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

Grieshaber, Nicole A.; Runac, Justin; Turner, Sierra; Dean, Marissa; Appa, Cody; Omsland, Anders; Grieshaber, Scott S.

doi:10.3389/fcimb.2021.692224

Cited by 7 publications

(15 citation statements)

References 51 publications

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“…The swarmer cell in the case of C. crescentus is non replicating and is out of the cell cycle (31). Our data support a similar role for the EB cell as the EB disseminates the infection, does not replicate and is out of the cell cycle (20).…”

Section: Discussionsupporting

confidence: 78%

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Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division

Cherelli¹,

Grieshaber²,

Appa³

et al. 2022

Preprint

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Chlamydia trachomatis is an obligate intracellular bacterium that progresses through an essential multi cell form developmental cycle. Infection of the host is initiated by the elementary body (EB). Once in the host, the EB cell differentiates into the non-infectious, but replication competent, reticulate body, or RB. After multiple rounds of replication, RBs undergo secondary differentiation eventually producing newly infectious EBs. Here we generated paired cell type promoter reporter constructs and determined the kinetics of the activities of the euo, hctA and hctB promoters. The paired constructs revealed that the developmental cycle produces at least three phenotypically distinct cell types; the RB (euoprom+), IB (intermediate body, hctAprom+) and EB (hctBprom+). The kinetic data from the three dual promoter constructs, was used to generate two computational agent-based models to reproduce the chlamydial developmental cycle. Both models simulated EB germination, RB amplification, IB formation and EB production but differed in the mechanism that generated the IB. The Direct Conversion and the Asymmetric Production models predicted different behaviors for the RB population which were experimentally testable. In agreement with the Asymmetric Production model, RBs acted as stem cells after the initial amplification stage, producing one IB and self renewing after every division. We also demonstrated that IBs are a transient cell population, maturing directly into EBs after formation without the need for cell division. The culmination of these results suggests that the developmental cycle can be described by a four stage model, EB germination, RB amplification/maturation, IB production, and EB formation.

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Section: Discussionsupporting

confidence: 78%

“…This index is a ratio of sequencing coverage near the origin of replication vs coverage near the terminus and represents the growth rate of the population. An RB replication index of 1.5 demonstrates the presence of partially replicated chromosomes (20).…”

Section: Resultsmentioning

confidence: 99%

Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division

Cherelli¹,

Grieshaber²,

Appa³

et al. 2022

Preprint

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“…In addition this induction was dose responsive providing an excellent tool for the control of ectopic gene expression. We recently published the use of the T5prom-E riboswitch for the ectopic expression of three chlamydial proteins, dbdA, hctA, and scc1 demonstrating its utility in dissecting the function of chlamydial proteins [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

“…Transformation of Ctr L2 was performed essentially as previously described [ 17 ]. Briefly, 1x10 8 EBs + >2μg DNA/well were used to infect a 6 well plate.…”

Section: Methodsmentioning

confidence: 99%

Translational gene expression control in Chlamydia trachomatis

et al. 2022

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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.

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“…Transformation of Ctr L2 was performed essentially as previously described [38]. Briefly, 1x10 8 EBs + >2µg DNA/well were used to infect a well plate.…”

Section: Introductionmentioning

confidence: 99%

Translational gene expression control in Chlamydia trachomatis

Grieshaber

Chiarelli

Appa

et al. 2021

Preprint

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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 pgp4plasmid 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

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

Cited by 7 publications

References 51 publications

Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division

Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division

Translational gene expression control in Chlamydia trachomatis

Translational gene expression control in Chlamydia trachomatis

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