Temporal profile of C. psittaci CCS formation correlates with a reduction in CERT recruitment

The recruitment of CERT to C. psittaci inclusions was exclusively studied for early and middle infections, and not for late infections¹³. Therefore, this study concentrated on the localization of CERT during late C. psittaci infections in the context of bacterial egress by CCS formation. At first, we analyzed CERT recruitment to C. psittaci inclusions by performing immunofluorescence staining using antibodies specific for CERT, and bacterial Hsp60. Hsp60-positive C. psittaci-inclusions strongly recruit CERT at 24 h post infection (h pi). However, this recruitment was reduced to weak or no recruitment at 48 h pi (Fig. 1A, right and left inclusion, respectively). Similar to this, ectopically expressed eGFP-CERT localized in close proximity to C. psittaci inclusions at 24 h pi, but not at 48 h pi (Fig. 1B).

In late infections, CERT recruitment to C. psittaci inclusions is reduced and CERT-KO induce early egress of C. psittaci by CCS formation at 24 h pi. (A) Representative immunofluorescence images of C. psittaci-infected HeLa cells (MOI 2) at 24 and 48 h pi. PFA-fixed cells were stained for C. psittaci and endogenous CERT using a mouse-anti-Hsp60 (Cy3) and a rabbit-anti-CERT (AF488) antibody, respectively. DNA was counterstained using DAPI. n = 3. (B) Representative fluorescence images of HeLa cells transiently expressing eGFP-CERT infected with C. psittaci (MOI 2) at 24 and 48 h pi. Cells were PFA-fixed and immunostained for C. psittaci using a mouse-anti-Hsp60 (Cy3) antibody and DNA was counterstained using DAPI. n = 3. (C) Quantification of CERT recruitment to C. psittaci inclusions at 24 and 48 h pi. Based on immunofluorescence images of C. psittaci-infected HeLa cells (MOI 2) at 24 and 48 h pi, the fluorescence intensity (in artificially units) of CERT at the inclusions was quantified. At least 59 inclusions per condition were analyzed. Data show mean ± SEM; n = 3; ***p < 0.001 (Student’s t‐test). (D) CCS formation in HeLa CERT-KO and KO control cells. Cells were infected with C. psittaci (MOI 2), medium was replaced at 20 h pi and CCS in the supernatant were visually quantified at 24 h pi. Data show mean ± SEM; n = 3; **p < 0.01 (Student’s t‐test).

Subsequently, the recruitment of CERT to C. psittaci inclusions was quantified at 24 and 48 h pi. The mean fluorescence intensity of CERT antibody labelling at C. psittaci inclusions was significantly reduced at 48 h pi in comparison to 24 h pi, from 30.7 to 14.6 relative units (Fig. 1C).

We therefore sought to determine whether the loss of CERT at the inclusion membrane is associated with egress by CCS formation. To this end, HeLa CERT-KO cells were infected with C. psittaci. We hypothesized that the absence of CERT at the inclusion membrane during intracellular development could lead to premature formation of CCS. We therefore quantified CCS in the supernatant of C. psittaci-infected cells at 24 h pi and compared CERT-KO with KO control cells. An almost tenfold increase in formed CCS was observed in the absence of CERT, from 2.0 × 10⁴ CCS/cm² in KO control cells to 19.6 × 10⁴ CCS/cm² in CERT-KO cells at 24 h pi (Fig. 1D). Interestingly, 84% of the inclusions detected in CERT-KO cells at 16 h pi formed CCS between 16 and 24 h pi (Supplemental Figure S1 A). In line with this observation, the number of CCS formed between 68 and 72 h pi is reduced under CERT-KO conditions compared to control cells (Supplemental Figure S1 B).

In conclusion, the data demonstrate that the absence of CERT at the inclusion membrane of C. psittaci correlates temporally with egress by CCS formation, indicating that alterations in CERT recruitment to C. psittaci inclusions regulate CCS formation.

Localization of CERT regulates CCS formation

CERT is composed of three functional domains: The N-terminal PH domain, mediating Golgi targeting in uninfected cells, the central FFAT motif, mediating ER targeting, and the C-terminal domain, which is a ceramide-specific lipid-binding START domain (Fig. 2A). During C. trachomatis, C. muridarum, and C. psittaci infections CERT is recruited to the inclusion by its PH-domain^13,20,30.

