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Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis mutants.

ABSTRACT: The pathogenic Neisseria species accumulate polyphosphate to levels between 10 and 20% of their total phosphate content. However, the significance of this compound for the growth and pathogenicity of these species is not understood. A previous report (C.R. Tinsley, B.N. Manjula, and E.C. Gotschlich, Infect. Immun. 61:3703-3710, 1993) describes the purification of polyphosphate kinase, the enzyme responsible for synthesis of polyphosphate, from Neisseria meningitidis BNCV. By use of probes based on the amino acid sequence of the purified enzyme, the structural gene ppk has been cloned and sequenced. The coding sequence is 2,055 bp long and codes for a protein of 77.2 kDa. The open reading frame of the cloned gene was interrupted by the insertion of a kanamycin resistance cassette, and ppk mutants were obtained in both Neisseria gonorrhoeae and N. meningitidis by transformation with the recombinant plasmid. Amounts of polyphosphate in the ppk mutants were reduced to between 2 and 10% of wild-type levels. The mutants grew less vigorously than wild-type organisms in vitro and showed a striking increase in sensitivity to killing by human serum.

SUBMITTER: Tinsley CR

PROVIDER: S-EPMC173201 | biostudies-other | 1995 May

REPOSITORIES: biostudies-other

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Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis mutants.

Tinsley C R CR Gotschlich E C EC

Infection and immunity 19950501 5

The pathogenic Neisseria species accumulate polyphosphate to levels between 10 and 20% of their total phosphate content. However, the significance of this compound for the growth and pathogenicity of these species is not understood. A previous report (C.R. Tinsley, B.N. Manjula, and E.C. Gotschlich, Infect. Immun. 61:3703-3710, 1993) describes the purification of polyphosphate kinase, the enzyme responsible for synthesis of polyphosphate, from Neisseria meningitidis BNCV. By use of probes based ...[more]

PMID: 7729865

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Project description:The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90 degrees C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95 degrees C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.

Project description:The Mycobacterium tuberculosis gene Rv3232c/MT3329 (ppk2) encodes a class II polyphosphate kinase, which hydrolyzes inorganic polyphosphate (poly P) to synthesize GTP. We assessed the role of ppk2 in M. tuberculosis poly P regulation, antibiotic tolerance, and virulence. A ppk2-deficient mutant (ppk2::Tn) and its isogenic wild-type (WT) and complemented (Comp) strains were studied. For each strain, the intrabacillary poly P content, 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 M. tuberculosis virulence was assessed in the murine model. The ppk2::Tn mutant was found to have significantly increased poly P content and a 4-fold increase in the MIC of isoniazid relative to the WT and Comp strains. The ppk2::Tn mutant showed reduced survival at day 7 in activated and naive J774 macrophages relative to the WT. Naive ppk2::Tn mutant-infected macrophages showed increased expression of interleukin 2 (IL-2), IL-9, IL-10, IL-12p70, and gamma interferon (IFN-?) relative to WT-infected macrophages. The ppk2::Tn mutant exhibited significantly lower lung CFU during acute murine infection compared to the control groups. ppk2 is required for control of intrabacillary poly P levels and optimal M. tuberculosis growth and survival in macrophages and mouse lungs. IMPORTANCE Mycobacterium tuberculosis, 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. In this study, we found that the mycobacterial enzyme polyphosphate kinase 2 (PPK2) is required for controlling intracellular levels of important regulatory molecules and for maintaining susceptibility to the first-line anti-TB drug isoniazid. In addition, PPK2 was found to be required for M. tuberculosis growth in the lungs of mice, at least in part by suppressing the expression of certain key cytokines and chemokines by inactivated lung macrophages.

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Dataset Information

Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis mutants.

Publications

Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis mutants.

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