca2+ re-addition assays were performed with Fura-2, AM to assess [Ca2+]i changes in HeLa cells infected with C. trachomatis L2 or uninfected. Thapsigargin (TG) was used to induce ER Ca2+ depletion and subsequent SOCE. DMSO was used as a vehicle control. A ratiometric assessment of Fura-2, AM fluorescence at 340 nm and 380 nm was taken to calculate the relative change in [Ca2+]i. (b) The peak Ca2+ efflux from the ER induced by TG for each time point was measured. The TG treatment was compared to DMSO for either uninfected or infected cells. Student’s T-test was used to compare the DMSO to the TG treatment, n = 3. (c) Peak SOCE for each time point was calculated. Student’s T-test was used to compare the uninfected TG treated condition to the infected TG treated conditions, n = 3. The SOCE peak for TG-treated samples was not significantly different between infected and uninfected cells at 1.5 hpi (p = 0.0929) or 8 hpi (p = 0.3491), however, it was significantly reduced in infected cells at the mid-cycle time point (24 hpi) (p = 0.002) and the 46 hpi time point (p = 0.0012). Data are presented as mean ± SEM.

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To interrogate changes in Ca2+ mobilization during C. trachomatis development, the Fura-2, AM Ca2+ re-addition assay was performed with HeLa cells infected with C. trachomatis L2 at early- (1.5 hpi), early-to-mid- (8 hpi), mid- (24 hpi), and late- (46 hpi) cycle developmental time points (Fig. 1a). TG induced a significant increase in cytosolic Ca2+ at all time points for uninfected and infected cells, except for 46 hpi, indicating TG triggered ER Ca2+ egress in infected and uninfected cells (Fig. 1b). While the SOCE peak was not significantly different between infected and uninfected cells at 1.5 hpi or 8 hpi, it was significantly depressed in infected cells at the mid-cycle time point (24 hpi) and the 46 hpi time point, indicating C. trachomatis impairs SOCE of the host cell by a mid-cycle developmental timepoint (Fig. 1c). At 46 hpi, the baseline level of F340/F380 was elevated in the infected vs the uninfected. The likely interpretation of this would be that membranes were compromised due to lysis resulting in elevated [Ca2+]i at this timepoint which is near the end of the C. trachomatis developmental cycle, and may explain why TG-induced ER Ca2+ egress and SOCE at this time point were impaired.

Intracellular pathogens depend upon viability of the host cell to complete their intracellular development2. We reconfirmed the viability of C. trachomatis infected cells at each of the time points analyzed. There was no significant difference in viability of uninfected versus infected cells at 8 h or 24 h post-infection. Viability was 89.3% ± 1.6% in uninfected versus 91.5% ± 2.4% (Mean ± SD; n = 6) in infected cells at 8 h post-infection and 90.8% ± 2.7% in uninfected versus 93.7% ± 2.0% in infected cells (Mean ± SD; n = 6) at 24 h post-infection. Viability dropped slightly in infected cells at 46 h post-infection from 88.0% ± 3.3% (Mean ± SD; n = 6) in uninfected to 80.7% ± 1.9% (Mean ± SD; n = 6) in infected cells (p < 0.013).

Verification of C. trachomatis-suppressed SOCE using single-cell analysis

The Fura-2, AM-based method measured changes in cytoplasmic Ca2+ at the cell population level. To assess the influence of C. trachomatis serovar L2 on host cell Ca2+ mobilization at a single cell level, we used the Ca2+ indicator Fluo-4, AM with live-cell microscopy to determine changes in [Ca2+]i in infected and uninfected cells. To quantify the normalized relative change in Fluo-4, AM fluorescence intensity (F) for each cell, ΔF/F0 was calculated. The baseline resting state fluorescence (F0) was the average of the first four mean intensity measurements in Ca2+-free Ringer’s solution, and ΔF = F − F0 was used to calculate the change in fluorescence.

