Cyclic di-AMP accumulation is linked to secondary differentiation in C. trachomatis.

A. Defining characteristics of chlamydial EBs and RBs. B. A hypothetical model representing the correlation between c-di-AMP levels and secondary differentiation. The dashed line represents a threshold level of c-di-AMP needed to drive secondary differentiation in a given RB. C. A schematic diagram of the constructs used in this study. All constructs used are anhydrotetracycline (aTc)-inducible as shown by the Ptet promoter. The location of the 6xH tag is shown as well as the approximate location of the crRNA for the CRISPRi vectors. Diagram is not to scale. D. Measurement of c-di-AMP concentrations in infected cell lysates from the strains shown in panel C. C. trachomatis serovar L2 (434/Bu) transformed with the indicated constructs were infected into HeLa cells. At 10 hpi, expression of the construct was induced or not with 5 nM aTc, and the infected cells were harvested at 16 or 24hpi. Levels of c-di-AMP in the supernatant were measured using ELISA. The left panel shows the increase from 16 to 24hpi during overexpression of the control mCherry protein. The middle and right panels show the levels of c-di-AMP in the indicated strains at 16 and 24hpi, respectively. EV: Empty vector *: p<0.05, **: p<0.001, NS: Not significant via one way ANOVA test. #: p<0.05 via ratio paired t-test as compared to the mCherry control.

Overexpression of DacA_6xH, but not YbbR_6xH, is detrimental to the chlamydial developmental cycle.

(A-D) HeLa cells were infected with C. trachomatis transformed with a plasmid encoding an anhydrotetracycline (aTc)-inducible DacA_6xH. At 10 hpi, expression of the construct was induced or not with 5 nM aTc, and DNA and RNA samples were collected at 10, 14, and 24 hpi. Immunofluorescence analysis (IFA) and inclusion forming units (IFU) samples were collected at 24hpi. For IFA images, the green color represents chlamydial major outer membrane protein (MOMP), which is showing the chlamydial cell morphology, and the red color represents DacA or YbbR tagged with six histidine. A. IFA images of the dacA_6xH strain at 24 hpi. Shown are individual panels of a representative inclusion for DacA_6xH and MOMP labeling as well as the merged image. B. Quantification of IFUs from uninduced and induced samples at 24 hpi. C. Quantification of genomic DNA by qPCR in uninduced and induced samples. D. Quantification of transcripts by RT-qPCR for dacA, ybbR, euo, hctA, and omcB from uninduced and induced samples. (E-H) HeLa cells were infected with C. trachomatis transformed with a plasmid encoding an aTc-inducible YbbR_6xH. At 10 hpi, expression of the construct was induced or not with 5 nM aTc, and IFA, IFU, DNA, and RNA samples were collected as above. E. IFA images of ybbR_6xH strain at 24 hpi. F. Quantification of IFU from uninduced and induced samples at 24 hpi. G. Quantification of genomic DNA by qPCR in uninduced and induced samples. H. Quantification of transcripts by RT-qPCR for dacA, ybbR, euo, hctA, and omcB. IFA images were acquired on a Zeiss AxioImager.Z2 equipped with an Apotome2 using a 100X lens objective. Scale bar: 1 µm. UI = uninduced (i.e., −aTc); I = induced (i.e., +aTc) for all sample types. *: p<0.05; **: p<0.001 via two sample equal variance t-test.

CRISPRi-mediated dacA-ybbR knockdown displays reduced levels of transcripts for late genes.

(A-D) HeLa cells were infected with C. trachomatis transformed with a plasmid encoding an anhydrotetracycline (aTc)-inducible CRISPRi-dCas12 system targeting the dacA promoter (i.e., dacA-KD; see Fig. 1C). At 10 hpi, knockdown was induced or not with 5 nM aTc, and DNA and RNA samples were collected at 10, 14, and 24 hpi. Immunofluorescence analysis (IFA) and inclusion forming units (IFU) samples were collected at 24hpi. A. IFA images of the dacA-KD strain at 24 hpi. Shown are individual panels of a representative inclusion for dCas12 and major outer membrane protein (MOMP) labeling as well as the merged image. B. Quantification of IFUs from uninduced and induced samples at 24 hpi. C. Quantification of genomic DNA by qPCR in uninduced and induced samples. D. Quantification of transcripts by RT-qPCR for dacA, ybbR, euo, hctA, and omcB from uninduced and induced samples. (E-H) HeLa cells were infected with C. trachomatis transformed with a plasmid encoding an aTc-inducible CRISPRi-dCas12 system targeting the dacA promoter and the complementing dacA and ybbR_6xH alleles (i.e., dacA-KDcom; see Fig. 1C). At 10 hpi, simultaneous knockdown and complementation were induced or not with 5 nM aTc, and immunofluorescence analysis (IFA), inclusion forming units (IFU), DNA, and RNA samples were collected as above. E. IFA images of dacA-KD-dacAop strain at 24 hpi. F. Quantification of IFU from uninduced and induced samples at 24 hpi. G. Quantification of genomic DNA by qPCR in uninduced and induced samples. H. Quantification of transcripts by RT-qPCR for dacA, ybbR, euo, hctA, and omcB. IFA images were acquired on a Zeiss AxioImager.Z2 equipped with an Apotome2 using a 100X lens objective. Scale bar: 1 µm. UI = uninduced (i.e., −aTc); I = induced (i.e., +aTc) for all sample types. *: p<0.05; **: p<0.001 via two sample equal variance t-test.

Overexpression of DacA and YbbR_6xH prematurely increases hctA transcript levels.

