Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis
ABSTRACT Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetr...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-04-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.00075-23 |
| _version_ | 1797839615034392576 |
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| author | Stefan Pan Aaron A. Jensen Nicholas A. Wood Beate Henrichfreise Heike Brötz-Oesterhelt Derek J. Fisher Peter Sass Scot P. Ouellette |
| author_facet | Stefan Pan Aaron A. Jensen Nicholas A. Wood Beate Henrichfreise Heike Brötz-Oesterhelt Derek J. Fisher Peter Sass Scot P. Ouellette |
| author_sort | Stefan Pan |
| collection | DOAJ |
| description | ABSTRACT Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and cell culture approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated β-casein. Cell culture experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, these data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here, again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of antichlamydial agents. IMPORTANCE Chlamydia trachomatis is an obligate intracellular pathogen and the world's leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new antichlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for antichlamydial compounds. |
| first_indexed | 2024-04-09T15:59:41Z |
| format | Article |
| id | doaj.art-857aa81bf9db41a79d73ef9b1e32e438 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-09T15:59:41Z |
| publishDate | 2023-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-857aa81bf9db41a79d73ef9b1e32e4382023-04-25T13:04:57ZengAmerican Society for MicrobiologymBio2150-75112023-04-0114210.1128/mbio.00075-23Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatisStefan Pan0Aaron A. Jensen1Nicholas A. Wood2Beate Henrichfreise3Heike Brötz-Oesterhelt4Derek J. Fisher5Peter Sass6Scot P. Ouellette7Department of Microbial Bioactive Compounds, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, GermanyDepartment of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USADepartment of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USAInstitute for Pharmaceutical Microbiology, University of Bonn, Bonn, GermanyDepartment of Microbial Bioactive Compounds, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, GermanySchool of Biological Sciences, Southern Illinois University Carbondale, Carbondale, Illinois, USADepartment of Microbial Bioactive Compounds, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, GermanyDepartment of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USAABSTRACT Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and cell culture approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated β-casein. Cell culture experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, these data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here, again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of antichlamydial agents. IMPORTANCE Chlamydia trachomatis is an obligate intracellular pathogen and the world's leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new antichlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for antichlamydial compounds.https://journals.asm.org/doi/10.1128/mbio.00075-23ChlamydiaClpCClpPAAA+ ATPaseClp proteasedifferentiation |
| spellingShingle | Stefan Pan Aaron A. Jensen Nicholas A. Wood Beate Henrichfreise Heike Brötz-Oesterhelt Derek J. Fisher Peter Sass Scot P. Ouellette Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis mBio Chlamydia ClpC ClpP AAA+ ATPase Clp protease differentiation |
| title | Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis |
| title_full | Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis |
| title_fullStr | Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis |
| title_full_unstemmed | Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis |
| title_short | Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis |
| title_sort | molecular characterization of the clpc aaa atpase in the biology of chlamydia trachomatis |
| topic | Chlamydia ClpC ClpP AAA+ ATPase Clp protease differentiation |
| url | https://journals.asm.org/doi/10.1128/mbio.00075-23 |
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