Analysis of [Ca2+]i mobilization in uninfected and C. trachomatis-infected cells was performed at a mid-cycle (24 h) developmental timepoint. In uninfected and infected HeLa cells treated with the DMSO carrier, stochastic Ca2+ elevations were observed in a small subset of cells, and when DMSO-treated control and infected cells were placed in Ca2+-Ringer’s solution, there was a modest increase in [Ca2+]i (Fig. 2a). When cells were treated with TG, a dramatic increase in ΔF/F0 was observed for both uninfected and infected cells, indicating the TG treatment mobilized Ca2+ from the ER into the cytoplasm. The transition of TG-treated cells to Ca2+-containing Ringer’s solution resulted in a striking increase in ΔF/F0 associated with SOCE in uninfected but not infected cells (Fig. 2a). The mean of the single-cell measurements provides a visualization of the single cell Ca2+ flux trends for each condition (Fig. 2b). TG-treated, C. trachomatis-infected cells demonstrated a significant increase in relative fluorescence of the Fluo-4, AM indicator compared to uninfected TG-treated cells (Fig. 2c). At 44 hpi, there was no significant difference in the TG peak between TG-treated uninfected and infected cells. (Extended Data Fig. 2a–c). These results indicate that TG induces Ca2+ egress from the ER of uninfected and infected cells.

Figure 2
figure 2

Fluo-4 assessment of C. trachomatis suppression of host cell SOCE. Fluo-4, AM, was used with live-cell microscopy to assess cytosolic Ca2+ mobilization in uninfected and C. trachomatis L2-infected cells. (a) Single-cell analysis of the relative change in Fluo-4 fluorescence during Ca2+ re-addition assay was performed for uninfected and C. trachomatis-infected cells at a mid-cycle developmental timepoint. For each condition, ≥ 39 cells were analyzed. (b) The mean relative change in Fluo-4 fluorescence was calculated for each condition (a). (c) The relative change in Fluo-4 fluorescence was assessed at the peak TG-induced ER Ca2+ egress. (d) The relative change in Fluo-4 fluorescence was calculated for peak SOCE. A Kruskal–Wallis test was performed with Dunn’s post-hoc multiple comparisons test to compare the conditions (c, d). Comparisons denoted with **** have a p value < 0.0001 and ns represents no significant difference. Data (b–d) are presented as mean ± SEM.

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The SOCE of uninfected and infected cells was also assessed using the Fluo-4, AM indicator. The mean ΔF/F0 at the SOCE peak for HeLa cells infected with C. trachomatis serovar L2 was severely reduced compared to uninfected cells. Furthermore, 53% of the uninfected, TG-treated cells had a ΔF/F0 greater than 0.5 compared with only 3% infected cells (Fig. 2d). Additionally, uninfected and infected DMSO treated cells had 5% and 2% of cells, respectively, with a ΔF/F0 of greater than 0.5 (Fig. 2d). At 44 hpi, infected cells had a significantly reduced ΔF/F0 mean at the SOCE peak compared to uninfected, and only 3% of the cells had a SOCE peak greater than 0.5 in the infected and TG-induced condition compared to 37% in the uninfected and TG-induced condition (Extended Data Fig. 2d). Collectively, the Fluo-4, AM single-cell analysis demonstrated that SOCE of the host cell is impaired by mid-cycle and remains suppressed at later developmental time points.

Genetically encoded Ca2+ indicator confirms SOCE inhibition in C. trachomatis infected cells

The genetically encoded Ca2+ indicator (GECI), GCaMP6m, was used to corroborate the results of the Fura-2, AM and Fluo-4, AM based quantification of [Ca2+]i mobilization in uninfected and C. trachomatis-infected cells. The GCaMP family of GECIs are single fluorescent protein indicators that operate by utilizing a circularly permuted GFP linked to calmodulin and the Ca2+-dependent calmodulin-interacting peptide M13 from myosin light chain kinase (MLCK). Ca2+ binding induces a conformational change in the circularly permuted GFP causing a large increase in fluorescence intensity30. Utilizing GCaMP6m, a relative change in GFP fluorescence can be used to determine changes in [Ca2+]i. C. trachomatis L2 expressing mScarlet permitted visualization of chlamydial inclusions during imaging. HeLa cells infected with mScarlet C. trachomatis L2 and transfected with pN1-GCaMP6m-XC were tested to assess Ca2+ mobilization.

