DRUG TOLERANCE AND PERSISTENCE MEDIATED BY INORGANIC POLYPHOSPHATE IN MYCOBACTERIUM TUBERCULOSIS
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Date
2013-09-23
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Johns Hopkins University
Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a highly successful human pathogen because it has developed mechanisms to multiply and survive in the lungs by circumventing the immune system. Identification of virulence factors responsible for M. tuberculosis growth and persistence in host tissues may assist in the development of novel strategies to treat TB. Stringent response pathways in bacteria, including (p)ppGpp and inorganic polyphosphate (poly P) signals, are believed to play a regulatory role in persistence during infection. We found that poly P levels increase sharply in wild-type Mtb as cultures entered stationary phase and then return to baseline. Transient increases in intrabacillary poly P also temporally correlate with growth restriction during nutrition starvation, progressive hypoxia, and inorganic phosphate-depleted conditions. The role of polyphosphate kinase and exopolyphosphatase in Mtb poly P regulation, antibiotic tolerance, and virulence in this study was assessed.
The Mtb gene Rv3232c/MT3329 (ppk2) encodes a class II polyphosphate kinase, which hydrolyzes poly P to synthesize GTP. We assessed the role of ppk2 in Mtb poly P regulation, antibiotic tolerance, and virulence. A ppk2-deficient mutant (ppk2::Tn), and its isogenic wild-type (WT) and complement (Comp) strains were studied. For each strain, the intrabacillary poly P content, minimum inhibitory concentration (MIC) of isoniazid, and growth kinetics during infection of J774 macrophages were determined. Multiplex immunobead assays were used to evaluate cytokines elaborated during macrophage infection. The requirement of ppk2 for Mtb virulence was assessed in the murine model. ppk2::Tn was found to have significantly increased poly P content and a four-fold increase in MIC of isoniazid relative to WT and Comp. ppk2::Tn showed reduced survival at Day 7 in activated and naïve J774 macrophages relative to WT. Naïve ppk2::Tn-infected macrophages showed increased expression of IL-2, IL-9, IL-10, IL-12p70, and IFN-γ relative to WT-infected macrophages. ppk2::Tn exhibited significantly lower lung bacillary counts during acute murine infection compared to control groups. Therefore, ppk2 is required for control of intrabacillary poly P levels and optimal Mtb growth and survival in macrophages and mouse lungs.
Poly P is synthesized by polyphosphate kinase (PPK), and hydrolyzed by exopolyphosphatase (PPX). ppk1 deficiency in Mtb leads to reduced poly P synthesis and bacillary survival in THP-1 macrophages. In order to study the role of poly P accumulation in Mtb growth, stress tolerance, and susceptibility to antibiotics, we used a tetracycline-inducible expression system to generate an Mtb ppk1 knock-in strain, as well as a ppx knock-down strain by cloning the Rv1026 sequence in reverse orientation. Increased expression of PPK1 in the ppk1 knock-in strain and decreased expression of Rv1026 in the Rv1026 knock-down were confirmed by Western blots. The poly P accumulation strains, Rv1026 knock-down and ppk1 knock-in, showed a greater than 4-fold increase in the MIC of isoniazid as compared to the empty vector control strain. Growth of the ppk1 knock-in and ppx knock-down strains in axenic cultures was retarded during log-phase, which was correlated with increased intrabacillary poly P content in each strain. The ppk1 knock-in and ppx knock-down strains showed increased survival during heat challenge, and exposure to acid and surfactant. Mice infected with the Rv1026 knock-down exhibited relatively lower mean lung bacillary burdens by Day 14 relative to the control strain and continued growth until Day 42 post-infection. Isoniazid showed reduced efficacy in Rv1026 knock-down-infected mice relative to those infected with empty vector control after 7 days of treatment. Global gene expression analysis using RNA-seq revealed 51 down-regulated genes and 23 up-regulated genes in common between the two poly P-accumulating strains, Rv1026 knock-down and ppk1 knock-in. Significantly down-regulated genes included several related to protein synthesis, consistent with a negative regulatory role of poly P on the rate of protein synthesis.
In summary, poly P plays an important role in Mtb growth restriction, antibiotic tolerance, and survival during stress conditions, including in mouse lungs.
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Keywords
Mycobacterium tuberculosis, inorganic polyphosphate, drug tolerance, persistence, polyphosphate kinase, exopolyphosphatase