The sRNA Regulated Protein DdbA Is Involved in Development and Maintenance of the Chlamydia trachomatis EB Cell Form

Abstract

The chlamydial small non coding RNA, IhtA, regulates the expression of both HctA and DdbA, the uncharacterized product of the C. trachomatis L2 CTL0322 gene. HctA is a small, highly basic, DNA binding protein that is expressed late in development and mediates the condensation of the genome during RB to EB differentiation. DdbA is conserved throughout the chlamydial lineage, and is predicted to express a small, basic, cytoplasmic protein. As it is common for sRNAs to regulate multiple mRNAs within the same physiological pathway, we hypothesize that DdbA, like HctA, is involved in RB to EB differentiation. Here, we show that DdbA is a DNA binding protein, however unlike HctA, DdbA does not contribute to genome condensation but instead likely has nuclease activity. Using a DdbA temperature sensitive mutant, we show that DdbAts creates inclusions indistinguishable from WT L2 in size and that early RB replication is likewise similar at the nonpermissive temperature. However, the number of DdbAts infectious progeny is dramatically lower than WT L2 overall, although production of EBs is initiated at a similar time. The expression of a late gene reporter construct followed live at 40°C indicates that late gene expression is severely compromised in the DdbAts strain. Viability assays, both in host cells and in axenic media indicate that the DdbAts strain is defective in the maintenance of EB infectivity. Additionally, using Whole Genome Sequencing we demonstrate that chromosome condensation is temporally separated from DNA replication during the RB to EB transition. Although DdbA does not appear to be directly involved in this process, our data suggest it is a DNA binding protein that is important in the production and maintenance of infectivity of the EB, perhaps by contributing to the remodeling of the EB chromosome.

Keywords: Chlamydia; bacterial cell development; bacterial replication; elementary bodies; reticulate body.

Figure 1

PSI-BLAST results showing distance tree of DdbA related proteins (highlighted). DdbA homologs are present in all the chlamydiaceae and are distantly related to an endonuclease in Clostridium saccharobutylicum.

Figure 2

DdbA binds to DNA. Confocal images of DdbA ectopically expressed in HeLa cells (A, B) and Biolayer interferometry of purified DdbA with DNA (C) indicate an affinity for DNA. (A) HctA and DdbA were cloned and expressed with GFP-N terminal tags in HeLa cells. GFP signal is in green and DNA was stained with Draq5 (Blue) to highlight the nucleus. (B) Cell transfected with DdbA undergoing mitosis. Arrows indicate co-localization of the GFP signal with the mitotic chromosomes. Size bar = 10µm. (C) His-tagged DdbA and HctA were bound to a biolayer interferometry glass probe and binding measured using dsDNA ranging from 20%, 40%, 60% and 80% GC content. Both HctA and DdbA showed significant DNA binding with a maximal binding kinetics of 2.3 nM KD and 7.3 nM KD respectively. A his-tagged Ctr protein library (Ct_control_lib) was used as a negative control and showed no DNA binding (representative binding kinetics from one of three independent binding experiments).

Figure 3

Ectopic expression of DdbA. (A) Cos-7 cells were infected with Ctr transformed with theophylline inducible expression plasmids controlling the production of HctA, DdbA, GFP (clover) or Scc1. Infected cells were induced for expression at 14 hpi and infectious progeny were isolated at 30 hpi. Progeny were quantified using an inclusion forming reinfection assay. Both HctA and DdbA induction resulted in a significant reduction in EB production (p < 0.01). While induction of Scc1 and GFP had no significant effect on EB production. (B) The morphological effects of ectopic expression was assessed by confocal microscopy. Cos-7 cells were infected with transformed Ctr expressing HctA, DdbA and Scc1, induced at 16 hpi, fixed at 24 hpi and stained with an anti-flag antibody to localize the expressed proteins. Additionally DNA morphology was assessed using DAPI staining. HctA expression resulted in a distinct change in the DAPI staining morphology, causing densely compacted regions of DNA in the RB. DdbA expression caused an increase in RB size but did not appreciatively change the compaction of the DNA. Scc1 expression had no effect on RB or DNA morphology. Size bar = 5µm. *p < 0.01.