The single-cell and mean analysis of the relative fluorescence change of GCaMP6m demonstrated similar trends as the Fluo-4 analysis with SOCE of infected cells impaired relative to uninfected cells (Fig. 3a, b). The single-cell analysis at the TG peak indicated that both uninfected and infected cells had elevated fluorescence upon TG addition compared to the DMSO vehicle control (Fig. 3c). The single-cell analysis of the SOCE peak indicated that uninfected cells treated with TG had an increased relative change in GCaMP6m fluorescence compared to the DMSO control, however, there was no significant difference in the SOCE peak between the 24 hpi cells treated with DMSO and TG (Fig. 3d). The GCaMP6m analysis of HeLa cells at a mid-to-late developmental cycle timepoint, 36 hpi, with C. trachomatis serovar L2 demonstrated that SOCE was suppressed in infected cells (Extended Data Fig. 3). The Fura-2, Fluo-4, and GCaMP6m Ca2+ indicators collectively demonstrated that SOCE is impaired in HeLa cells infected with C. trachomatis L2 by mid-cycle developmental time point.

Figure 3
figure 3

Genetically encoded Ca2+ indicator verification of impaired SOCE of host cell. Live-cell microscopy with the genetically encoded Ca2+ indicator, GCaMP6m, was used to measure changes in [Ca2+]i. (a) Relative change in GCaMP6m fluorescence was calculated throughout Ca2+ re-addition assay for individual HeLa cells at a mid-cycle (24 h) developmental timepoint. For each condition, ≥ 67 cells were analyzed. (b) The mean relative change of GCaMP6m fluorescence was measured from the single-cell analysis (a). (c) The relative change in GCaMP6m fluorescence at peak TG-induced ER Ca2+ efflux was calculated. (d) The relative change in GCaMP6m fluorescence was measured at peak ΔF/F0 during SOCE. Because GCaMP6m measurements were a non-Gaussian data set, a Kruskal–Wallis test was performed with Dunn’s post-hoc multiple comparisons test to compare the conditions (c, d). Comparisons denoted with **** have a p value < 0.0001 and * have a p value < 0.05. Data (b–d) are presented as mean ± SEM.

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To determine if a urogenital C. trachomatis serovar also impairs SOCE of the host cell, the Ca2+ re-addition assay was performed using GCaMP6m in HeLa cells infected with C. trachomatis serovar D at a chlamydia developmental midpoint (24 h). Similar to the C. trachomatis L2 results, C. trachomatis D impaired SOCE of the host cell by the mid-cycle developmental timepoint (Extended Data Fig. 4).