(A-C) HeLa cells were infected with C. trachomatis transformed with an aTc-inducible plasmid encoding wild-type DacA and YbbR_6xH (i.e., dacAop; see Fig. 1C). At 10 hpi, expression of the constructs were induced or not with 5 nM aTc, and DNA and RNA samples were collected at 10, 14, and 24 hpi. Immunofluorescence analysis (IFA) samples were collected at 24hpi. A. IFA images of the dacAop strain at 24 hpi. Shown are individual panels of a representative inclusion for DacA, YbbR_6xH, and major outer membrane protein (MOMP) labeling as well as the merged image. The arrowheads represent the co-localization of DacA and YbbR. B. Quantification of genomic DNA by qPCR in uninduced and induced samples. C. Quantification of transcripts by RT-qPCR for dacA, ybbR, euo, hctA, and omcB. (D-F) HeLa cells were infected with C. trachomatis transformed with an aTc-inducible plasmid encoding DacA(D164N) and YbbR_6xH (i.e., dacAopMut; see Fig. 1C). At 10 hpi, expression of the constructs were induced or not with 5 nM aTc, and DNA and RNA samples were collected at 10, 14, and 24 hpi. IFA samples were collected at 24hpi. D. IFA images of dacAopMut strain at 24 hpi. Shown are individual panels for DacA, YbbR_6xH, and MOMP labeling as well as the merged image. E. Quantification of genomic DNA by qPCR in uninduced and induced samples. F. Quantification of transcripts by RT-qPCR for dacA, ybbR, euo, hctA, and omcB from uninduced and induced samples. IFA images were acquired on a Zeiss AxioImager.Z2 equipped with an Apotome2 using a 100X lens objective. Scale bar: 1 µm. UI = uninduced (i.e., −aTc); I = induced (i.e., +aTc) for all sample types. *: p<0.05; **: p<0.001 via two sample equal variance t-test.

The levels of c-di-AMP are correlated with late gene transcripts.

RNA sequencing was performed from HeLa cells infected with either the dacAop or dacA-KD strains. RNA samples were collected at 16hpi for dacAop and 24hpi for dacA-KD after inducing expression of the relevant constructs at 10hpi. Shown is a volcano plot of the RNA sequencing results with the vertical dashed lines indicating a two-fold change in transcript levels as compared to the respective uninduced control for the given strain and the horizontal lines indicating a p-value of 0.05. The plot was made using GraphPad Prism software. Green spots represent genes demonstrating a statistically significant two-fold change in transcription levels between uninduced and induced samples. Red dots represent genes with significant changes in transcript levels less than two-fold. Blue dots represent genes not significantly different but more than two-fold changed between the conditions. Black dots represent genes not significantly different and less than two-fold changed between the conditions. See also Table 1 and Supplemental Tables 2 and 3 for more details.

Genes regulated by cyclic di-AMP levels.

Late genes transcripts are increased by high levels of c-di-AMP in penicillin-treated conditions.

HeLa cells were infected with a C. trachomatis strain transformed with a dacAop construct encoding spectinomycin resistance. At 10 hpi, expression of the construct was induced or not with 5 nM aTc and treated with 1 U/mL of penicillin G (PenG). DNA and RNA samples were collected at 10, 14, and 24 hpi, and immunofluorescence analysis (IFA) samples were collected at 24hpi. A. IFA images of the dacAop strain in 1 U/mL PenG at 24 hpi. Shown are individual panels of a representative inclusion for DacA, YbbR_6xH, and major outer membrane protein (MOMP) labeling as well as the merged image. IFA images were acquired on a Zeiss AxioImager.Z2 equipped with an Apotome2 using a 100X lens objective. Scale bar: 2 µm. B. Quantification of genomic DNA by qPCR in uninduced and induced samples. C. Quantification of transcripts by RT-qPCR for dacA, ybbR, euo, hctA, and omcB. UI = uninduced (i.e., −aTc); I = induced (i.e., +aTc) for all sample types. *: p<0.05; **: p<0.001 via two sample equal variance t-test.

EB production is induced by high levels of c-di-AMP.

(A-C) HeLa cells were infected with the chlamydial transformants dacAop (A), dacA-KD (B), and dacAopMut (C). Expression of the constructs was induced or not with 5 nM aTc at 10hpi. At 18, 20, 22, 24, 32, and 48 hpi, infected cell lysates were harvested for IFU quantification. UI = uninduced (i.e., −aTc); I = induced (i.e., +aTc) for all sample types. #: Non-detected from the induced samples. *: p<0.05; **: p<0.001 via two sample equal variance t-test.

A working model illustrating how c-di-AMP impacts the onset of secondary differentiation in C. trachomatis.

A. During RB division, there is a gradient of c-di-AMP that forms between the mother and daughter cell. The lighter blue represents lower levels of c-di-AMP, the darker blue represents higher levels of c-di-AMP. In the depicted scenario, the daughter cell is able to divide again, having not reached a critical threshold of c-di-AMP. In contrast, the mother cell accumulates sufficient c-di-AMP to trigger secondary differentiation to an EB. B. A model illustrating how c-di-AMP levels impact secondary differentiation progression. Shown is the “normal” condition of wild-type bacteria, bacteria overexpressing DacA and YbbR (higher cyclic di-AMP), and bacteria expressing the CRISPRi system targeting the dacA promoter (lower cyclic di-AMP). Depending on the amount of c-di-AMP produced, secondary differentiation can either be triggered earlier or later as shown. The black dots represent EBs; the bigger and blue-colored circles show the RB cells. The brightness of blue reflects the c-di-AMP level.