Exploring the mechanisms of chlamydial cell division, peptidoglycan turnover, and persistence
Exploring the mechanisms of chlamydial cell division, peptidoglycan turnover, and persistence
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DissertationDate of Exam
27.03.2025Date of Publication
29.04.2025Advisor
Schneider, TanjaCo-Referee
Dahl, ChristianeMetadata
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Abstract
The obligate intracellular pathogen C. trachomatis is an important human pathogen and retained only a minimal, yet sufficient biosynthetic pathway needed for peptidoglycan biosynthesis. The genome-reduced organism divides by a unique mechanism which depends on the synthesis of a PGN ring at the division site and requires constant remodeling and recycling of the PGN structure. This work aimed to provide novel insights on the role that PGN amidase AmiA plays in the tightly connected interplay of PGN synthesis, cell division, PGN turnover and recycling in the major human pathogen C. trachomatis.
Functional conservation of AmiA activity in C. trachomatis was confirmed by means of cell lysis and complementation experiments employing surrogate host systems. Moreover, amidase activity on PGN and precursor molecule lipid II could be validated in in vitro analyses with the purified enzyme. Experiments revealed a differential active site functional composition compared to the AmiA homolog of the closely related organism C. pneumoniae and, in contrast to C. pneumoniae AmiA, only monofunctional amidase activity. PGN binding affinity of C. trachomatis AmiA was shown to be independent of enzymatic activity. In vitro, as expected for a zinc-coordinating enzyme, the enzyme was shown to be inhibited by chelators of bivalent cations such as the antibiotic clioquinol in a pH dependent manner. Reaction products of amidase activity are likely recycled by the chlamydial homolog of NlpC/P60 domain-containing enzyme YkfC, for which the substrate spectrum was further specified as a part of this work.
Chelators were also used in tissue culture-based infection models and proved to induce nutrient starvation-induced persistence in C. trachomatis. Thereby, novel opportunities of inducing nutrient starvation and concomitant persistence in Chlamydia, equal to state of the art compound 2,2-bipyridyl, were identified. Leveraging CRISPRi as a novel tool of genetic manipulation of Chlamydia, knockdown of amiA led to an impaired cell division phenotype and visibly distorted PGN localization and metabolism. These results highlight the role AmiA plays in the closed-loop system of PGN biosynthesis, remodeling and recycling in Chlamydia already in the early stages of cell division. In contrast, the cell division amidase AmiA of organism E. coli mediates cell separation in the final stages.
Furthermore, CRISPRi was employed to investigate RodA and NamZ homologs in Chlamydia, providing first evidence on the important role of SEDS protein RodA within chlamydial cell division. Knockdown of namZ yielded a phenotype alluding to inhibited cell division and indicated that C. trachomatis NamZ, the homolog of a exo-β-N-acetylmuramidase in B. subtilis is contributing to the interconnected chlamydial PGN metabolism, likely by performing recycling processes. Also overexpression of rodA and namZ in C. trachomatis led to distinct phenotypes with chlamydial development and division processes being severely impaired, emphasizing the impact these enzymes have on Chlamydia.
All in all, this work provides new insights into the tightly interlinked processes comprised within the chlamydial PGN metabolism and contributes to the characterization of enzymes involved in PGN ring biosynthesis and degradation and cell division in a genetically streamlined and optimized organism.
Functional conservation of AmiA activity in C. trachomatis was confirmed by means of cell lysis and complementation experiments employing surrogate host systems. Moreover, amidase activity on PGN and precursor molecule lipid II could be validated in in vitro analyses with the purified enzyme. Experiments revealed a differential active site functional composition compared to the AmiA homolog of the closely related organism C. pneumoniae and, in contrast to C. pneumoniae AmiA, only monofunctional amidase activity. PGN binding affinity of C. trachomatis AmiA was shown to be independent of enzymatic activity. In vitro, as expected for a zinc-coordinating enzyme, the enzyme was shown to be inhibited by chelators of bivalent cations such as the antibiotic clioquinol in a pH dependent manner. Reaction products of amidase activity are likely recycled by the chlamydial homolog of NlpC/P60 domain-containing enzyme YkfC, for which the substrate spectrum was further specified as a part of this work.