C. trachomatis prevents SOCE-induced NFAT nuclear localization

Extended increases in [Ca2+]i caused by SOCE result in the activation of various Ca2+-dependent pathways. To gain insights into physiological consequences of impaired SOCE in C. trachomatis infected cells, a specific SOCE-dependent pathway was investigated. Sustained elevated [Ca2+]i activates the calcineurin-NFAT signaling pathway via the Ca2+-mediated binding of calmodulin to calcineurin, the calcineurin-dependent dephosphorylation of the NFAT transcription factor to expose its nuclear localization signal, and the subsequent cytosol-to-nucleus translocation of NFAT. We investigated the SOCE-induced nuclear localization of NFAT1 in C. trachomatis-infected cells at the mid-cycle time point to determine if a SOCE-dependent pathway is abrogated during C. trachomatis infection. HeLa cells infected with mScarlet-expressing C. trachomatis L2 were transfected with the HA-NFAT1(4-460)-GFP plasmid. A pilot study indicated that the optimal time following TG treatment and Ca2+ incubation for NFAT-GFP nuclear translocation in HeLa cells was 18 min post-Ca2+ addition (Supplementary Video 1). Therefore, imaging for NFAT-GFP nuclear translocation was performed immediately following TG or DMSO treatment, and again at 18 min post-Ca2+ incubation. Imaging of NFAT-GFP expressing HeLa cells treated with DMSO demonstrated no noticeable change in nuclear NFAT-GFP fluorescence following the 18 min Ca2+ incubation, while the TG treatment caused a dramatic increase in nuclear NFAT-GFP following the Ca2+ incubation (Fig. 4a). However, neither the DMSO nor TG treatment caused a noticeable increase of nuclear NFAT-GFP in C. trachomatis-infected cells at 24 hpi (Fig. 4a). A representative time course video of NFAT-GFP-expressing HeLa cells infected with mScarlet C. trachomatis L2 did not demonstrate NFAT-GFP nuclear localization in the presence of TG, however, NFAT-GFP-expressing cells in the same field of view without a chlamydial inclusion showed nuclear translocation of NFAT-GFP (Supplementary Video 2).

Figure 4
figure 4

NFAT nuclear localization is diminished in C. trachomatis-infected cells. Nuclear translocation of NFAT-GFP was assessed in C. trachomatis L2 infected and uninfected HeLa cells. (a) Cells expressing NFAT-GFP were induced with thapsigargin (TG) to trigger SOCE or treated with the DMSO vehicle control for 5 min. Cells were imaged at 0 min post-addition of TG or DMSO, and then imaged at 18 min after administering Ca2+-containing Ringer’s solution. Asterisks denote the nuclei of C. trachomatis-infected cells. (b) Single-cell analysis of the NFAT-GFP nuclear-to- cytoplasmic mean fluorescence ratio (N/C) was calculated for each cell immediately following TG or DMSO treatment and following the 18 min Ca2+ incubation. For each condition, ≥ 134 cells were analyzed. A Kruskal–Wallis test was performed with Dunn’s post-hoc multiple comparisons test. Comparisons denoted with **** have a p value < 0.0001 and ns represents no significant difference. The data are presented as mean ± SEM. (c) The percent change in NFAT-GFP N/C ratio was calculated from the N/C ratios before and after induction (b) for each individual cell.

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Image analysis was performed to calculate the NFAT-GFP nuclear-to-cytoplasmic fluorescence ratio (N/C) for each condition. The NFAT-GFP N/C was measured as the mean fluorescence intensity of nuclear NFAT-GFP / the mean fluorescence intensity of cytoplasmic NFAT-GFP for individual cells. Cells with an observable inclusion containing mScarlet C. trachomatis were calculated for infected cells. The only significant difference identified between the pre- and post-incubation with Ca2+-containing Ringer’s solution was for the uninfected + TG condition, which demonstrated a substantial increase in the NFAT-GFP N/C ratio following the 18 min Ca2+ incubation (p value < 0.0001). No significant difference in NFAT-GFP N/C was observed for C. trachomatis-infected cells treated with TG post-Ca2+ incubation. (Fig. 4b). To visualize how the NFAT-GFP N/C ratio changed from post-TG or -DMSO treatment to post-Ca2+ incubation for each cell, a percent change in NFAT-GFP N/C ratio was calculated per cell (Fig. 4c). Collectively, the NFAT-GFP N/C ratio assessment demonstrated a severe reduction in NFAT-GFP nuclear localization in C. trachomatis L2 infected HeLa cells when induced to undergo SOCE.

