To verify that EBs have higher levels of c-di-AMP compared to RBs, we infected HeLa cells at a multiplicity of infection (MOI) of 1 with a transformant of C. trachomatis L2 carrying a shuttle plasmid with inducible mCherry. This strain serves as a control for subsequent overexpression experiments, and we induced mCherry expression with 5 nM aTc at 10 hr post-infection (hpi). We collected uninfected and infected HeLa cell lysates at 16 and 24 hpi and measured c-di-AMP levels by ELISA (Figure 1C). As expected, only basal amounts (<100 pg/mL) of c-di-AMP were detected in uninfected HeLa cells at either timepoint (Figure 1C). In infected cells, the 16 hpi timepoint is characterized by predominantly an RB population whereas the 24 hpi timepoint is characterized by ongoing secondary differentiation and a mixture of RBs, IBs, and EBs. We observed that c-di-AMP levels were significantly higher at 24 hpi (~1700 pg/mL) compared to 16 hpi (~250 pg/mL) and increased approximately sevenfold over this timeframe (Figure 1C). These results are in agreement with the Barker et al. study indicating higher levels of c-di-AMP in EBs (Barker et al., 2013). From these data, we infer that the concentration of c-di-AMP in our culture conditions necessary to trigger conversion of RBs to EBs is at most 1700 pg/mL.

To test the link between c-di-AMP levels and chlamydial developmental cycle progression, we made a collection of C. trachomatis strains carrying anhydrotetracycline (aTc)-inducible constructs (Figure 1D). These included strains to overexpress a wild-type or a catalytically dead (D164N) DacA isoform (Girardin et al., 2003), wild-type or mutant isoforms of DacA lacking transmembrane domains (ΔTM), YbbR_6xH, or both wild-type or mutant DacA and YbbR_6xH (dacAop or dacAopMut). For all of these overexpression strains, we emphasize that the chromosomal expression of wild-type dacA and ybbR is maintained. In addition to the overexpression constructs, we also made a conditional knockdown construct for dacA (dacA-KD), targeting its promoter region, to decrease the expression of dacA-ybbR using a dCas12/crRNA-based CRISPRi system that our group developed for Chlamydia (Ouellette et al., 2021). Finally, we made a complementation construct for dacA-KD by introducing dacA-ybbR_6xH (dacA-KDcom) 3’ to dCas12 such that induction of dCas12 results in the coexpression of DacA and YbbR_6xH during knockdown of endogenous dacA-ybbR transcripts.

To assess whether we could alter c-di-AMP levels in our various strains, we first measured c-di-AMP levels from infected cell lysates at 16 and 24 hpi after inducing overexpression or knockdown of the target genes at 10 hpi (Figure 1E). Overexpressing DacA resulted in ~ fourfold increase in c-di-AMP levels as compared to the mCherry-expressing strain at 16 hpi (~1,000 pg/mL) and ~ threefold increase at 24 hpi (~5000 pg/mL). Of note, the level of c-di-AMP in the DacA overexpressing condition at 16 hpi was below the 24 hpi level for EBs (EB threshold line in graphs: ~1700 pg/mL). Overexpressing YbbR_6xH did not impact c-di-AMP production. When overexpressing the catalytically inactive mutant of DacA(D164N), c-di-AMP levels were ~ twofold lower at 16 hpi (~115 pg/mL) and, at 24 hpi (~320 pg/mL), was similar to the 16 hpi mCherry control, indicating a severe reduction in c-di-AMP accumulation. Overexpressing ΔTMDacA phenocopied the effect of overexpressing full-length DacA on c-di-AMP production. Interestingly, the negative effects of overexpressing full-length DacA(D164N) associated with reduced c-di-AMP levels were lost when expressing ΔTMDacA(D164N), which phenocopied the mCherry-expressing control.

When DacA and YbbR_6xH were co-overexpressed, c-di-AMP levels increased by approximately 30-fold at 16 hpi (~8000 pg/mL) and 120-fold at 24 hpi (~340,000 pg/mL) compared to that of the mCherry-expressing control strain. Of note, the levels of c-di-AMP at 16 hpi in the dacAop strain (~8000 pg/mL) were higher even than the control strain at 24 hpi (~1700 pg/mL; EB threshold in Figure 1E). To confirm whether DacA enzyme activity is critical for the high levels of c-di-AMP in the dacAop strain, we co-overexpressed DacA(D164N) and YbbR_6xH (dacAopMut). Here, c-di-AMP levels were reduced compared to the control at 16 (~100 pg/mL) and 24 hpi (~400 pg/mL), suggesting that c-di-AMP synthase activity was blocked in this strain similar to overexpressing the DacA(D164N) alone.

