Sigma 54-Regulated Transcription Is Associated with Membrane Reorganization and Type III Secretion Effectors during Conversion to Infectious Forms of Chlamydia trachomatis

Abstract

Chlamydia bacteria are obligate intracellular organisms with a phylum-defining biphasic developmental cycle that is intrinsically linked to its ability to cause disease. The progression of the chlamydial developmental cycle is regulated by the temporal expression of genes predominantly controlled by RNA polymerase sigma (σ) factors. Sigma 54 (σ⁵⁴) is one of three sigma factors encoded by Chlamydia for which the role and regulon are unknown. CtcC is part of a two-component signal transduction system that is requisite for σ⁵⁴ transcriptional activation. CtcC activation of σ⁵⁴ requires phosphorylation, which relieves inhibition by the CtcC regulatory domain and enables ATP hydrolysis by the ATPase domain. Prior studies with CtcC homologs in other organisms have shown that expression of the ATPase domain alone can activate σ⁵⁴ transcription. Biochemical analysis of CtcC ATPase domain supported the idea of ATP hydrolysis occurring in the absence of the regulatory domain, as well as the presence of an active-site residue essential for ATPase activity (E242). Using recently developed genetic approaches in Chlamydia to induce expression of the CtcC ATPase domain, a transcriptional profile was determined that is expected to reflect the σ⁵⁴ regulon. Computational evaluation revealed that the majority of the differentially expressed genes were preceded by highly conserved σ⁵⁴ promoter elements. Reporter gene analyses using these putative σ⁵⁴ promoters reinforced the accuracy of the model of the proposed regulon. Investigation of the gene products included in this regulon supports the idea that σ⁵⁴ controls expression of genes that are critical for conversion of Chlamydia from replicative reticulate bodies into infectious elementary bodies.IMPORTANCE The factors that control the growth and infectious processes for Chlamydia are still poorly understood. This study used recently developed genetic tools to determine the regulon for one of the key transcription factors encoded by Chlamydia, sigma 54. Surrogate and computational analyses provide additional support for the hypothesis that sigma 54 plays a key role in controlling the expression of many components critical to converting and enabling the infectious capability of Chlamydia These components include those that remodel the membrane for the extracellular environment and incorporation of an arsenal of type III secretion effectors in preparation for infecting new cells.

Keywords: Chlamydia trachomatis; gene regulation; regulon; sigma factors.

FIG 1

C. trachomatis CtcC domain organization and in vitro ATPase activity of CtcC protein constructs. (A) Graphic domain structure of the canonical σ⁵⁴-regulatory protein showing that the native chlamydial homolog, CtcC, does not contain a DNA-binding domain and that the ATPase domain-only variant eliminates the regulatory domain. (B) Triplicate ATPase hydrolysis activity of full-length, ATPase domain, and active-site-defective σ⁵⁴-regulatory protein CtcC. The ATPase domain-only recombinant protein depleted approximately four times as much ATP on average as the full-length CtcC protein. When the E242A substitution was introduced into the ATPase domain, there was a significant decrease in the amount of ATP depletion compared to the full-length CtcC, supporting the hypothesis that this single amino acid substitution disrupts the ability for the protein to perform its normal activity. *, P value of <0.05 by Student's t test.

FIG 2

CtcC construct overexpression with inducible vector. (A) Vector map with ctcC gene variants under the control of an ATc-inducible promoter on C. trachomatis native plasmid backbone (light gray) (49). (B) Western blot showing overexpression of full-length CtcC, CtcC ATPase domain-only CtcC, and CtcC with an ATPase domain with an E242A substitution induced with addition of ATc after infection at 12 hpi and harvest at 24 hpi.

FIG 3

Beta-galactosidase assays of σ⁵⁴- and CtcC-dependent activation on selected promoters. Promoter regions upstream of selected σ⁵⁴-regulated genes were tested for their ability to induce expression of lacZ on the pACYC vector with or without the chlamydial rpoN gene (σ⁵⁴) and ctcC ATPase gene (pRSF-DUET). (A) WebLogo alignment of the −12/−24 promoter elements shows the relative frequencies of nucleotides of the predicted σ⁵⁴ promoter regions upstream of the genes that were found by RNA-seq to be differentially regulated. IFU, inclusion-forming units. (B) Promoters were used to test the induction of lacZ expression without either σ⁵⁴ or CtcC present (vector control) and with each protein expressed individually in order to assess the system for induction from the E. coli σ⁵⁴. (C) Additional promoters were tested from the predicted σ⁵⁴ regulon, looking at the difference between the constitutively active CtcC ATPase and the inactivated E242A mutant. The predicted regulatory sigma factor is indicated below the promoter region. Experiments were performed in triplicate. * and **, P values of <0.05 and <0.01, respectively, by Student's t test comparing the CtcC ATPase to the E242A ATPase mutant; †, P value of <0.05 by Student's t test comparing the CtcC E242A ATPase mutant to a promoterless pACYC-lacZ negative control. ns, not significant. (D) Two point mutations were introduced into the ct620 promoter (alignment below graph). The level of LacZ activity was decreased significantly compared to the wild-type promoter but was still increased significantly compared to the inactive conditions. *, P value of <0.05 by Student's t test. All beta-galactosidase assays were performed in triplicate.

FIG 4

Phenotypic analysis of CtcC variant overexpression during host cell infection. (A) Progeny assay showing a significant decrease in progeny production with overexpression of the full-length and ATPase-only CtcC protein variants. The vector control (pL2) showed no difference in progeny production with the addition of ATc. Induction of full-length CtcC at 12 hpi resulted in a decrease in the number of progeny passaged at 30 hpi compared to the results seen with the pL2 control and the uninduced conditions. Overexpression of the CtcC ATPase domain-only construct resulted in less progeny as well. Additionally, the uninduced CtcC ATPase domain-only infection resulted in a significant difference in progeny produced compared to the pL2 vector control. No difference in progeny production was observed with the E242A substitution in the ATPase domain. *, P value of <0.05. (B) Immunofluorescent microscopy of C. trachomatis-infected cells following expression induction of CtcC variants. Induction of full-length CtcC appears to have affected the observable chlamydial cell density inside the center of the inclusion. Overexpression of the ATPase domain-only variant caused the chlamydial cells to have an enlarged appearance, in addition to having fewer cells apparent in center of the inclusion. Infections were induced with 10 ng/ml of ATc at 12 hpi, and the infected cell monolayers were fixed and stained at 24 hpi. Green, OmpA; red, host cytosol; blue, DAPI—DNA. Representative images show at least 5 inclusions each; each image represents an average of 3 projections of z-stack images. Scale bar = 2 μm.