Initial characterization of the two ClpP paralogs of chlamydia trachomatis suggests unique functionality for each

Nicholas A. Wood, Krystal Y. Chung, Amanda M. Blocker, Nathalia Rodrigues de Almeida, Martin Conda Sheridan, Derek J. Fisher, Scot P Ouellette

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX, clpC, two clpP paralogs, and clpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen. IMPORTANCE Chlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

Original languageEnglish (US)
Article numbere00635-18
JournalJournal of bacteriology
Volume201
Issue number2
DOIs
StatePublished - Jan 1 2019

Fingerprint

Chlamydia
Chlamydia trachomatis
Endopeptidase Clp
Bacteria
Proteins
Peptide Hydrolases
Protein Unfolding
Two-Hybrid System Techniques
Bacterial Proteins
Blindness
Sexually Transmitted Diseases
Computational Biology
Recombinant Proteins
Immunoblotting
Proteomics
Proteolysis
Genes
Adenosine Triphosphatases
Gels
Electrons

Keywords

  • Chlamydia
  • Clp protease
  • ClpP
  • Differentiation
  • Protein quality control
  • Protein turnover

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Initial characterization of the two ClpP paralogs of chlamydia trachomatis suggests unique functionality for each. / Wood, Nicholas A.; Chung, Krystal Y.; Blocker, Amanda M.; Rodrigues de Almeida, Nathalia; Conda Sheridan, Martin; Fisher, Derek J.; Ouellette, Scot P.

In: Journal of bacteriology, Vol. 201, No. 2, e00635-18, 01.01.2019.

Research output: Contribution to journalArticle

Wood, Nicholas A. ; Chung, Krystal Y. ; Blocker, Amanda M. ; Rodrigues de Almeida, Nathalia ; Conda Sheridan, Martin ; Fisher, Derek J. ; Ouellette, Scot P. / Initial characterization of the two ClpP paralogs of chlamydia trachomatis suggests unique functionality for each. In: Journal of bacteriology. 2019 ; Vol. 201, No. 2.
@article{f6b30ca5d2e0459499fd9468e6d4a76f,
title = "Initial characterization of the two ClpP paralogs of chlamydia trachomatis suggests unique functionality for each",
abstract = "Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX, clpC, two clpP paralogs, and clpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen. IMPORTANCE Chlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.",
keywords = "Chlamydia, Clp protease, ClpP, Differentiation, Protein quality control, Protein turnover",
author = "Wood, {Nicholas A.} and Chung, {Krystal Y.} and Blocker, {Amanda M.} and {Rodrigues de Almeida}, Nathalia and {Conda Sheridan}, Martin and Fisher, {Derek J.} and Ouellette, {Scot P}",
year = "2019",
month = "1",
day = "1",
doi = "10.1128/JB.00635-18",
language = "English (US)",
volume = "201",
journal = "Journal of Bacteriology",
issn = "0021-9193",
publisher = "American Society for Microbiology",
number = "2",

}

TY - JOUR

T1 - Initial characterization of the two ClpP paralogs of chlamydia trachomatis suggests unique functionality for each

AU - Wood, Nicholas A.

AU - Chung, Krystal Y.

AU - Blocker, Amanda M.

AU - Rodrigues de Almeida, Nathalia

AU - Conda Sheridan, Martin

AU - Fisher, Derek J.

AU - Ouellette, Scot P

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX, clpC, two clpP paralogs, and clpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen. IMPORTANCE Chlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

AB - Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX, clpC, two clpP paralogs, and clpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen. IMPORTANCE Chlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

KW - Chlamydia

KW - Clp protease

KW - ClpP

KW - Differentiation

KW - Protein quality control

KW - Protein turnover

UR - http://www.scopus.com/inward/record.url?scp=85058902556&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85058902556&partnerID=8YFLogxK

U2 - 10.1128/JB.00635-18

DO - 10.1128/JB.00635-18

M3 - Article

C2 - 30396899

AN - SCOPUS:85058902556

VL - 201

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 2

M1 - e00635-18

ER -