Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

ABSTRACT Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory...

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Autores principales:	Yu-Min Chuang, Nirmalya Bandyopadhyay, Dalin Rifat, Harvey Rubin, Joel S. Bader, Petros C. Karakousis
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a3187c98a97a4104bf853ed7623dc7ae
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spelling	oai:doaj.org-article:a3187c98a97a4104bf853ed7623dc7ae2021-11-15T15:41:34ZDeficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>10.1128/mBio.02428-142150-7511https://doaj.org/article/a3187c98a97a4104bf853ed7623dc7ae2015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02428-14https://doaj.org/toc/2150-7511ABSTRACT Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here that M. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identify M. tuberculosis Rv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found that M. tuberculosis poly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of the ppx2 knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role in M. tuberculosis growth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability. IMPORTANCE The stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediate Mycobacterium tuberculosis persistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in the ppx2 gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis.Yu-Min ChuangNirmalya BandyopadhyayDalin RifatHarvey RubinJoel S. BaderPetros C. KarakousisAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Yu-Min Chuang Nirmalya Bandyopadhyay Dalin Rifat Harvey Rubin Joel S. Bader Petros C. Karakousis Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
description	ABSTRACT Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here that M. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identify M. tuberculosis Rv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found that M. tuberculosis poly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of the ppx2 knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role in M. tuberculosis growth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability. IMPORTANCE The stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediate Mycobacterium tuberculosis persistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in the ppx2 gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis.
format	article
author	Yu-Min Chuang Nirmalya Bandyopadhyay Dalin Rifat Harvey Rubin Joel S. Bader Petros C. Karakousis
author_facet	Yu-Min Chuang Nirmalya Bandyopadhyay Dalin Rifat Harvey Rubin Joel S. Bader Petros C. Karakousis
author_sort	Yu-Min Chuang
title	Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_short	Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full	Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_fullStr	Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full_unstemmed	Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_sort	deficiency of the novel exopolyphosphatase rv1026/ppx2 leads to metabolic downshift and altered cell wall permeability in <named-content content-type="genus-species">mycobacterium tuberculosis</named-content>
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/a3187c98a97a4104bf853ed7623dc7ae
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Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

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