Chelators were also used in tissue culture-based infection models and proved to induce nutrient starvation-induced persistence in C. trachomatis. Thereby, novel opportunities of inducing nutrient starvation and concomitant persistence in Chlamydia, equal to state of the art compound 2,2-bipyridyl, were identified. Leveraging CRISPRi as a novel tool of genetic manipulation of Chlamydia, knockdown of amiA led to an impaired cell division phenotype and visibly distorted PGN localization and metabolism. These results highlight the role AmiA plays in the closed-loop system of PGN biosynthesis, remodeling and recycling in Chlamydia already in the early stages of cell division. In contrast, the cell division amidase AmiA of organism E. coli mediates cell separation in the final stages.
Furthermore, CRISPRi was employed to investigate RodA and NamZ homologs in Chlamydia, providing first evidence on the important role of SEDS protein RodA within chlamydial cell division. Knockdown of namZ yielded a phenotype alluding to inhibited cell division and indicated that C. trachomatis NamZ, the homolog of a exo-β-N-acetylmuramidase in B. subtilis is contributing to the interconnected chlamydial PGN metabolism, likely by performing recycling processes. Also overexpression of rodA and namZ in C. trachomatis led to distinct phenotypes with chlamydial development and division processes being severely impaired, emphasizing the impact these enzymes have on Chlamydia.
All in all, this work provides new insights into the tightly interlinked processes comprised within the chlamydial PGN metabolism and contributes to the characterization of enzymes involved in PGN ring biosynthesis and degradation and cell division in a genetically streamlined and optimized organism.
Classification (DDC)
500 Naturwissenschaften 570 Biowissenschaften, BiologieRelated Publications
https://doi.org/10.1371/journal.ppat.1011047Dannenberg, Julia: Exploring the mechanisms of chlamydial cell division, peptidoglycan turnover, and persistence. - Bonn, 2025. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-82392
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-82392
@phdthesis{handle:20.500.11811/13028,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-82392,
author = {{Julia Dannenberg}},
title = {Exploring the mechanisms of chlamydial cell division, peptidoglycan turnover, and persistence},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2025,
month = apr,
note = {The obligate intracellular pathogen C. trachomatis is an important human pathogen and retained only a minimal, yet sufficient biosynthetic pathway needed for peptidoglycan biosynthesis. The genome-reduced organism divides by a unique mechanism which depends on the synthesis of a PGN ring at the division site and requires constant remodeling and recycling of the PGN structure. This work aimed to provide novel insights on the role that PGN amidase AmiA plays in the tightly connected interplay of PGN synthesis, cell division, PGN turnover and recycling in the major human pathogen C. trachomatis.
Functional conservation of AmiA activity in C. trachomatis was confirmed by means of cell lysis and complementation experiments employing surrogate host systems. Moreover, amidase activity on PGN and precursor molecule lipid II could be validated in in vitro analyses with the purified enzyme. Experiments revealed a differential active site functional composition compared to the AmiA homolog of the closely related organism C. pneumoniae and, in contrast to C. pneumoniae AmiA, only monofunctional amidase activity. PGN binding affinity of C. trachomatis AmiA was shown to be independent of enzymatic activity. In vitro, as expected for a zinc-coordinating enzyme, the enzyme was shown to be inhibited by chelators of bivalent cations such as the antibiotic clioquinol in a pH dependent manner. Reaction products of amidase activity are likely recycled by the chlamydial homolog of NlpC/P60 domain-containing enzyme YkfC, for which the substrate spectrum was further specified as a part of this work.
Chelators were also used in tissue culture-based infection models and proved to induce nutrient starvation-induced persistence in C. trachomatis. Thereby, novel opportunities of inducing nutrient starvation and concomitant persistence in Chlamydia, equal to state of the art compound 2,2-bipyridyl, were identified. Leveraging CRISPRi as a novel tool of genetic manipulation of Chlamydia, knockdown of amiA led to an impaired cell division phenotype and visibly distorted PGN localization and metabolism. These results highlight the role AmiA plays in the closed-loop system of PGN biosynthesis, remodeling and recycling in Chlamydia already in the early stages of cell division. In contrast, the cell division amidase AmiA of organism E. coli mediates cell separation in the final stages.
