ClgR regulation of chaperone and protease systems is essential for Mycobacterium tuberculosis parasitism of the macrophage
Megan Estorninho1, Hilde Smith2, Jelle Thole2, Jose Harders-Westerveen2, Andrzej Kierzek1, Rachel E. Butler1, Olivier Neyrolles3, Graham R. Stewart1
Affiliations: 1 1Division of Microbial Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, UK 2 2Central Veterinary Institute of Wageningen UR, Division of Infectious Diseases, Animal Sciences Group, Edelhertweg 15, 8219 PH Lelystad, The Netherlands 3 3Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique and Université Paul Sabatier (UMR 5089), 205 Route de Narbonne, 31000 Toulouse, France
CorrespondenceGraham R. Stewart
[email protected]
First Published: 01 November 2010 https://doi.org/10.1099/mic.0.042275-0
Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the clp gene regulator (ClgR) of Mycobacterium tuberculosis activates the transcription of at least ten genes, including four that encode protease systems (ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, ClpB and the chaperonin Rv3269). Thus, M. tuberculosis ClgR controls a larger network of protein homeostatic and regulatory systems than ClgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for M. tuberculosis to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as M. tuberculosis lacking ClgR is deficient in the ability to control phagosome pH 1 h post-phagocytosis.
Keyword(s): CDS, coding sequences , ClgR, Clp gene regulator , EMSA, electrophoretic mobility shift assay , FAM, 5-carbofluorescein , GST, glutathione S-transferase , LC, liquid chromatography , pfp, percentage of false positives , qRT-PCR, quantitative real-time PCR and TAMRA, N,N,N′,N′-tetramethyl-6-carboxyrhodamine
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Microbiology 156, 3445 (2010); https://doi.org/10.1099/mic.0.042275-0
Supplementary Fig. S2. Expression ratios of clpC1 , clpP1 and acr2 compared by microarray and qRT-PCR. Supplementary Figs [PDF]
Supplementary Table. S4. Primers and probes used for quantitative real-time PCR of gene targets. [PDF]
Supplementary Table. S1. Downregulated genes in the Δ clgR mutant M. tuberculosis versus wild-type M. tuberculosis . [Excel file]
Supplementary Table. S2. Transcription of genes in complemented Δ clgR mutant versus wild-type M. tuberculosis . [Excel file]
Supplementary Table. S3. LC-MS/MS proteomic data of Δ clgR mutant versus wild-type M. tuberculosis . [Excel file]
10.1099/mic.0.042275-0