For the dacA-KD strain, the c-di-AMP level was unchanged at 16 hpi and reduced a statistically significant ~ fivefold at 24 hpi (~500 pg/mL; roughly twice the level of the 16 hr mCherry-expressing control but below the EB threshold). The loss of c-di-AMP production in this strain was restored in the complemented dacA-KDcom strain, which showed a phenotype similar to the dacAop overexpression strain with c-di-AMP levels above the EB threshold at both timepoints assessed (~3700 pg/mL at 16 hpi;~10,000 pg/mL at 24 hpi). Based on these data, we conclude that both DacA and YbbR are necessary for optimal c-di-AMP synthesis in Chlamydia. Importantly, our collection of strains that are high or low producers of c-di-AMP give us an opportunity to test our overarching hypothesis (Figure 1B).

To begin exploring the impact of altering dacA and/or ybbR expression on chlamydial growth, we performed a series of experiments to assess their protein localization in chlamydiae. We also measured impacts of overexpressing each individually on chlamydial growth and development. To observe the localization of DacA and YbbR, we infected HeLa cells with transformants encoding dacA or ybbR_6xH alone and induced expression of the constructs at 10 hpi with 5 nM aTc. At 24 hpi, infected cells were fixed, and we performed an indirect immunofluorescence assay (IFA) by labeling the chlamydial major outer membrane protein (MOMP) and DacA or 6xH. We observed that both DacA and YbbR localized at the bacterial membrane as expected (Figure 2A and Figure 2—figure supplement 1). When DacA was overexpressed, the inclusion size was significantly reduced in area (~ sixfold) with larger individual organisms (~ twofold) than those in the uninduced control (Figure 2—figure supplement 2). Similarly, overexpressing the inactive DacA(D164N) mutant resulted in smaller inclusions similar to overexpressing wild-type DacA (Figure 2B). We did not quantify effects of YbbR_6xH overexpression on inclusion or bacterial size since the measured phenotypes indicated no differences from the uninduced control (see below; Figure 2—figure supplement 3).

To investigate the effects of DacA isoforms or YbbR_6xH overexpression on chlamydial growth, we measured EB progeny production using an inclusion-forming unit (IFU) assay and genomic DNA copy number (a proxy for total bacteria, i.e., EB+RB) by qPCR. To quantify IFUs, a lysate from a primary infection is prepared and used to infect a fresh monolayer of cells. Any inclusions in the secondary infection are derived from viable EBs present in the primary infection. The vector control strain showed no differences in IFUs when overexpressing mCherry (Figure 2—figure supplement 4). At 24 hpi, both IFUs and genome copy numbers were significantly decreased in the DacA overexpression strain (Figure 2C and D). We obtained similar results when overexpressing wild-type DacA with a 6xHis tag in wild-type or STING KO cells, indicating that a C-terminal tag does not impact these phenotypes in Chlamydia (Figure 2—figure supplement 5 and Figure 2—figure supplement 6). We also performed the same experiments with the inactive isoform of DacA, DacA(D164N). Like overexpression of the wild-type DacA, overexpression of DacA(D164N) also negatively affected IFU production and resulted in reduced genome copy numbers even though c-di-AMP levels were reduced under these conditions (Figure 2E and F). YbbR_6xH overexpression did result in a statistically significant, ~ twofold decrease in IFU production (Figure 2—figure supplement 3). This reflects one division cycle difference from the uninduced control and, in the absence of any other phenotypic discrepancy, is not considered biologically relevant. No differences were noted in genome copy number when overexpressing YbbR_6xH (Figure 2—figure supplement 3).