In contrast to full CERT and CERT variants lacking the START- or FFAT-domain, a CERT variant lacking the PH domain is not recruited to C. psittaci inclusions and cannot prevent premature CCS formation. (A) CERT is composed of the N-terminal PH domain, mediating Golgi targeting in uninfected cells, the central FFAT motif, mediating ER targeting, and the C-terminal START-domain, that is a ceramide-specific lipid binding domain. (B) Representative fluorescence images of HeLa CERT-KO cells transiently expressing eGFP-CERT or eGFP-CERT variants lacking the START, FFAT or PH domain and controls infected with C. psittaci (MOI 2) at 24 h pi. Cells were PFA-fixed and immunostained for C. psittaci using a mouse-anti-Hsp60 (Cy3) antibody; DNA was counterstained using DAPI; n = 4. (C) CCS formation in HeLa CERT-KO cells transiently expressing eGFP‐CERT or eGFP-CERT variants lacking the START, FFAT or PH domain and controls. Cells were infected with C. psittaci (MOI 2), medium was replaced at 20 h pi and CCS in the supernatant were visually quantified at 24 h pi. CCS number was normalized on the transfection efficiency. Data show mean ± SEM; n = 4; *p < 0.05, **p < 0.01 (Student’s t‐test).

Initially, we infected CERT-KO cells ectopically expressing distinct eGFP-CERT variants with C. psittaci and examined the localization of the eGFP-CERT variants through immunofluorescence staining at 24 h pi. In cells that had not been transfected with the CERT-GFP construct, no GFP signal was observed (Fig. 2B). In CERT-KO cells transfected with full-length eGFP-CERT, the protein was observed to localize in close proximity to C. psittaci inclusions (Fig. 2B), a finding that is comparable to those observed in wildtype HeLa cells (Fig. 1B). A comparable staining pattern was observed following transfection with eGFP-CERT lacking the START or FFAT domain (Fig. 2B). As published, eGFP-CERT lacking the PH domain was not recruited to C. psittaci inclusions¹³.

CERT-KO-induced early CCS are formed by an analogous sequence of events as mature CCS. (A) During early CCS formation, plasma membrane blebbing occurs and the inclusion membrane destabilizes. HeLa CERT-KO cells transiently expressing eGFP were infected with C. psittaci (MOI 2) and monitored live starting at 20 h pi using a CLSM. Panels show representative images of a section plane of a CCS-forming CERT-KO cell; The inclusion is marked with an asterisk; n = 3. (B) EqtSM is recruited to C. psittaci inclusions during early CCS formation. CERT-KO cells transiently expressing EqtSM-HaloTag were infected with C. psittaci (MOI 2). At 20 h pi, cells were labeled with 200 nM Janelia Fluor 585 HaloTag Ligand (Promega) and monitored live. Panel shows representative images of an EqtSM-HaloTag-recruiting C. psittaci inclusion during early CCS formation; n = 3. (C) A DEVD-cleaving protease is activated during early CCS formation. C. psittaci-infected CERT-KO and KO control cells (MOI 2) were stained with Incucyte Caspase-3/7 Dye for Apoptosis at 16 h pi and monitored using a CLSM for 8 h. Mean fluorescence intensity of infected cells at 16, 20, and 24 h pi was normalized to mean fluorescence intensity of uninfected cells at respective time points. Data show mean ± SEM; n = 3; *p < 0.05 (Student’s t-test). (D) Early CCS formation depends on the extracellular calcium concentration. CERT-KO cells were infected with C. psittaci (MOI 2). At 20 h pi, culture medium was replaced by serum-free medium supplemented with the indicated concentrations of calcium chloride. CCS in the supernatant were visually quantified at 24 h pi. Data show mean ± SEM; n = 3; *p < 0.05 (Student’s t‐test). (E, F) CERT-KO cells were infected with C. psittaci (MOI 2). At 20 h pi, medium was replaced by serum-free medium supplemented with 0 mM calcium chloride (E) or 1.8 mM calcium chloride (F). At 22 h pi, cells were labeled with the calcium sensor Rhod-3 for 1 h. At 24 h pi, DNA was counterstained using Hoechst and images were acquired. Representative images of intact cells (0.0 mM CaCl₂) and CCS formation (1.8 mM CaCl₂) are shown; n = 2.

