c. trachomatis l2 wild type  (INCF)

Journal: bioRxiv

Article Title: A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress

doi: 10.1101/2025.05.30.657042

Figure Lengend Snippet: (A) Under non-stressed conditions, HrcA binds to CIRCE elements and represses transcription from the KP1 and EP promoters. (B) Heat shock disrupts HrcA repression, allowing RNA polymerase (RNAP) to transcribe the hrcA-grpE-dnaK and groES-groEL operons. Arrows denote the extent of transcription. Note that dnaK is regulated by both the operon promoter and its own internal promoter (KP2). (C) Consensus and putative CIRCE sequences upstream of indicated heat shock genes in C. trachomatis (Ct) and Rhizobium meliloti (Rm) . Blue letters indicate nucleotide deviations from the consensus; lowercase letters denote mismatches between inverted repeats. The-35 core promoter elements for C. trachomatis genes are shown. (D) HrcA binds the ctl0271 promoter in chlamydiae. ChIP-qPCR was performed in biological triplicate using L2/NH-HrcA cultures treated with 10 nM ATC for 30 min at 37 °C. Anti-His antibody enrichment of the ctl0271 promoter was comparable to that of known HrcA targets ( groESL and dnaK P1), and significantly higher than enrichment at non-target promoters KP2 and E2P ( groEL2 promoter) , . (E) Expression of ctl0271 , dnaK , and groEL was reduced upon ATC-induced NH-HrcA overexpression in L2/NH-HrcA, as determined by qRT-PCR. (F) ctl0271 expression is induced in wild-type L2 cultured at 45 °C, as measured by qRT-PCR. (D–F) Data represent the mean ± SD of biological triplicates. (G, H) Based on results from panels B–F, the updated HrcA regulon includes ctl0271 (renamed hagF ) as its sixth target gene. (A, B, G, H) These panels were generated using a paid subscription to bioRender.

Article Snippet: Wild-type C. trachomatis serovar L2 strain 434/BU (L2) was obtained from the American Type Culture Collection (ATCC) and expanded in our laboratory using L929 cells as host cells.

Techniques: ChIP-qPCR, Expressing, Over Expression, Quantitative RT-PCR, Cell Culture, Generated

Journal: bioRxiv

Article Title: A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress

doi: 10.1101/2025.05.30.657042

Figure Lengend Snippet: (A) Effective silencing of hagF in C. trachomatis using deactivated CRISPR-associated protein 12 (dC12). L2/dC12-ntg expresses an ATC-inducible dC12 and a non-targeting guide RNA. L2/dC12-hagF-g1 and L2/dC12-hagF-g2 express dC12 and a guide RNA targeting hagF (g1 or g2). The left panel shows elevated dC12 transcript levels upon ATC induction in all three transformants. The right panel shows reduced hagF expression in ATC-treated L2/dC12-hagF-g1 and L2/dC12-hagF-g2 but not in L2/dC12-ntg. RNA levels were quantified by qRT-PCR. (B) ATC-induced dC12 expression has no effect on genome replication (left) or EB formation (right) in 37 °C cultures of L2/dC12-ntg. (C, D) ATC-induced hagF silencing has no detectable effect on genome replication (left) or EB formation (right) in 37 °C cultures of L2/dC12-hagF-g1 (C) and L2/dC12-hagF-g2 (D). (E) ATC-induced dC12 expression has no effect on genome replication (left) or EB formation (right) in 40.5 °C cultures of L2/dC12-ntg. (F, G) ATC-induced hagF silencing does not affect genome replication (left) but causes a substantial reduction in EB formation (right) in 40.5 °C cultures of L2/dC12-hagF-g1 (F) and L2/dC12-hagF-g2 (G). (B-G) ATC was added at 0 hpi. Genome copy number was quantified at the indicated times. EB yields were measured at 30 hpi for 37 °C cultures and at 40 hpi for 40.5 °C cultures.

Article Snippet: Wild-type C. trachomatis serovar L2 strain 434/BU (L2) was obtained from the American Type Culture Collection (ATCC) and expanded in our laboratory using L929 cells as host cells.

