A Survey of Two-Component Systems in Coxiella burnetii Reveals Redundant Regulatory Schemes and a Requirement for an Atypical PhoBR System in Mammalian Cell Infection

Wachter, Shaun; Larson, Carl L.; Virtaneva, Kimmo; Kanakabandi, Kishore; Darwitz, Benjamin; Crews, Ben; Storrud, Keelee; Heinzen, Robert A.; Beare, Paul A.

doi:10.1128/jb.00416-22

Cited by 4 publications

(3 citation statements)

References 61 publications

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“…CRISPRi has provided inducible knockdown of gene expression and enabled genetic approaches to study essential gene function in several bacterial pathogens ( 51 – 53 ). Only a handful of studies have reported the use of CRISPRi in intracellular bacteria ( 22 , 54 , 55 ). We have demonstrated successful knockdown of topA in C. trachomatis using a plasmid-based CRISPRi system that relies on the combination of inducible dCas12 and a topA -specific crRNA ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Targeted repression of topA by CRISPRi reveals a critical function for balanced DNA topoisomerase I activity in the Chlamydia trachomatis developmental cycle

Shen,

Gao,

Swoboda

et al. 2024

mBio

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Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infection. 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 contributes to controlling 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 developmental cycle 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 developmental 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 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 developmental 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 mechanisms by which well-balanced topoisomerase functions in adaptation of C. trachomatis to unfavorable growth conditions imposed by antibiotics.

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Section: Discussionmentioning

confidence: 99%

Targeted repression of topA by CRISPRi reveals a critical function for balanced DNA topoisomerase I activity in the Chlamydia trachomatis developmental cycle

Shen,

Gao,

Swoboda

et al. 2024

mBio

View full text Add to dashboard Cite

show abstract

“…CRISPRi has provided inducible knockdown of gene expression and enabled genetic approaches to studying essential gene function in several bacterial pathogens (49-51). Only a handful of studies have reported the use of CRISPRi in intracellular bacteria (20, 52, 53). We have demonstrated successful knockdown of topA in C. trachomatis using a plasmid-based CRISPRi system that relies on the combination of inducible dCas12 and a topA -specific crRNA.…”

Section: Discussionmentioning

confidence: 99%

“…Together, these results imply disparities in the responses of C. trachomatis to the sub-MIC of Mox between L2/topA-kd, L2/Nt, and L2/topA-kd, suggesting that the levels of topA expression influences the chlamydial response to moxifloxacin. Only a handful of studies have reported the use of CRISPRi in intracellular bacteria (20,52,53) . We have demonstrated successful knockdown of topA in C. trachomatis using a plasmid-based CRISPRi system that relies on the combination of inducible dCas12 and a topA-specific crRNA.…”

Section: Targeted Topa Knockdown Affects the Response Of C Trachomati...mentioning

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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Coxiella burnetii protein CBU2016 supports CCV expansion

Thomas,

Garnish,

Khoo

et al. 2024

Pathogens and Disease

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Coxiella burnetii is a globally distributed obligate intracellular pathogen. Although often asymptomatic, infections can cause acute Q fever with influenza-like symptoms and/or severe chronic Q fever. C. burnetii develops a unique replicative niche within host cells called the Coxiella-containing vacuole (CCV), facilitated by the Dot/Icm type IV secretion system translocating a cohort of bacterial effector proteins into the host. The role of some effectors has been elucidated; however, the actions of the majority remain enigmatic and the list of true effectors is disputable. This study examined CBU2016, a unique C. burnetii protein previously designated as an effector with a role in infection. We were unable to validate CBU2016 as a translocated effector protein. Employing targeted knock-out and complemented strains, we found that the loss of CBU2016 did not cause a replication defect within Hela, THP-1, J774, or iBMDM cells or in axenic media, nor did it affect the pathogenicity of C. burnetii in the Galleria mellonella infection model. Absence of CBU2016 did, however, result in a consistent decrease in the size of CCVs in HeLa cells. These results suggest that although CBU2016 may not be a Dot/Icm effector, it is still able to influence the host environment during infection.

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A Survey of Two-Component Systems in Coxiella burnetii Reveals Redundant Regulatory Schemes and a Requirement for an Atypical PhoBR System in Mammalian Cell Infection

Cited by 4 publications

References 61 publications

Targeted repression of topA by CRISPRi reveals a critical function for balanced DNA topoisomerase I activity in the Chlamydia trachomatis developmental cycle

Targeted repression of topA by CRISPRi reveals a critical function for balanced DNA topoisomerase I activity in the Chlamydia trachomatis developmental cycle

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

Coxiella burnetii protein CBU2016 supports CCV expansion

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