The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence

Abstract

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions.

Figure 1. Protein modeling predicts that M. tuberculosis MT0516 is an exopolyphosphatase.

The protein backbone ribbon structure was modeled by PHYRE , showing the conserved hydrolase fold associated with exopolyphosphatases. Left-angled, top-down view of the interface canyon between domains I and II with the α helix 4 (within box) that houses the highly conserved active site E112 side chain opening into the canyon floor. The canyon walls are lined with β sheets.

Figure 2. Complementation of transposon mutant MT0516::Tn.

A. Diagram of expected recombination between integrating plasmid and genomic DNA. B. PCR analysis of genomic DNA. A list of relevant primers is included in Table 1. Lane 1: Molecular weight markers. Lanes 2-4: Amplification of the MT0516 gene from wild-type, MT0516::Tn, and MT0516::Tn Comp, respectively. The expected 1035-bp PCR product is present in Lanes 2 and 4 but absent in Lane 3. Lane 5: Molecular weight markers. Lanes 6-8: Amplification of the kanamycin resistance cassette from wild type, MT0516::Tn and MT0516::Tn Comp, respectively, yielding the expected 226-bp product only in Lanes 7 and 8. Lane 9: Molecular weight markers. Lanes 10-11: Amplification of the hygromycin resistance cassette from MT0516::Tn and MT0516::Tn Comp, respectively, yielding the expected 319-bp product only in Lane 11. C. Diagram of expected sizes of EcoRI-digested genomic fragments expected to hybridize to probe recognizing region of MT0516 coding sequence by Southern blot (D). Note that MT0516::Tn Comp is expected to have two different size fragments hybridizing to the MT0516 probe, including that found in MT0516::Tn (1.8 Kb) and a distinct fragment unique to the attB integration site (1.7 Kb). D. Southern blot detecting the presence of DNA fragments bound to the MT0516 hybridization probe. Lane 1: Molecular weight markers. Lane 2: Blank. Lanes 3-5: Wild type, MT0516::Tn, and MT:0516::Tn Comp, respectively.

Figure 3. MT0516 deficiency results in persistently elevated intrabacillary levels of poly P and impaired growth in axenic cultures and macrophages.

A. Intrabacillary poly P content expressed as ng of poly P per µg of bacterial protein in each strain during different stages of the growth cycle in nutrient-rich broth (**p<0.01). B. RT-PCR analysis of gene expression in MT0516::Tn expressed as change in cycle threshold relative to that of the isogenic wild-type strain during mid-log phase growth in nutrient-rich broth. C. Growth of each strain in nutrient-rich 7H9 broth. D. Colony size of each strain on 7H10 solid media determined 28 days after plating. E. Growth and survival of each strain in THP-1 cells (**p<0.001). F. Growth and survival of each strain during exposure to progressive hypoxia in vitro (**p <0.001). CDC1551 =  wild type; MT0516::Tn =  MT0516-deficent mutant; MT0516::Tn Comp =  MT0516::Tn complement strain. CFU  =  colony-forming units. NRP-2 =  Nonreplicating persistence stage 2, as determined by color change of methylene blue dye.

Figure 4. MT0516 is required for full virulence of Mtb in guinea pigs.

A. Growth and survival of the mutant following low-dose aerosol infection in guinea pig lungs relative to wild-type and complement strains (*p≤0.01; **p≤0.001 for differences in bacillary counts between mutant- and wild-type-infected lungs). B. Gross pathology of Mtb-infected guinea pig lungs at Day 84 after aerosol infection. C. Microscopic examination of Mtb-infected lungs at Day 84 (H&E stain, 2× magnification. Insets: Ziehl-Neelsen staining, 50x magnification. CDC1551 =  wild-type strain; MT0516::Tn =  MT0516-deficent mutant; MT0516::Tn Comp =  MT0516::Tn complement strain.

Figure 5. Recombinant MT0516 exhibits concentration- and time-dependent exopolyphosphatase activity.

A. Polyacrylamide gel electrophoresis showing the fractions of recombinant His-tagged MT0516 protein (36.6 KDa), expressed in E.coli Arctic Express (DE3) BL 21. Lane 1: Molecular weight markers; Lanes 2-3: Supernatant and pellet, respectively, prior to Ni⁺⁺NTA column binding; Lanes 4-5: Elution fraction 1 and 2, respectively, eluted sequentially from Ni⁺⁺NTA column. B. Western blot using Penta-His antibody. Lane 1: Molecular weight markers. Lane 2: Elution fraction 2 after native purification demonstrating the expected size band (36.6 KDa). C. Exopolyphosphatase activity of recombinant MT0516 expressed as % hydrolysis of substrate (65-mer poly P) as a function of protein concentration (μg/ml). **p <0.001. D. Exopolyphosphatase activity of recombinant MT0516 expressed as % hydrolysis of substrate (65-mer poly P) as a function of time (hours). E =  elution fraction 2; AP =  alkaline phosphatase (positive control); NC =  negative control comprising supernatant obtained following IPTG induction of Arctic E coli transformed with empty vector. In each reaction, 1 μg/ml poly P was used as substrate. Data for Fig. 5C and 5D are derived from individual experiments, each of which was performed three times with separate samples yielding similar results.