To further investigate impacts of overexpression of these proteins on developmental cycle progression, we quantified transcripts by RT-qPCR for a canonical early-cycle gene, euo, and two late-cycle genes, hctA (an ‘early’ late gene Chiarelli et al., 2020) and omcB (a canonical late gene Belland et al., 2003). Consistent with the genome copy numbers and IFU data, overexpression of DacA resulted in elevated euo transcripts and a reduction in the amounts of the late gene transcripts at 24 hpi (Figure 2G). Similar effects on these transcripts were noted when overexpressing DacA(D164N) (Figure 2H). We measured no effect of YbbR_6xH overexpression on transcript levels for euo, hctA, or omcB (Figure 2—figure supplement 3).

Given that overexpression of either the wild-type or mutant isoform of DacA gave the same phenotype yet yielded differences in c-di-AMP levels, we next explored the need for DacA or DacA(D164N) to be membrane-localized to effect these changes. Therefore, we evaluated the effects of overexpression of wild-type or mutant DacA lacking its transmembrane domains (ΔTM). As noted in Figure 1, overexpression of wild-type ΔTM DacA yielded the same level of c-di-AMP as overexpression of the full-length wild-type DacA. In contrast, c-di-AMP levels measured from chlamydiae overexpressing the mutant ΔTM DacA(D164N) were the same as the mCherry-expressing strain but reduced in chlamydiae overexpressing the full-length DacA(D164N). By IFA, both ΔTM isoforms localized to the cytosol, and there were no observable differences in inclusion or bacterial morphology as compared to the uninduced control (Figure 3A and B). We did not quantify effects of overexpression on inclusion or bacterial size since the measured phenotypes indicated no differences from the uninduced control. Overexpression of the ΔTM isoforms resulted in no statistical differences in IFU production or genome copy numbers, and, similarly, there were no differences in transcript levels for ybbR, euo, hctA, or omcB as compared to the uninduced controls (Figure 3C–H). These data indicate that the negative impacts of DacA overexpression are linked to its membrane localization and are independent of c-di-AMP production.

As mentioned above, the dacA-KD strain displayed a lower c-di-AMP level compared to that of the control strain at 24 hpi, whereas the dacA-KDcom complemented strain exhibited high levels of c-di-AMP at this timepoint and at 16 hpi (Figure 1E). The complemented strain encodes not only the dacA-knockdown system but also the dacA-ybbR_6xH operon as a transcriptional fusion with dCas12. To observe effects of knockdown or complementation on the developmental cycle, we first examined inclusion and bacterial morphology by IFA. When the knockdown system was induced, bacterial cell size was enlarged (~ twofold), but we observed no change in inclusion area as compared to that of the uninduced control (Figure 4A, Figure 4—figure supplement 1). In the complemented strain, induction of dCas12 and YbbR_6xH was confirmed by IFA under inducing conditions (Figure 4B). We expected that co-expressing DacA and YbbR_6xH would complement the dacA-KD to the wild-type phenotype. However, the inclusion area was reduced (~ threefold) with slightly larger organisms (~1.4 fold) as compared to the uninduced control (Figure 4—figure supplement 1), indicating this is not the case.

In addition, we also measured the amount of EB progeny (IFUs) and genome copy numbers. Although genome copy numbers were the same, IFUs decreased by approximately 80% compared to that of the uninduced sample at 24 hpi in the knockdown strain (Figure 4C and D), suggesting more RBs are present in the sample. IFUs and genome copies showed an approximate twofold reduction at 24 hpi in the complemented strain (Figure 4E and F). The data for the knockdown strain suggest that low levels of c-di-AMP are detrimental for secondary differentiation. As genome copies alone cannot assess the ratio of RBs to EBs, we performed RT-qPCR to quantify transcripts of relevant gene targets. When dCas12 was induced, both dacA and ybbR transcript levels decreased (Figure 4G). Since dacA and ybbR are transcribed in an operon, this result is not surprising. Consistent with reduced EB yields, transcripts of the late genes omcB and hctA were decreased at 24 hpi. In contrast, transcripts of the early gene euo were slightly elevated at 24 hpi. We have previously observed no effects on genome levels or transcription of these genes when overexpressing the dCas12 gene alone (Ouellette et al., 2021), indicating these effects are specific to dacA-KD and that dacA knockdown reduces or delays expression of late gene transcripts. For the complemented strain, both dacA and ybbR transcripts were increased compared to the dacA-KD strain (Figure 4H), and transcript levels for these genes were increased beyond the ‘wild-type’ uninduced control levels, again suggesting the ‘complemented’ strain did not return to a wild-type phenotype. Nonetheless, transcripts for the early gene euo were indistinguishable to levels measured in the uninduced strain (Figure 4H). Surprisingly, hctA transcripts were increased ~10 fold at 14 hpi compared to that of the uninduced sample, and omcB transcripts were slightly, but not significantly, increased at 14 and 24 hpi (Figure 4H). These data suggest that overexpressing DacA and YbbR_6xH, with associated increases in c-di-AMP levels (Figure 1), may alter the timing of secondary differentiation.

