Context-Dependent Action of Scc4 Reinforces Control of the Type III Secretion System

Gao, Leiqiong; Cong, Yanguang; Plano, Gregory V.; Rao, Xiancai; Gisclair, Lyndsey N.; Bartra, Sara Schesser; Macnaughtan, Megan A.; Shen, Li

doi:10.1128/jb.00132-20

Cited by 8 publications

(7 citation statements)

References 59 publications

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“…C. trachomatis infected HeLa cells were cultured in medium without or with aTC (at the concentration of 10 ng/mL or ~5nM) for 40 hrs. The small amount of aTC used did not have a significant inhibitory effect on the growth of C. trachomatis (20,22,25). Expression of dCas12 was examined by indirect immunofluorescence assay (IFA) in single cells and by immunoblotting analysis with the lysates of the cell population.…”

Section: Resultsmentioning

confidence: 99%

Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in theChlamydia trachomatisdevelopmental cycle

Shen

Gao

Swoboda

et al. 2023

Preprint

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Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infections. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases (Topos) contributes to Chlamydia developmental processes. Utilizing catalytically inactivated Cas12 (dCas12) based-clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomal topA transcription in C. trachomatis without detected toxicity of dCas12. Repression of topA impaired the growth of C. trachomatis mostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes of C. trachomatis was downregulated while early genes maintained their expression. Importantly, the growth defect associated with topA knockdown was rescued by overexpressing topA at an appropriate degree and time, directly linking the growth patterns to the levels of topA expression. Interestingly, topA knockdown had pleiotropic effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency. C. trachomatis with topA knocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential development and transcriptional processes of C. trachomatis. Importance: We used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential gene topA with a CRISPRi approach, using dCas12, in C. trachomatis indicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanism(s) by which well-balanced topoisomerase activities enable C. trachomatis to adapt to unfavorable growth conditions imposed by antibiotics.

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Section: Resultsmentioning

confidence: 99%

Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in theChlamydia trachomatisdevelopmental cycle

Shen

Gao

Swoboda

et al. 2023

Preprint

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“…The chaperone function of Scc4 occurs primarily in elemental bodies between the late stage of development and initiation of infection (when CopN is secreted into the host cell). The activity of Scc4 as a transcription factor occurs in reticulate bodies during the middle stage of development, when σ 66 -dependent transcription of housekeeping genes are active (Gao et al 2020). The stage-specific activity and multiple protein-protein interactions of Scc4 offer several pathways to approach drug development.…”

Section: Biological Contextmentioning

confidence: 99%

Backbone and sidechain resonance assignments and secondary structure of Scc4 from Chlamydia trachomatis

2020

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Chlamydia trachomatis is an obligate intracellular bacterium that causes the most common sexually transmitted bacterial diseases in the world. With a biphasic developmental cycle, the bacteria utilize a type III secretion system (T3SS) to invade host cells as infectious elemental bodies, which then differentiate into actively dividing reticulate bodies. The regulation of the developmental cycle and the T3SS are linked by the bi-functional protein, specific Chlamydia chaperone 4 (Scc4). Scc4 is a class I T3SS chaperone forming a heterodimer with specific Chlamydia chaperone 1 (Scc1) to chaperone the essential virulence effector, Chlamydia outer membrane protein N (CopN). Scc4 also functions as a transcription factor by binding to the RNA polymerase holoenzyme between the flap region of the β subunit and region 4 of σ 66 . In order to investigate the mechanism behind Scc4's dual functions and target its protein-protein interactions Terms of use and reuse: academic research for non-commercial purposes, see here for full terms. https://www.springer.com/aamterms-v1

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“…Scc4 switches between two functions that depend on different protein-protein interactions at specific stages of the developmental cycle with impacts on chlamydial development, CopN secretion, and selective gene expression [7]. A model of these protein-protein interactions is shown in Figure 1.…”

Section: Introductionmentioning

confidence: 99%

“…Scc4 regulates CT's σ 66 -dependent transcription by directly interacting with σ 66 region 4 and the flap tip domain of the β subunit of RNA polymerase (RNAP) [15]. Overexpression of Scc4 in CT accelerates the development of the inclusions by regulating T3SS gene expression [7]. During the middle to late stages of the cycle, Scc1 and CopN are expressed [16,17] (Figure 1, (iv)) and RBs redifferentiate to EBs.…”

Section: Introductionmentioning

confidence: 99%

“…The T3SS and transcriptional control of the developmental cycle are essential for infection, and Scc4 (specific chlamydia chaperone 4, formerly CT663, UniprotKB O84670) is a bi-functional protein with implications to both systems. Scc4 switches between two functions that depend on different protein-protein interactions at specific stages of the developmental cycle with impacts on chlamydial development, CopN secretion, and selective gene expression [7]. A model of these protein-protein interactions is shown in Figure 1.…”

Section: Introductionmentioning

confidence: 99%

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Chain-Selective Isotopic Labeling of the Heterodimeric Type III Secretion Chaperone, Scc4:Scc1, Reveals the Total Structural Rearrangement of the Chlamydia trachomatis Bi-Functional Protein, Scc4

et al. 2020

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Scc4 is an unusual bi-functional protein from Chlamydia trachomatis (CT) that functions as a type III secretion system (T3SS) chaperone and an RNA polymerase (RNAP)-binding protein. Both functions require interactions with protein partners during specific stages of the CT developmental cycle. As a T3SS chaperone, Scc4 binds Scc1 during the late stage of development to form a heterodimer complex, which chaperones the essential virulence effector, CopN. During the early-middle stage of development, Scc4 regulates T3SS gene expression by binding the σ66-containing RNAP holoenzyme. In order to study the structure and association mechanism of the Scc4:Scc1 T3SS chaperone complex using nuclear magnetic resonance (NMR) spectroscopy, we developed an approach to selectively label each chain of the Scc4:Scc1 complex with the 15N-isotope. The approach allowed one protein to be visible in the NMR spectrum at a time, which greatly reduced resonance overlap and permitted comparison of the backbone structures of free and bound Scc4. 1H,15N-heteronuclear single quantum coherence spectra of the 15N-Scc4:Scc1 and Scc4:15N-Scc1 complexes showed a total structural rearrangement of Scc4 upon binding Scc1 and a dynamic region isolated to Scc1, respectively. Development of the chain-selective labeling approach revealed that the association of Scc4 and Scc1 requires partial denaturation of Scc1 to form the high affinity complex, while low affinity interactions occurred between the isolated proteins under non-denaturing conditions. These results provide new models for Scc4′s functional switching mechanism and Scc4:Scc1 association in CT.

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Context-Dependent Action of Scc4 Reinforces Control of the Type III Secretion System

Cited by 8 publications

References 59 publications

Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in theChlamydia trachomatisdevelopmental cycle

Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in theChlamydia trachomatisdevelopmental cycle

Backbone and sidechain resonance assignments and secondary structure of Scc4 from Chlamydia trachomatis

Chain-Selective Isotopic Labeling of the Heterodimeric Type III Secretion Chaperone, Scc4:Scc1, Reveals the Total Structural Rearrangement of the Chlamydia trachomatis Bi-Functional Protein, Scc4

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