Discussion

We examined Ca2+ dynamics in C. trachomatis-infected cells and demonstrate that C. trachomatis inhibits SOCE of the host cell by a mid-cycle (24 h) developmental time point. This inhibition of SOCE was confirmed using three independent methods of intracellular Ca2+ quantitation. High concentrations of intracellular Ca2+ resulting from SOCE induction can act as a second messenger to activate numerous signaling pathways18,31. Among the pathways activated is the calcineurin/NFAT pathway. Sustained high concentrations of intracellular Ca2+ activates the phosphatase calcineurin which, in turn, dephosphorylates the cytoplasmic components of the NFAT transcription complex to trigger NFAT translocation into the nucleus32. NFAT transcription complexes regulate genes encoding immunomodulatory proteins or involved in developmental cellular differentiation19,33,34. C. trachomatis inhibition of SOCE had the downstream effect of inhibiting the calcineurin/NFAT pathway, thus providing a biological confirmation of the intracellular Ca2+ quantitation. The impairment of NFAT1 nuclear translocation demonstrate a specific signaling pathway that is affected by suppressing SOCE in C. trachomatis infected cells and likely has a multifaceted impact on host cell physiology and chlamydial pathogenesis.

At the end of their developmental cycle, chlamydiae are released by one of two distinct mechanisms for infection of adjacent cells and subsequent cycles of infection. Infectious EBs are released either by lysis of the infected cell, or intact or partially intact membrane bound inclusions are released by a process known as extrusion35. The cellular requirements for the two different release mechanisms are unique. Myosin II and Ca2+ are essential for chlamydial extrusion-based dissemination14,17,35. Ca2+ appears to be an important determinant for chlamydial release mechanism from the host cell, however, the suppression of SOCE indicates that other sources of Ca2+ must be utilized. The lack of synchrony and the fact that all infected cells do not undergo extrusion may present challenges in characterizing the formation of Ca2+ microenvironments near the inclusion microdomain.

Although the detailed mechanisms of chlamydial inhibition of SOCE remain unknown, at least two host components involved in calcium homeostasis are inappropriately localized in C. trachomatis infected cells. STIM1 and IP3R are recruited to and enriched in microdomains on the inclusion membrane17,26. IP3R is recruited to the inclusion membrane through interactions with the chlamydial protein, MrcA17. ATP-induced IP3R activation in C. trachomatis infected cells36,37 suggests MrcA does not have an inhibitory effect on IP3R. The mechanism of STIM1 recruitment is unknown though STIM1 is known to form complexes with IP3R38. Although STIM1 is recruited to the chlamydial inclusion, Orai1 was not and remained localized to the host plasma membrane26. A model for possible interactions and events associated with inhibition of SOCE and NFAT translocation is depicted in Fig. 5.

Figure 5
figure 5

Working model for C. trachomatis impaired SOCE. Typical NFAT mobilization induced by SOCE in uninfected cells (left side) compared to a working model for C. trachomatis impaired SOCE (right side) and subsequent inhibition of calcineurin/NFAT signaling of the host cell. See text for details. The figure was created by the NAID Visual and Medical Arts Unit of the Research Technologies Branch as part of their official duties using Adobe Illustrator v26.0 https://www.adobe.com/products/illustrator.html.

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TG treatment induces puncta formation and co-localization of Orai1 and STIM1 in uninfected cells39. Puncta formation was previously observed in cells overexpressing fluorescent protein tagged STIM1 and Orai126. While different pools of mCherry-STIM1 localized to the inclusion or the ER, Orai1-GFP did not associate with the inclusion membrane. When these cells were induced with TG to undergo SOCE, the inclusion membrane-associated mCherry-STIM1 remained at the inclusion, but the pool of ER-distributed mCherry-STIM1 demonstrated punctate staining indicative of ER-PM junctions necessary for the formation of CRAC channels. This suggested that an unknown chlamydial factor retains STIM1 at the inclusion membrane when CRAC channel formation is induced and likely limits the amount of available STIM1 for formation of these channels. Some caution must be exercised in interpretation of these results since overexpression of STIM1 or Orai1 can dramatically increase the number of CRAC channels40. The stoichiometry of STIM1 to the C. trachomatis sequestering agent will thus be important when assessing the influence of C. trachomatis on SOCE since STIM1 binding to the inclusion membrane presumably lowers the available STIM1 for binding Orai1 and could lower the endogenous STIM1:Orai1 molecular ratio. Re-analysis of the data from Dzakah41 revealed that none of the isoforms of STIM1 or Orai1 were significantly up- or down-regulated at 20 h post chlamydia infection. As to whether or not STIM1 expressed at endogenous levels are able to form puncta in infected cells remains unclear.

