Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in &lt;em&gt;Chlamydia trachomatis&lt;/em&gt;

Chiarelli, Travis J; Grieshaber, Nicole A.; Grieshaber, Scott S.

doi:10.3791/61365

Cited by 9 publications

(14 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It divides asymmetrically, resulting in a stalked cell that immediately enters S phase and a swarmer cell that arrests in G1 phase until it differentiates into a stalked cell. C. crescentus chromosomal replication is tightly regulated and occurs only in stalked cells and only once per cell cycle (O'Neill and Bender, 1989;Ozaki et al, 2014). Our iRep data is suggestive of a cell cycle control system in Chlamydia that allows replication in the RB cell type but suppresses DNA replication initiation upon RB differentiation to the EB cell.…”

Section: Data Availability Statementmentioning

confidence: 55%

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

Grieshaber

Runac

Turner

et al. 2021

Front. Cell. Infect. Microbiol.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Data Availability Statementmentioning

confidence: 55%

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

Grieshaber

Runac

Turner

et al. 2021

Front. Cell. Infect. Microbiol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Therefore we assessed the dose responsiveness of the E riboswitch to its ligand theophylline. Gene expression was measured using live-cell time-lapse microscopy and particle tracking to quantify the fluorescent expression of individual inclusions over time [ 19 , 30 ]. This technique allows for the tracking of gene expression in multiple individual inclusions over the entire developmental cycle.…”

Section: Resultsmentioning

confidence: 99%

Translational gene expression control in Chlamydia trachomatis

et al. 2022

Self Cite

View full text Add to dashboard Cite

The human pathogen Chlamydia trachomatis proceeds through a multi phenotypic developmental cycle with each cell form specialized for different roles in pathogenesis. Understanding the mechanisms regulating this complex cycle has historically been hampered by limited genetic tools. In an effort to address this issue, we developed a translational control system to regulate gene expression in Chlamydia using a synthetic riboswitch. Here we demonstrate that translational control via a riboswitch can be used in combination with a wide range of promoters in C. trachomatis. The synthetic riboswitch E, inducible with theophylline, was used to replace the ribosome binding site of the synthetic promoter T5-lac, the native chlamydial promoter of the pgp4 plasmid gene and an anhydrotetracycline responsive promoter. In all cases the riboswitch inhibited translation, and high levels of protein expression was induced with theophylline. Combining the Tet transcriptional inducible promoter with the translational control of the riboswitch resulted in strong repression and allowed for the cloning and expression of the potent chlamydial regulatory protein, HctB. The ability to control the timing and strength of gene expression independently from promoter specificity is a new and important tool for studying chlamydial regulatory and virulence genes.

show abstract

“…This technique allows for the tracking of gene expression in multiple individual inclusions over the entire developmental cycle while avoiding the inherent variability of whole-population studies on an asynchronous infection. A detailed description of the system is described in our recently published paper (23). To verify that the fluorescent reporters accurately reflected the developmental cycle, total chlamydial growth was determined by measuring genomic copies by quantitative PCR (qPCR) and EB production by a replating assay to quantify inclusion-forming units (IFU).…”

Section: Figmentioning

confidence: 99%

Single-Inclusion Kinetics of Chlamydia trachomatis Development

et al. 2020

Self Cite

View full text Add to dashboard Cite

The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an “intrinsic” cell-autonomous program inherent to each RB and an “extrinsic” environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium’s pathogenesis.

show abstract

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in <em>Chlamydia trachomatis</em>

Cited by 9 publications

References 19 publications

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

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

Translational gene expression control in Chlamydia trachomatis

Single-Inclusion Kinetics of Chlamydia trachomatis Development

Contact Info

Product

Resources

About