Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

Abstract

bInorganic polyphosphate (polyP), a linear polymer of hundreds of phosphate residues linked by ATP-like phosphoanhydride bonds, is found in all organisms and performs a wide variety of functions. This study shows that polyP accumulation occurs in Mycobacterium tuberculosis upon exposure to various stress conditions. M. tuberculosis possesses a single homolog of ppk-1, and we have disrupted ppk-1 in the M. tuberculosis genome by allelic replacement. The mutant strain exhibited negligible levels of intracellular polyP, decreased expression of sigF and phoP, and reduced growth in the stationary phase and displayed a survival defect in response to nitrosative stress and in THP-1 macrophages compared to the wild-type strain. We report that reduction in polyP levels is associated with increased susceptibility of M. tuberculosis to certain TB drugs and impairs its ability to cause disease in guinea pigs. These results suggest that polyP contributes to persistence of M. tuberculosis in vitro and plays an important role in the physiology of bacteria residing within guinea pigs.

MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

“…For histopathological analysis, liver and lung sections were fixed in 10% formalin and stained with hematoxylin and eosin. The extent of tissue damage in these sections was determined by calculating the total granuloma score and cellular infiltration as described previously 68 .…”

“…E. coli strains were cultured in Luria-Bertani broth or Luria-Bertani agar at 37°C, 200 r.p.m. Mycobacterial strains were cultured in MB7H9 supplemented with ADS (0.81 mg ml À 1 NaCl, 5 mg ml À 1 BSA fraction V, 2 mg ml À 1 glucose), 0.2% glycerol and 0.05% Tween-80 or minimal medium, or MB7H11 agar plates supplemented with OADS (ADS with 0.0006% v/v oleic acid) enrichment, as per standard protocols 68 . In this study, Mtb H 37 Rv and M. bovis BCG Danish were used as parental strains for the construction of various overexpression and mutant strains.…”

“…RT-PCR studies. The relative transcript levels of mazE3, mazF3, mazE6, mazF6, mazE9 and mazF9 were quantified at different growth stages or on exposure to various stress conditions or drugs as previously described 68 . For RT-PCR analysis, total mRNA was isolated using RNAeasy columns (Qiagen), treated with DNase I and complementary DNA was synthesized using Superscript III reverse transcriptase, according to standard protocols (Life Technologies).…”

“…The growth kinetics of various strains in either MB7H9 medium or minimal medium was determined by OD 600nm measurement. The susceptibility of the parental and mutant strains to oxidative and nitrosative stress was determined as described previously 68 . To understand the role of MazF toxins in the adaptation of Mtb to nutritional stress, various strains were grown until early-log phase, washed twice with 1 Â TBST and resuspended in TBST for either 7 or 14 days.…”

“…For RT-PCR analysis, total mRNA was isolated using RNAeasy columns (Qiagen), treated with DNase I and complementary DNA was synthesized using Superscript III reverse transcriptase, according to standard protocols (Life Technologies). The synthesized cDNA was subjected to PCR analysis using gene-specific primers as previously described 68 .…”