The primary sites of C. trachomatis infection for genital infections in women and ocular infections are the endocervical epithelium and conjunctival epithelium, respectively4. As the first targets of infection, mucosal epithelial cells act as first responders to pathogen challenge by the secretion of cytokines and chemokines, and thus play a key role in the innate immune response42. In an immortalized primary human endocervix-derived epithelial cell line, a productive C. trachomatis infection mitigated a pro-inflammatory cytokine and chemokine response. Although the mechanism was not defined, it was proposed that the circumvention of a robust cytokine and chemokine response represented a potential evasion strategy promoting the establishment of a favorable intracellular niche within the endocervix epithelium43. SOCE impacts cytokine and chemokine signaling events31. The influence of C. trachomatis-mediated suppression of SOCE warrants further investigation.

The impairment to calcineurin activation and NFAT1 nuclear translocation demonstrates a specific signaling pathway that is affected by chlamydial suppression of SOCE. Although the major functions of NFAT are often considered in innate immune cells such as lymphocytes, macrophages, dendritic cells, or neutrophils, NFAT plays multiple additional roles in development and cellular differentiation19. NFAT is also expressed in multiple cell types19, including epithelial cells44,45,46 and endothelial cells47, however, direct information on how disruption of NFAT signaling might impact pathogen interaction with epithelial cells is limited. African swine fever virus inhibits NFAT-regulated transcription of immunomodulatory proteins by synthesis of a protein, A238L, that directly binds to and inhibits the calcineurin phosphatase activity required for NFAT activation48. Reactivation of latent Epstein-Barr virus to a lytic state via a Ca2+/calcineurin dependent activation of NFAT by a complex mechanism has been proposed to represent a negative feedback loop involving a viral protein, Zta, that directly binds to and attenuates NFAT49. H. pylori has the capacity to influence NFAT activity either positively or negatively by the bacterial proteins CagA or VacA, respectively, in gastric epithelial cells46. VacA forms an anion selective channel in the plasma membrane50, which is believed to deregulate membrane depolarization and inhibit SOCE. Although the mechanism of SOCE inhibition differs, C. trachomatis also inhibits SOCE and subsequent NFAT nuclear translocation. NFAT is a transcriptional regulator that, upon translocation to the nucleus, induces expression of several genes important in innate immune responses51. Presumably, inhibition of SOCE by chlamydiae and the resultant inhibition of NFAT translocation blocks transcription of NFAT regulated genes. We hypothesize that this inhibition of SOCE and NFAT signaling promotes chlamydial survival, possibly by downregulation of chemokine or cytokine production. Further studies in human primary cervical epithelial cells or animal model systems will undoubtedly be necessary to fully elucidate the benefits to chlamydial and host survival.

Methods

Bacterial and mammalian cell culture

HeLa 229 human cervical epithelial-like cells (American Type Culture Collection) were cultivated in RPMI-1640 (Gibco) supplemented with 5% fetal bovine serum (HyClone) at 37 °C and 5% CO2 in a humidified incubator. The C. trachomatis D (UW-3-Cx) and L2 (LGV 434) serovars were cultured in HeLa cells and EBs purified by density gradient centrifugation as previously described52. Infectious titers were determined by Inclusion Forming Unit (IFU) assays53 with inclusions detected by indirect immunofluorescence. Viability of infected and uninfected cells was determined by Trypan Blue exclusion.