To clarify the effect of DacA and YbbR_6xH overexpression on secondary differentiation, we next evaluated the phenotype of the dacAop strain in contrast with the dacAopMut strain, in which the active site residue of DacA has been mutated. When we induced expression of the wild-type constructs, we confirmed the induction of DacA and YbbR_6xH and their colocalization at the membrane Figure 5A; Pearson correlation coefficient of 0.713±0.109 from 20 inclusions measured by JACoP Plugin of ImageJ; values near 1 indicate colocalization (Bolte and Cordelières, 2006; Dunn et al., 2011). This is not surprising as both proteins are critical for c-di-AMP synthesis based on our c-di-AMP measurements (Figure 1E). Organism and inclusion morphology were similar to the dacA-KDcom complemented strain (Figures 4B and 5A; Figure 5—figure supplement 1). The bacterial morphology of the dacAopMut strain indicated larger organisms (~1.8 fold) in smaller inclusions (~2.5 fold) after inducing expression (Figure 5B; Figure 5—figure supplement 1). We next assessed whether the overexpressed DacA and YbbR_6xH affected IFU production and replication. IFUs were reduced roughly twofold for the dacAop strain and 1000-fold in the dacAopMut strain after inducing overexpression (Figure 5C&E). Again, the genome copy data for dacAop overexpression closely phenocopied the complemented knockdown strain, showing a decrease at 24 hpi (Figure 5D). Similarly, genome copy numbers for the dacAopMut strain showed a significant drop at 24 hpi after inducing expression (Figure 5F).

We then quantified transcripts for the developmentally regulated genes euo, hctA, and omcB as well as for dacA and ybbR (Figure 5G&H). Not surprisingly, dacA and ybbR transcripts were elevated at the timepoints assessed under inducing conditions for both the dacAop and dacAopMut strains. Transcripts for euo were not statistically changed but trended higher at the 24 hpi timepoint, whereas omcB transcripts were slightly, but not significantly, increased at 14 and 24 hpi during dacAop overexpression. Once again, we observed that hctA transcripts were increased over 10-fold at 14 hpi in the developmental cycle (Figure 5G), similar to what we measured for the complemented knockdown strain (Figure 4H). These data reinforce that elevated c-di-AMP levels (Figure 1E) in these strains lead to increased expression of the late gene hctA at an earlier timepoint (14 hpi) in the developmental cycle. As further validation of this dacAop overexpression strain that was constructed in a beta-lactamase producing background, we also generated a spectinomycin-resistant dacAop overexpression strain and validated that induction of the dacA operon resulted in earlier accumulation of hctA transcripts (Figure 5—figure supplement 2). In assessing developmentally regulated transcripts for the dacAopMut strain, euo levels were maintained, albeit not significantly so, at 24 hpi (Figure 5H). In contrast to the c-di-AMP overproducing strains, transcripts for hctA and omcB were decreased at 24 hpi (Figure 5H). The transcriptional results of the developmentally regulated genes in the dacAopMut strain were very similar to the dacA-KD strain (Figure 4G), suggesting that blocking c-di-AMP accumulation interferes with developmental cycle progression.