Next, we analyzed CCS formation after infecting CERT-KO cells ectopically expressing different eGFP-CERT variants with C. psittaci. In untransfected CERT-KO cells, 36.0 × 10⁴ CCS/cm² were formed compared to 4.9 × 10⁴ CCS/cm² in untransfected KO control cells (Fig. 2C). In the conditions in which the CERT variants were introduced by transient transfection, the CCS numbers were normalized to the transfection efficiencies. Upon transfection of CERT-KO cells with full length eGFP-CERT, the normalized CCS formation (4.5 × 10⁴ CCS/cm²) resembles that detected in the control cells, indicating that full length CERT can complement the premature egress phenotype seen in the CERT-KO cells. Similarly, transfection of CERT-KO cells with eGFP-CERT lacking the START or FFAT domain significantly decreased CCS formation to 7.9 × 10⁴ and 6.1 × 10⁴ CCS/cm², respectively. In contrast, transfection of CERT-KO cells with eGFP-CERT lacking the PH domain did not decrease CCS formation (40.7 × 10⁴ CCS/cm²) (Fig. 2C).

Taken together, expression of CERT variants revealed that the ability of CERT to be recruited to the inclusion correlates with its capacity to prevent premature egress and suggest that CERT recruitment to the inclusion controls CCS formation.

CERT-KO induces early CCS formation by a sequence of events analogous to mature CCS formation

Next, we asked if the early egress seen in infected CERT-KO cells has the same mechanistic characteristics as the (mature) CCS formation that occurs in wildtype cells at the culmination of C. psittaci development. Mature CCS are formed by a process with distinct characteristics¹⁴. An increase in intracellular calcium precedes protease activity that can be detected by cleavage of a DEVD tetrapeptide-containing substrate. Loss of inclusion membrane integrity can be detected by recruitment of EqtSM caused by exposure of sphingomyelin to the cytoplasm. Plasma membrane blebbing then leads to eventual detachment of the CCS¹⁴. Thus, we examined the sequence of events described for mature CCS during the early CCS formation induced in CERT-KO cells.

At first, we monitored early CCS formation using HeLa CERT-KO cells transiently expressing eGFP starting at 20 h pi. Before CCS formation, the bacterial inclusion excluded the cytosolic eGFP (Fig. 3A). Then in all analyzed cells, CCS formation initiated with blebbing of the cellular plasma membrane and influx of eGFP into the inclusion lumen. This was followed by enlargement of the plasma membrane blebs and subsequent detachment of the entire host cell, which completed the formation of the CCS in the supernatant of the cell culture.

Premature, CERT-KO-induced CCS contain mainly non-infectious reticulate bodies. (A) CCS in the supernatant of C. psittaci-infected CERT-KO and KO control cells (MOI 2) were separated from free bacteria by centrifugation (300 x g, 5 min, RT) at 24 h pi. Infectious progeny was titrated after glass bead lysis and numbers were normalized to genome copy numbers determined by qRT-PCR. Data show mean ± SEM; n = 3. (B) Transmission electron microscopy (TEM) of a thin section through the chemically fixed supernatant of C. psittaci-infected HeLa CERT-KO cells (MOI 2, 24 h pi). Numerous Chlamydia, mostly in RB stage, were found; n = 3. The image shows a group of RBs (*) which are associated with cellular debris, which is the main component of the supernatant. (C) Representative fluorescence images of an early live CCS isolated of the supernatant of C. psittaci-infected HeLa CERT-KO cells (MOI 2, 24 h pi). The surrounding membrane was visualized using the membrane marker FM 4–64 and DNA was counterstained by Hoechst; n = 3. (D) Representative fluorescence images of a PFA-fixed early CCS isolated from C. psittaci-infected HeLa CERT-KO cells (MOI 2, 24 h pi). Bacteria inside the CCS were detected using a chlamydial Hsp60 (Cy3) antibody and the DNA was counterstained using DAPI; n > 3.