Cell transfection

For siRNA transfections, HeLa cells were seeded at 1 × 104 cells/well in 96-well, black-wall, clear-bottom plates (Costar). Transfection complexes, either ON-TARGETplus Non-Targeting Control Pool siRNA (Dharmacon) or ON-TARGETplus SMARTpool STIM1 siRNA (Dharmacon) were diluted to a 1 µM working concentration in Opti-MEM. An equivalent volume of the diluted DharmaFECT 1 reagent and diluted siRNA were combined and incubated at room temperature for 30 min to form siRNA/DharmaFECT complexes. For the mock transfection, DharmaFECT 1 reagent in Opti-MEM was used. HeLa growth medium was exchanged with Opti-MEM while siRNA transfection complexes were generated. Following incubation, 0.1 mL of the diluted transfection complex was added per well. Cells were incubated for 48 h prior to cell infection.

For mammalian expression vector transfections, HeLa cells that were either infected with C. trachomatis, L2 for the stated times or uninfected were transfected with the desired mammalian expression vector using X-tremeGENE HP DNA transfection reagent (Sigma) according to the manufacturer’s instructions.

Fura-2, AM Ca2+ measurement

HeLa cells were seeded in 96-well, black-walled plates at a density of 2 × 104 cells/well. Cells were infected the following day with C. trachomatis serovar L2 at an MOI of 1. At the desired time post infection, medium was removed and cells were washed with PBS. Cells were loaded with 5 µM Fura-2, AM in Ca2+-containing Ringer’s Solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 2 mM CaCl2, 10 mM glucose, pH 7.4) for 1 h at room temperature. Cells were washed twice with PBS and incubated in Ca2+-free Ringer’s Solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 3 mM EGTA, 10 mM glucose, pH 7.4) for 30 min at room temperature. Following incubation, Fura-2, AM readings were performed by measuring fluorescence at 340/11 nm and 380/20 nm excitation and 508/20 emission every 20 s for 5 min using a Cytation 5 (BioTek). The solution was replaced with Ca2+-free Ringer’s solution containing either 2 µM thapsigargin (TG) or an equivalent volume of DMSO (vehicle control). Fluorescence readings were repeated every 20 s for 5 min. Each solution was then exchanged with Ca2+-containing Ringer’s solution, and fluorescence readings were taken every 20 s for 5 min. Each condition was performed in triplicate.

Fluo-4, AM Ca2+ measurement

HeLa cells were seeded at 2 × 104 cell/well in a 24-well, glass-bottom Cellvis imaging plate. The following day, HeLa cells were infected with C. trachomatis serovar L2 at an MOI of 1. At the desired time post-infection, cultures were washed with PBS and loaded with 2.5 µM Fluo-4, AM in Ca2+-containing Ringer’s Solution for 1 h at room temperature. Cells were washed twice with PBS and incubated in Ca2+-free Ringer’s solution (140 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4) for 30 min at room temperature. Following incubation, cells were imaged using the Nikon Ti2e with a CFI60 Super Plan Fluor Phase Contrast ADM ELWD 40 × Objective Lens (N.A. 0.6,; Nikon). ND acquisition (NIS-Elements 64-bit version 5.11.02 software (Nikon) was set to acquire images from 4 locations per well for infected and uninfected condition. ND acquisition was programmed to image every 30 s for 5 min. At the end of the Ca2+-free buffer imaging, the Ca2+-free buffer was exchanged with Ca2+-free buffer containing either 2 µM thapsigargin (TG) or DMSO carrier. Cells were imaged for another 5 min imaging interval. At the end of that interval, buffers were exchanged with a Ca2+-containing Ringer’s solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 3 mM EGTA, 10 mM glucose, pH 7.4), and images aquired for another 5 min interval. Huygens Essential software version 20.04 (Scientific Volume Imaging) was used to stabilize frames between time points and Imaris × 64 software version 9.6.0 (Oxford Instruments) was used to measure the mean fluorescence intensity of Fluo-4 per cell at each time point.