Given the surprising finding that, in strains overproducing c-di-AMP, hctA transcripts were 10-fold higher at a timepoint not associated with secondary differentiation, we asked the question whether all genes related to secondary differentiation were increased after inducing production of c-di-AMP. Conversely, we wanted to explore whether reducing c-di-AMP levels would delay expression of genes related to secondary differentiation. Thus, to further investigate how c-di-AMP affects transcription of such genes, we performed RNA sequencing on both the dacAop overexpression and dacA-KD strains and compared the transcriptome between uninduced and induced samples within the given strain at the given timepoint. HeLa cells were infected with these transformants, and overexpression or knockdown was induced or not at 10 hpi with 5 nM aTc. For the dacAop strain, RNA was collected at 16 hpi, a time at which late genes are beginning to be expressed (as opposed to 14 hpi) but remain near a basal level of transcription (Belland et al., 2003). The rationale for this was to determine whether high c-di-AMP levels result in increased late gene transcripts at this timepoint above and beyond the levels of the control, uninduced condition. For the dacA-KD strain, RNA was collected at 24 hpi, a time at which late genes are peaking in their expression. The rationale for this was to determine whether late gene transcription was decreased, which could not otherwise be reliably assessed at the 16 hpi timepoint.

RNA sequencing results were statistically analyzed by the UNMC Bioinformatics Core (Supplementary file 1 and Supplementary file 2) . Figure 6 shows a volcano plot of the results for the dacAop overexpression and dacA-KD strains. Of note, many late genes were evident in the upregulated quadrant for the dacAop strain whereas these genes were present in the downregulated quadrant for the dacA-KD strain. We further characterized the upregulated or downregulated gene sets for the dacAop overexpression and dacA-KD strains, respectively, based on significant difference (p<0.05) and fold-change (>1.5). A summary of these results is presented in Table 1 (all data are presented in Supplementary file 2). We grouped the differentially expressed genes into five categories: (1) canonical late genes for which the literature has associated them with EB function, (2) outer membrane-associated, (3) gene regulation-associated, (4) glycogen synthesis-associated, and (5) type III secretion system associated. Recent work from our group and the Hefty group to define the regulons of the alternative sigma factors in Chlamydia demonstrated that these sigma factors regulate some late gene expression associated with outer membrane remodeling, type III secretion, and other processes (Hatch and Ouellette, 2023; Soules et al., 2020b). Consistent with our RT-qPCR data, we observed that all canonical late genes, as well as all the other genes listed in these categories, showed an increase in expression after c-di-AMP production was induced. In contrast, all the genes, and particularly the canonical late genes, showed a decrease in expression under conditions where c-di-AMP production was impaired. Overall, these RNA sequencing data confirm the direct influence of c-di-AMP on expression of genes related to secondary differentiation.

The phenotypic effect of increased late gene transcription should be an increase in EB production. However, if EB production is initiated at an earlier time in the developmental cycle when there are fewer non-infectious RBs to convert, then overall EB production should be decreased with a concomitant decrease in genome copies since only the RB replicates DNA. Conversely, delayed expression of late genes should be associated with a delay in EB production. Therefore, to test these predictions, we quantified EB production at 2 hr intervals from 18 to 24 hpi to assess EB production during earlier phases of the developmental cycle, as well as at 32 and 48 hpi to assess overall EB yields (Figure 7). We did not detect any EBs at 16 hpi or earlier (not shown). Cells were infected with the dacAop, dacAopMut, and dacA-KD strains and induced or not at 10 hpi with 5 nM aTc as previously noted. Consistent with the transcriptional data, we measured higher EB yields at 18 and 20 hpi during dacAop overexpression that quickly plateaued by 24 hpi (Figure 7A). Conversely, when c-di-AMP accumulation was blocked by reducing the expression of DacA and YbbR (dacA-KD), we observed delayed EB production (Figure 7B). We also measured lower EB yields when c-di-AMP levels were decreased by overexpression of the DacA(D164N) isoform and YbbR (dacAopMut; Figure 7C). The uninduced control conditions for each strain showed a steady accumulation of EBs throughout the course of the experiment, as expected. The vector control strain expressing mCherry showed no differences as previously noted (Figure 2—figure supplement 4). From these data, we conclude that earlier production of c-di-AMP results in earlier production of EBs.