Figure 4

Purified DdbA displays nuclease activity. (A) The indicated concentrations of DdbA-6xHis, HctA-6xHis and a cocktail of purified proteins from a his tagged chlamydial protein expression library ectopically expressed and purified from E coli were incubated with plasmid DNA for 5 minutes at 37°C. Deproteinated, purified DNA was analyzed by agarose gel electrophoresis. EDTA was used as a control to demonstrate the nuclease activity was Mg++ dependent. HctA and the chlamydial library did not significantly change the migration of the DNA in the agarose gel. Purified DdbA cleaved DNA in a concentration dependent manner and was inhibited by EDTA. (B) Euo-6xHis, DdbA-6xHis and Clover-6xHis ectopically expressed in Ctr L2 were purified and incubated with 1µg of plasmid DNA for 5 minutes at 37°C. Deproteinated, purified DNA was analyzed by agarose gel electrophoresis. Only DNA incubated with DdbA-6xHis was cleaved.

Figure 5

Growth and infectivity characteristics of the ddbAts mutant. (A) The ddbAts chlamydial mutant was grown at 37°C or 40°C and production of infectious progeny (IFUs) and genomic copy number, were compared to the isogenic strain L2(G5). There was little difference in growth and progeny production between L2(G5) and the ddbAts mutant at 37°C but at 40°C there was a dramatic difference in IFU counts late in the infection. (B) Live cell analysis of ddbAts late gene expression and comparison of inclusion size. ddbAts and L2(G5) were transformed with a plasmid expressing the fluorescent protein Clover driven by the hctA promoter and imaged every 15 minutes for 30 hours at either 37°C or 40°C. The size of the chlamydial inclusions was determined by measurement of inclusions from the microscopy images at the nonpermissive temperature of 40°C. The cloud represents 95% confidence intervals and n > 50 inclusions were measured per experiment. (C) The ddbAts defective phenotype is only observable during EB production. Confocal images of ddbAts growth showed that the inclusions are nearly identical to L2(G5) at 18 hpi but that at 36 hpi the ddbAts mutant inclusion contains less chlamydial organisms. L2(G5) and ddbAts mutants grown at 40°C for 18 and 36 hours were fixed and stained with DAPI to label the DNA (blue) and an anti-MOMP antibody to label Chlamydia (green). Insert pictures highlight the DAPI channel to better visualize the EBs. Size bar = 10µm.

Figure 6

EBs produced by ddbAts mutant cells have a defect in maintenance of EB infectivity at the non-permissive temperature. (A) The ability of EBs to maintain infectivity after formation was determined by treating infected cells grown at 37°C with penicillin at 24 hpi and then shifting the temperature to 40°C. The premade EBs were harvested at 48 hours and maintenance of infectivity measured by IFU assay at 37°C. (B) Maintenance of infectivity was also measured using an axenic growth media. EBs from G5 or the ddbAts mutant were incubated in CIP-1 axenic growth media at 37°C or 40°C for 24 hours before measuring infectivity by an IFU assay. *p < 0.01.

Figure 7

DdbA is not involved in the RB to EB replication check point. The EB cell type index of replication suggests a DNA replication initiation checkpoint is a component of the chlamydial developmental cycle. The index of replication was determined using the iRep script which calculates the sequence read bias at the origin of replication as compared to the terminus. The iRep index for L2 wt RBs was temperature dependent; RBs grown at 35°C had an iRep index of 1.57, at 37°C an index of 1.66 and at 40°C an index of 1.70. These differences were statistically significant with p < 0.001. The iRep index for L2 wt EBs was much lower and showed no temperature dependent changes (1.35, 1.33, and 1.34 respectively). The ddbAts mutant showed the same significant difference in the iRep between the RB cell type and EB cell types (1.63 and 1.33). This difference was also evident at the nonpermissive temperature (RB = 1.56 and EB = 1.36). *p < 0.001. NS, not significant.