GCaMP6m Ca2+ measurement

HeLa cells were seeded at 1 × 104 cell/well in a 24-well, glass-bottom Ibidi imaging plate. The following day, HeLa cells were infected with C. trachomatis serovar L2 expressing mScarlet at an MOI of 1 and were transfected with pGP-CMV-GCaMP6m 4 h post-infection. The pGP-CMV-GCaMP6m plasmid was a gift from Douglas Kim and the GENIE project (Addgene plasmid # 40,754; RRID:Addgene_40754)54. At the desired time post infection, medium was removed from cells, and cells were washed with PBS. Cells were incubated in Ca2+-free Ringer’s solution for 15 min. Imaging, solution exchanges, image processing, and fluorescence measurements were performed as described in the Fluo-4, AM Ca2+ measurements section. Imaging was performed at 37 °C, 5% CO2, 92% relative humidity using a stage-top incubation chamber (Okolab).

NFAT1-GFP nuclear translocation assay

Infections and transfections were performed as stated in the GCaMP6m Ca2+ measurement section, except that cells that were transfected with the HA-NFAT1(4-460)-GFP plasmid. The HA-NFAT1(4-460)-GFP plasmid was a gift from Anjana Rao (Addgene plasmid # 11,107 ;; RRID:Addgene_11107)55. At the desired time post infection, cells were washed twice with PBS, and then incubated in Ca2+ -free Ringer’s solution containing 1 ug/mL Hoechst stain for 30 min. Buffer was then exchanged with either DMSO or 2 µM TG Ca2+-free Ringer’s solution. Immediately following treatment, an image was acquired using the Nikon Ti2e with a CFI60 Super Plan Fluor Phase Contrast ADM ELWD 40 × Objective Lens. ND acquisition was programmed to acquire images from 4 locations per well for each condition. After incubating cells with either DMSO or TG for 5 min, the solution was exchanged with the Ca2+-containing Ringer’s solution, and then imaged at 18 min post Ca2+ addition. Imaging was performed at 37 °C, 5% CO2, 92% relative humidity using a stage-top incubation chamber (Okolab). Following imaging, Huygens Essential software version 20.04 (Scientific Volume Imaging) was used to stabilize frames between time points and Imaris × 64 software version 9.6.0 (Oxford Instruments) was used to measure the mean fluorescence intensity of NFAT-GFP in the nucleus and the cytoplasm of individual cells before and after SOCE induction. The NFAT-GFP N/C ratio was calculated as the mean nuclear NFAT-GFP fluorescence intensity ∕ the mean cytoplasmic NFAT-GFP fluorescence intensity.

Transcriptional analysis of ORAI1 and STIM1

The RNAseq dataset from Dzakah et al. (PMID 33,397,284) was analyzed. The runs for uninfected or 20 hpi C. trachomatis serovar E infected samples were accessed through the Sequence Read Archive (BioProject PRJNA666640). Paired reads were mapped to STIM1 (Gene ID 6786), ORAI1 (Gene ID 84,876), and nine housekeeping genes56 using the Geneious read mapper (Geneious Prime version 2022.1.1). Expression levels were calculated for each gene simultaneously with ambiguously mapped reads counting as partial matches and transcripts were compared across all samples normalizing to the median gene expression ratios using the DESeq2 method. Significant differences in expression were determined as greater than 1 or less than − 1 log2 fold change with a p-value of < 0.05.

Statistics

Statistical analysis was conducted using Prism version 9.1.1 software for Windows (GraphPad). An unpaired Student’s T test was performed for Fura-2, AM analysis, and Kruskal–Wallis test with Dunn’s posttest was performed for Fluo-4, AM relative change, GCaMP6m relative change, and NFAT-GFP N/C ratios. The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.