Wild-type (ATCC, Manassas, VA) and STING KO (Dr. Frank van Kuppeveld; Utrecht University) HeLa, human cervical epithelial-derived, and McCoy (kind gift of Dr. Harlan Caldwell, NIH), mouse fibroblast-derived, cell lines were cultured at 37 °C with 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Waltham, MA, #10-569-044) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, #SH30396.03) and 10 μg/mL gentamicin (Gibco, Waltham, MA, #15710072). Cell lines were verified by STR profiling (ATCC) and monitoring of cell morphology. C. trachomatis serovar L2 (434/Bu) lacking the endogenous plasmid (-pL2; kind gift of Dr. Ian Clarke, Univ. Southampton) was used for transformation. All cell cultures and chlamydial stocks were routinely tested for Mycoplasma contamination using the Mycoplasma PCR detection kit and confirmed negative (Sigma, St. Louis, MO, #MP0035-1KT). For E. coli, NEB10β competent cells (New England Biolabs, Ipswich, MA, #C3019H) were used for the amplification of pBOMB-derivative vectors. E. coli was grown at 30 °C in LB media. All chemicals and antibiotics were obtained from Sigma unless otherwise noted.

The list of the vectors and primers used in this study is detailed in Supplementary file 3. Target genes were amplified by PCR with Phusion DNA polymerase (NEB, #M0530L) using 10 ng C. trachomatis L2 genomic DNA or appropriate vectors as a template. Some DNA segments were directly synthesized as a gBlock fragment (Integrated DNA Technologies, Coralville, IA). If plasmids were used as a template, then we treated the PCR product with DpnI enzyme to remove templates. The PCR products were purified using a PCR purification kit (Qiagen, Hilden, Germany, #28506). The HiFi Assembly reaction master mix (NEB, #E2621X) was used following the manufacturer’s manual in conjunction with plasmids pBOMB (Ouellette et al., 2021) linearized with EagI and KpnI or pBOMBL12CRia (empty vector) linearized with BamHI. The linearized plasmids were also dephosphorylated with FastAP (ThermoFisher, #EF0652). The products of the HiFi reaction were transformed into NEB10β competent cells (NEB) and plated on LB agar with appropriate antibiotics. Plasmids were subsequently isolated using a mini-prep kit (Qiagen, #27106) and verified by plasmid digest and sequencing from individual colonies grown overnight in LB broth with appropriate antibiotic selection.

McCoy cells were plated in a six-well plate the day before beginning the transformation procedure. C. trachomatis serovar L2 without plasmid (-pL2) resuspended with Tris-CaCl₂ buffer (10 mM Tris-Cl pH 7.5, 50 mM CaCl₂) was incubated with 2 μg plasmid at room temperature for 30 min. During this step, McCoy cells were washed with 2 mL Hank’s Balanced Salt Solution (HBSS) media containing Ca²⁺ and Mg²⁺ (Corning, Corning, NY #21–023-CV). After that, McCoy cells were infected with the transformants in 2 mL HBSS per well. The plate was centrifuged at 400 × g for 15 min at room temperature and incubated at 37 °C for 15 min. The inoculum was aspirated, and 2 mL 1 X DMEM containing 10% FBS and 10 μg/mL gentamicin was added per well. At 8 hr post- infection (hpi), 1 μg/mL cycloheximide and either 1 or 2 U/mL penicillin G or 500 μg/mL spectinomycin were added, and the plate was incubated at 37 °C until 48 hpi. At 48 hpi, the transformants were harvested and infected onto a new McCoy cell monolayer. These harvest and infection steps were repeated every 44–48 hpi until fluorescent, antibiotic-resistant inclusions were observed.

HeLa cells were infected with chlamydial transformants at an MOI of 0.5. At 10 hpi, 5 nM anhydrotetracycline (aTc) was added or not to the culture medium. Total RNA and DNA were harvested at this time point from duplicate wells not treated with aTc. At 14 and 24 hpi, total RNA and DNA were collected using Trizol (Invitrogen, #15596018) and DNeasy Tissue (Qiagen, #69506) kit, respectively, as described elsewhere (Hatch and Ouellette, 2023). After DNase (Invitrogen, #69506) treatment of total RNA, cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen, #18-080-085). After diluting the cDNA 10-fold, 5 μl of the diluted cDNA was used as a template for qPCR. Equal masses of genomic DNA were used from each of the samples to quantify chlamydial genomes, which were used to normalize transcript data as described (Ouellette et al., 2005). For both cDNA and gDNA samples, triplicate qPCR reactions were prepared using 2 X SYBR Green (Applied Biosystems, #A25778) in a total volume of 25 μL per well. Standard cycling conditions were used with a melting curve analysis to verify products. Transcripts and genome copies were assessed from at least three biological replicates.