Techniques: CRISPR, Expressing, Quantitative RT-PCR

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: Venn diagrams showing numbers of up- and down-regulated genes detected in ATC-treated C. trachomatis L2 (CtL2) transformants of full-length GrgA (CtL2/GrgA), GrgAΔσ 66 BD (CtL2/GrgAΔσ 66 BD), and GrgAΔσ 28 BD (CtL2/GrgAΔσ 28 BD). CtL2/GrgA-, CtL2/GrgAΔσ 66 BD-, or CtL2/GrgAΔσ 28 BD-infected L929 cells in biological duplicates were treated with 10 nM ATC at 12 hpi or left untreated. Cultures were terminated at 16 hpi and processed for RNA-seq analysis as described in Materials and Methods. Venn diagrams were derived from RNA-seq data presented in in the supplemental material. Identities of genes commonly upregulated or downregulated by all three GrgA constructs are shown. Genes commonly upregulated or downregulated by any two of the constructs are listed in . Up- or down-regulation was defined as a ≥ 1.33-fold change with an adjusted P < 0.05.

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: Infection, RNA Sequencing, Derivative Assay, Construct

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: Temporal patterns of transcriptomic changes induced by GrgA overexpression. CtL2/GrgA-infected L929 cultures in biological triplicates were treated with 10 nM ATC at 14, 15, or 15.5 hpi or left untreated. Cultures were terminated at 16 hpi and processed for RNA-seq analysis. C. trachomatis genes were clustered into six groups of temporal expression change patterns by analyzing RNA-seq data presented in . (A) Six target genes were increased by 0.5 h of ATC induction. RNAs whose increases were statistically significant (i.e., with an adjusted P < 0.05) are shown in solid lines. RNAs that increased with an adjusted P > 0.05 by 0.5 h are shown in dashed lines. (B) RNAs of 175 genes are stimulated by GrgA overexpression only after 1 h of ATC induction. (C) RNAs of 444 genes remained relatively constant. (D to F) Genes are downregulated following different kinetics. In panels A to F, solid black lines are trend lines in the respective groups.

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: Over Expression, Infection, RNA Sequencing, Expressing

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: qRT-PCR detection or confirmation of genes upregulated by GrgA within 10 to 30 min of ATC treatment. CtL2/GrgA-infected L929 cultures in biological triplicates were treated with 10 nM ATC for 10, 20, or 30 min or left untreated. Cultures were terminated at 16 hpi. The levels of expression of individual genes were determined using qRT-PCR. (A) Descriptions of the genes studied in this figure. (B) Three of four nonoperon genes from Fig. 2A showed increased mRNA levels by 10 to 30 min. The color code to the right of the graph applies to panels B to F. (C) Expression of lplA and its cotranscribed gene ctl0536 was increased at 10 and 30 min, respectively. (D) Transcripts of both aroC and its cotranscribed gene aroB were increased by 30 min. aroL and aroDE from the same operon were not analyzed. (E) Expression of the nonoperon gene murE was significantly increased at 30 min. (F) Transcripts of hrcA and its cotranscribed genes grpE and dnaK were increased by 10 min, although the increases in grpE were less pronounced with only trending significant adjusted P values. In panels B to F, data are averages plus standard deviations (error bars). All P values were adjusted for multiple comparison. Upregulation detected by RNA-seq at 0.5 h after ATC treatment is presented as a black bar for comparison.

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: Quantitative RT-PCR, Infection, Expressing, Comparison, RNA Sequencing

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: GrgA-, Euo-, and HrcA-coregulated genes. RNA-seq data generated from biologically duplicated cultures of CtL2/GrgA , CtL2/Euo , and CtL2/HrcA treated with 10 nM ATC from 12 to 16 hpi were analyzed to reveal genes coregulated by GrgA, Euo, and HrcA. (A) Venn diagrams showing numbers of genes upregulated by overexpression of each of the 3 TFs as shown in . (B) List of genes commonly upregulated by GrgA, Euo, and HrcA overexpression. Note that genes commonly upregulated by two TFs are shown in . (C) Venn diagrams showing numbers of genes downregulated by overexpression of each of the three TFs as shown in . (D) List of genes commonly downregulated by GrgA, Euo, and HrcA overexpression. Note that genes commonly downregulated by two TFs are shown in . Up- or downregulation was defined as a ≥1.33-fold change with an adjusted P < 0.05.