Next, we asked if EqtSM is recruited to the C. psittaci-inclusion when early CCS formation starts. To test this, we transiently expressed EqtSM-HaloTag in CERT-KO cells, labeled them with HaloTag Ligand and performed live cell imaging starting at 20 h pi. We observed many cells recruiting EqtSM to the inclusion coincident with the start of plasma membrane blebbing (Fig. 3B) and followed by CCS formation.

In addition, we tested if proteolytic cleavage of a DEVD-containing substrate is increased in adherent CERT-KO cells during early CCS formation. Indeed, we detected a significant increase in fluorescence intensity of a cleaved DEVD-containing substrate compared to uninfected cells from 1.0-fold at 16 h pi to 1.8-fold and 1.9-fold at 20 and 24 h pi, respectively (Fig. 3C). In comparison, in control cells the intensity was only slightly increased from 1.1-fold at 16 h pi to 1.2-fold and 1.3-fold at 20 and 24 h pi, respectively.

Furthermore, the role of calcium signaling for early CCS formation was investigated. During mature CCS formation, CCS numbers increased with extracellular calcium concentrations¹⁴. Similarly, during early CCS formation in CERT-KO cells, we observed a dose-dependent increase in CCS numbers from 3.4 × 10⁴ CCS/cm² to 26.1 × 10⁴ CCS/cm² when increasing the extracellular calcium from 0.0 to 1.8 mM CaCl₂ (Fig. 3D).

Subsequently, we analyzed the intracellular calcium concentration during early CCS formation using the Rhod-3 membrane-permeant cytosolic calcium sensor under conditions containing 0.0 and 1.8 mM calcium chloride. At 0.0 mM calcium chloride, we did not detect an increase in cytosolic calcium concentration in C. psittaci-infected CERT-KO cells at 24 h pi (Fig. 3E). However, at 1.8 mM calcium chloride, we observed C. psittaci-infected CERT-KO cells with blebbing membranes and an increased cytosolic calcium concentration (Fig. 3F).

In sum, this data revealed that CERT-KO induced early CCS are formed by an analogous sequence of events as mature CCS including plasma membrane blebbing, inclusion membrane destabilization, EqtSM recruitment, activation of a DEVD cleaving protease, dependency on the extracellular calcium concentration and intracellular calcium increase. This further supports that the presence and absence of CERT at the inclusion regulates CCS formation.

CERT-KO induces the formation of RB-containing, non-infectious CCS

At 24 h pi, C. psittaci inclusions contain mostly RBs, with only few bacteria starting to perform secondary differentiation to EBs³¹. Thus, we asked if bacteria in early-formed CCS are infectious. To test this, we separated CCS from free bacteria present in the supernatant of C. psittaci-infected CERT-KO and control cells at 24 h pi using differential centrifugation. We then quantified the infectious progeny (IFU) relative to the bacterial genome copy numbers associated with the harvested CCS. Although more CCS are released in the supernatants from CERT-KO cells than from control cells 24 h pi, the percentage of the progeny that are infectious are comparable (0.003 ± 0.0016% and 0.006 ± 0.0038%, respectively) (Fig. 4A). These values 24 h pi are extremely reduced compared to the percentage of infectious progeny in mature CCS released from HeLa cells at 48 h pi (3.2 ± 0.9%)¹⁴.

To assess the presence and morphology of the C. psittaci released from infected CERT-KO cells, we analyzed the culture supernatants using thin-section transmission electron microscopy (TEM). Among cellular debris, which is the main component of the supernatant, we found numerous Chlamydia, which were, with few exceptions, in RB stage (Fig. 4B). We further analyzed the morphology of early CCS by staining with the membrane dye FM 4–64 and with Hoechst. Fluorescence live cell confocal microscopy revealed that early CCS were surrounded by a membrane and contained concentrated DNA, which are both features of mature CCS (Fig. 4C)¹⁴. Bacteria could be detected in fixed early CCS with antibodies specific for bacterial Hsp60 (Fig. 4D). Bacteria were also labeled with the DNA marker DAPI, which also labeled DNA we identified as the host cell nucleus, which is also found within mature CCS¹⁴.

These data show that CCS released early in the absence of CERT rarely contain any infectious EBs although the CCS are morphologically similar to mature CCS. In wildtype infections, CERT recruitment is reduced after RB perform secondary differentiation to the EB stage, indicating that stage-controlled changes of the localization of CERT allow release of infectious progeny containing CCS.