HeLa cells were infected with chlamydial transformants as above. At 10 hpi, 5 nM aTc was added or not, and the infected cells were fixed with fixing solution (3.2% formaldehyde and 0.022% glutaraldehyde in 1 X DPBS) for 2 min and permeabilized with 90% MeOH for 1 min at 10.5 hpi or 24 hpi. The fixed cells were labeled with primary antibodies, including rabbit anti-DacA (custom anti-peptide antibody targeting the C-terminal TRNERKTNPIISWMRKK prepared by Pacific Immunology, Ramona, CA), goat anti-major outer-membrane protein (MOMP; Meridian, Memphis, TN), and mouse or rabbit anti-six histidine tag (Genscript, Piscataway, NJ, #A00186-100 and Abcam, Cambridge, UK, #AB213204, respectively). To visualize the primary antibodies, donkey anti-goat (488) (Invitrogen, #A32814) donkey anti-mouse (405) (Invitrogen, #A48257), or donkey anti-rabbit antibodies (594) (Invitrogen, #A21207) were used as secondary antibodies. Coverslips were observed using a Zeiss AxioImager.Z2 with Apotome2 as noted in the figure legends.

HeLa cells were infected with chlamydial transformants as above. At 10 hpi, 5 nM aTc was added or not to the culture medium. At the indicated times, infected cells were harvested in 1 mL 2SP media then frozen at –80 °C. After thawing the lysates, the samples were serially 1:10 diluted and used to infect HeLa cells seeded in 24-well plates. At 24 hpi, the number of GFP- expressing inclusions was counted from 30 fields of view to calculate the IFUs from the original sample. Three biological replicates were performed.

HeLa cells were infected with the indicated chlamydial transformants as above. At 10 hpi, expression of the constructs was induced or not with 5 nM aTc, and, at 16 and 24 hpi, samples were prepared for measuring the level of cyclic di-AMP. After aspirating the media, the infected cell monolayers were washed with 1 X PBS and resuspended with B-Per Bacterial Cell Lysis Buffer (Pierce, Appleton, WI, #PI90078). Samples were vortexed for 1 min and then centrifuged at 13,300×g for 15 min at 4℃. The levels of cyclic di-AMP from the supernatants of the cell lysates were quantified using the Cyclic di-AMP ELISA kit (Cayman, Ann Arbor, MI, #501960-STRIP) following the manufacturer’s instructions (Underwood et al., 2014).

Samples were prepared as reported previously (Hatch and Ouellette, 2023). Briefly, the transformants of dacAop and dacA-KD were infected into HeLa cells. At 10 hpi, the constructs were induced or not with 5 nM aTc. RNA samples were prepared from dacAop-infected cells and dacA-KD-infected cells at 16 hpi and 24 hpi, respectively. 20 μg RNA samples were treated with DNase to remove DNA contamination using DNA-free Turbo kit (Invitrogen, #AM1907) according to the manufacturer’s instructions. Ribosomal RNA was depleted from samples using the MICROBEnrich (Thermo, #AM1901) and MICROBExpress kits (Thermo, AM1905) following the manufacturer’s instructions. RNA samples were processed for sequencing by the UNMC Genomics Core Facility. The resultant libraries from the individual samples were multiplexed and subjected to 100 bp paired-read sequencing to generate approximately 60 million pairs of reads per sample on an Illumina NovaSeq 6000 sequencer in the UNMC Genomics Core facility. The original fastq format reads were trimmed by fqtirm tool (https://ccb.jhu.edu/software/fqtrim) to remove adapters, terminal unknown bases (Ns) and low quality 3’ regions (Phred score <30). The trimmed fastq files were processed by FastQC (Andrews, 2010) for quality control. Chlamydia trachomatis 434/Bu bacterial reference genome and annotation files were downloaded from Ensembl (http://bacteria.ensembl.org/Chlamydia_trachomatis_434_bu/Info/Index). Sequencing data were analyzed by the Bioinformatics and Systems Biology Core (BSBC). The trimmed fastq files were mapped to Chlamydia trachomatis 434/Bu by CLC Genomics Workbench 23 for RNAseq analyses.

To analyze the statistical significance between uninduced and induced samples of qPCR and IFU data, we used two-sample equal variance Student’s t-test. For the levels of cyclic di-AMP data, we used two-sample equal variance Student’s t-test.