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: RNA Sequencing, Generated, Over Expression

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: GrgA-regulated TRN. (A) TRN established within 10 to 30 min of ATC-induced GrgA overexpression. , which contains RNA-seq data extracted from (biological triplicates), and qRT-PCR data (biological triplicates) ( Fig. 3 ), were used to generate the TRN using STRING v11. Light blue and red nodes are genes upregulated and downregulated by GrgA, respectively. Black lines connect functional or physical associations identified by the STRING v11 database. Solid and dashed lines connect GrgA to upregulated and downregulated genes, respectively. (B) Interregulatory relationship among grgA , euo , and hrcA deduced following 15-min and 30-min ATC treatments of transformants. qRT-PCR data obtained from biological triplicates of CtL2/GrgA ( Fig. 3B and ), CtL2/Euo ( Fig. S3A ), and CtL2/HrcA ( Fig. S3B ) were used to manually generate the TRNs. Arrows signify direction of regulation. Solid and dotted line indicate upregulation and downregulation, respectively. (C) TRN developed by 4 h of ATC-induced GrgA overexpression. , which contains a subset of RNA-seq data extracted from (biological duplicates of CtL2/GrgA), (biological duplicates of CtL2/Euo), and (biological duplicates of CtL2/HrcA), were used to generate the TRN using STRING v11. See panel A for information for node colors and black lines. Solid and dashed lines connect TFs to upregulated and downregulated genes, respectively. Blue, purple, and green lines connect GrgA, Euo, and HrcA, respectively, to the genes that the TFs regulate. (A and C) Functional groups are labeled. Abbreviations: AA, amino acid; metab., metabolism; Nt.M, nucleotide metabolism; post-transl., posttranslational protein modification; recom, recombination; replic, replication. An interactive version of this figure is available at the Figshare public data repository ( https://doi.org/10.6084/m9.figshare.14195447 ).

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: Over Expression, RNA Sequencing, Quantitative RT-PCR, Functional Assay, Labeling, Modification

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: Genome copy numbers and temporary expression patterns of endogenous GrgA, Euo, and HrcA during the CtL2 developmental cycle. L929 cells infected with wild-type CtL2 at the multiplicity of infection of 0.5 inclusion-forming unit per cell were harvested at 0, 1, 3, 5, 8, 12, 18, 24, 30, and 36 hpi. Genomic DNA (gDNA) was quantified using qPCR (biological triplicates). RNAs of grgA , euo , and hrcA were quantified using qRT-PCR (biological triplicates). GrgA protein was quantified using ELISA (biological duplicates). All expression data were normalized with the amount of gDNA. See Fig. S5 for genome copy number data and expression results of individual genes with error bars.

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: Expressing, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: Effects of overexpression of GrgA forms, Euo and HrcA on CtL2 growth in L929 cells. (A) Differential effects of 10 nM ATC treatment on inclusion morphology and RFP intensity in cultures of CtL2/GrgA, CtL2/GrgAΔσ 66 BD, and CtL2/GrgAΔσ 28 BD. ATC was added at 0 hpi. Images were acquired at 35 hpi. (B) Genome copy reduction in CtL2/GrgA after treatment with 10 nM ATC for 4 h (from 12 to 16 hpi). Genome copy was determined using qPCR. (C) Moderate increases in euo and hrcA expression in CtL2/Euo and CtL2/HrcA, respectively, after treatment with a low concentration of ATC for 4 h (from 12 to 16 hpi). RNAs of euo and hrcA were quantified using qRT-PCR. Note that mRNA expression values have been normalized with gDNA due to (possible) overexpression-induced growth inhibition (D) Genome copy reduction in CtL2/Euo but not CtL2/HrcA after treatment with a low concentration of ATC for 4 h. Genome copy was determined using qPCR. In panels B to D, all quantitative data were obtained with biological triplicates.

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: Over Expression, Expressing, Concentration Assay, Quantitative RT-PCR, Inhibition

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: Upregulated euo and hrcA expression and growth inhibition in CtL2/GrgA cultured in human HeLa cells following ATC treatment. (A) Increased euo and hrcA RNA levels following treatment with 10 nM ATC for 30 min (from 15.5 to 16 hpi). RNAs of euo and hrcA in triplicated cultures were quantified using qRT-PCR. (B) Reduced inclusion size and RFP intensity of inclusions formed by CtL2 treated with 10 nM ATC from 0 to 35 hpi.

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: Expressing, Inhibition, Cell Culture, Quantitative RT-PCR

Journal: mSystems

Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

doi: 10.1128/mSystems.00738-21

Figure Lengend Snippet: Plasmids

Article Snippet: Wild-type C. trachomatis L2 (CtL2) (strain 434/BU) was purchased from ATCC ( ).

Techniques: Plasmid Preparation, Control, Over Expression, Expressing