Furthermore, CRISPRi was employed to investigate RodA and NamZ homologs in Chlamydia, providing first evidence on the important role of SEDS protein RodA within chlamydial cell division. Knockdown of namZ yielded a phenotype alluding to inhibited cell division and indicated that C. trachomatis NamZ, the homolog of a exo-β-N-acetylmuramidase in B. subtilis is contributing to the interconnected chlamydial PGN metabolism, likely by performing recycling processes. Also overexpression of rodA and namZ in C. trachomatis led to distinct phenotypes with chlamydial development and division processes being severely impaired, emphasizing the impact these enzymes have on Chlamydia.
All in all, this work provides new insights into the tightly interlinked processes comprised within the chlamydial PGN metabolism and contributes to the characterization of enzymes involved in PGN ring biosynthesis and degradation and cell division in a genetically streamlined and optimized organism.},
url = {https://hdl.handle.net/20.500.11811/13028}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-82392,
author = {{Julia Dannenberg}},
title = {Exploring the mechanisms of chlamydial cell division, peptidoglycan turnover, and persistence},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2025,
month = apr,
note = {The obligate intracellular pathogen C. trachomatis is an important human pathogen and retained only a minimal, yet sufficient biosynthetic pathway needed for peptidoglycan biosynthesis. The genome-reduced organism divides by a unique mechanism which depends on the synthesis of a PGN ring at the division site and requires constant remodeling and recycling of the PGN structure. This work aimed to provide novel insights on the role that PGN amidase AmiA plays in the tightly connected interplay of PGN synthesis, cell division, PGN turnover and recycling in the major human pathogen C. trachomatis.
Functional conservation of AmiA activity in C. trachomatis was confirmed by means of cell lysis and complementation experiments employing surrogate host systems. Moreover, amidase activity on PGN and precursor molecule lipid II could be validated in in vitro analyses with the purified enzyme. Experiments revealed a differential active site functional composition compared to the AmiA homolog of the closely related organism C. pneumoniae and, in contrast to C. pneumoniae AmiA, only monofunctional amidase activity. PGN binding affinity of C. trachomatis AmiA was shown to be independent of enzymatic activity. In vitro, as expected for a zinc-coordinating enzyme, the enzyme was shown to be inhibited by chelators of bivalent cations such as the antibiotic clioquinol in a pH dependent manner. Reaction products of amidase activity are likely recycled by the chlamydial homolog of NlpC/P60 domain-containing enzyme YkfC, for which the substrate spectrum was further specified as a part of this work.
Chelators were also used in tissue culture-based infection models and proved to induce nutrient starvation-induced persistence in C. trachomatis. Thereby, novel opportunities of inducing nutrient starvation and concomitant persistence in Chlamydia, equal to state of the art compound 2,2-bipyridyl, were identified. Leveraging CRISPRi as a novel tool of genetic manipulation of Chlamydia, knockdown of amiA led to an impaired cell division phenotype and visibly distorted PGN localization and metabolism. These results highlight the role AmiA plays in the closed-loop system of PGN biosynthesis, remodeling and recycling in Chlamydia already in the early stages of cell division. In contrast, the cell division amidase AmiA of organism E. coli mediates cell separation in the final stages.
Furthermore, CRISPRi was employed to investigate RodA and NamZ homologs in Chlamydia, providing first evidence on the important role of SEDS protein RodA within chlamydial cell division. Knockdown of namZ yielded a phenotype alluding to inhibited cell division and indicated that C. trachomatis NamZ, the homolog of a exo-β-N-acetylmuramidase in B. subtilis is contributing to the interconnected chlamydial PGN metabolism, likely by performing recycling processes. Also overexpression of rodA and namZ in C. trachomatis led to distinct phenotypes with chlamydial development and division processes being severely impaired, emphasizing the impact these enzymes have on Chlamydia.
All in all, this work provides new insights into the tightly interlinked processes comprised within the chlamydial PGN metabolism and contributes to the characterization of enzymes involved in PGN ring biosynthesis and degradation and cell division in a genetically streamlined and optimized organism.},
url = {https://hdl.handle.net/20.500.11811/13028}
}
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