Mary Ko

The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo

Summary The function of the Mycobacterium tuberculosis eukaryotic‐like protein serine/threonine kinase PknG was investigated by gene knock‐out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C‐terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient‐depleted media. The PknG‐deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar‐containing molecules, GlcN‐Ins and mycothiol, which are derived from glutamate, were detected in the ΔpknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.

Mycothiol-Deficient Mycobacterium smegmatis Mutants Are Hypersensitive to Alkylating Agents, Free Radicals, and Antibiotics

ABSTRACT Mycothiol (MSH; 1d-myo-inosityl 2-[N-acetyl-l-cysteinyl]amido-2-deoxy-α-d-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc2155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1d-myo-inosityl 2-deoxy-α-d-glucopyranoside to levels 20- to 25-fold the level found in the parent strain. The cysteine:1d-myo-inosityl 2-amino-2-deoxy-α-d-glucopyranoside ligase (MshC) activities of the three mutant strains were ≤2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics.

Slow Release of Nitric Oxide from Charged Catheters and Its Effect on Biofilm Formation by Escherichia coli

ABSTRACT Catheter-associated urinary tract infection is the most prevalent cause of nosocomial infections. Bacteria associated with biofilm formation play a key role in the morbidity and pathogenesis of these infections. Nitric oxide (NO) is a naturally produced free radical with proven bactericidal effect. In this study, Foley urinary catheters were impregnated with gaseous NO. The catheters demonstrated slow release of nitric oxide over a 14-day period. The charged catheters were rendered antiseptic, and as such, were able to prevent bacterial colonization and biofilm formation on their luminal and exterior surfaces. In addition, we observed that NO-impregnated catheters were able to inhibit the growth of Escherichia coli within the surrounding media, demonstrating the ability to eradicate a bacterial concentration of up to 104 CFU/ml.

Signalling Inhibitors Against Mycobacterium tuberculosis – Early Days of a New Therapeutic Concept in Tuberculosis

Tuberculosis causes nearly two million deaths per year world-wide. In addition multidrug-resistant mycobacterial strains rapidly emerge so novel therapeutic approaches are needed. Recently, several promising mycobacterial target molecules were identified, which are involved in bacterial or host cell signalling e.g. the serine/threonine protein kinases, PknB and PknG, NAD kinase and the NAD synthetase. Here we describe some early efforts in the development of novel signal transduction inhibitory anti-mycobacterial drugs using a multiple target approach, with special emphasis on the kinase inhibitory field. Initially, we are using the Nested Chemical Library (NCL) technology and pharmacophore modelling. A hit-finding library, consisting of approximately 19000 small molecules with a bias for prototypic kinase inhibitors from our NCL library and commercial sources was virtually screened against these validated target molecules. Protein structures for the virtual screening were taken from the published three dimensional crystal structures of the enzymes. The hits from the virtual screening were subsequently tested in enzymatic assay systems. Potent hits were then tested for biological activity in macrophages, infected with mycobacteria. The final goal of this exercise is not only to identify potent anti-mycobacterial substances, but also a common pharmacophore for the mycobacterial target PknG in combination with PknB, NAD kinase and/or NAD synthetase. This common pharmacophore still needs to be a unique pharmacophore for the mycobacterial target proteins over human off-targets. Such a pharmacophore might then drive the optimization of a completely new profile of an antibiotic agent with activity against latent mycobacteria and resistance mycobacterial strains.

Comparative efficacy of commercially available and emerging antimicrobial urinary catheters against bacteriuria caused by E. coli in vitro.

OBJECTIVES To compare the efficacy of both commercially available and emerging urinary catheter technologies in relation to their effects on bacteriuria caused by Escherichia coli in vitro. Antiseptic urinary catheters have recently become commercially available and others are in the developmental stage. METHODS Silver alloy-coated catheters, antibiotic Nitrofurazone (NF)-coated catheters, and nitric oxide (NO)-coated catheters were tested against a noncoated control for their antiseptic ability. Inhibition of bacterial growth, biofilm formation, and the number of live bacteria within the biofilm, using up to 10(3) bacterial load were evaluated. Experiments were performed either in E. coli containing Luria broth media or in urine infected with E. coli. RESULTS NF- and NO-coated catheters had equivalent antimicrobial activity and eradicated all bacteria in planktonic and biofilm states. Silver-coated catheters had no effect on E. coli growth or biofilm formation compared with the control, although silver-coated catheters did inhibit bacterial levels within the biofilm by 50%. CONCLUSIONS NF- and NO-coated catheters are highly effective in preventing planktonic growth and biofilm formation. Silver-coated catheters were not found to be effective in this study.

Antimycobacterial activity of UDP-galactopyranose mutase inhibitors

The galactofuran region of the mycobacterial cell wall consists of alternating 5- and 6-linked beta-d-galactofuranose (beta-D-Galf) residues, essential for viability. UDP-galactofuranose (UDP-Galf), the donor for Galf, is synthesised from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (UGM), which is not found in humans, rendering it a therapeutic target. The in vitro properties, i.e. enzymatic activity, antimycobacterial activity, cellular toxicity, activity in mycobacterial-infected macrophages and activity against non-replicating persistent mycobacteria, of (4-chlorophenyl)-[1-(4-chlorophenyl)-3-hydroxy-5-methyl-1H-pyrazol-4-yl]-methanone and 3-(4-iodophenyl)-2-[4-(3,4-dichlorophenyl)-thiazol-2-ylamino]-propionic acid were studied. The former compound, a pyrazole, was an inhibitor of UGM from Mycobacterium tuberculosis and Klebsiella pneumoniae and was effective against Mycobacterium smegmatis, Mycobacterium bovis BCG and M. tuberculosis but ineffective against other bacterial strains tested. This compound showed potency against mycobacteria in infected macrophages but exhibited moderate cellular toxicity and was ineffective against non-replicating persistent mycobacteria. This is the first report of a compound both with UGM inhibitory properties and broad antimycobacterial activities. The latter compound, an aminothiazole, was active against UGM from K. pneumoniae and M. tuberculosis but was ineffective against M. bovis BCG or M. tuberculosis as well as demonstrating higher cellular toxicity. These data validate the choice of UGM as a target for active antimycobacterial therapy and confirm the pyrazole compound as a viable lead candidate.

Aminorifamycins and Sporalactams Produced in Culture by a Micromonospora sp. Isolated from a Northeastern-Pacific Marine Sediment Are Potent Antibiotics

The new ansa macrolide antibiotics 1 to 4 have been isolated from cultures of a Micromonospora sp. obtained from a marine sediment. Rifamycins 1 and 2 are the first natural ansa macrolides to have a 3-amino substituent. Sporalactams A (3) and B (4) are comprised of a heterocylic core 5 and a 14-membered ansa bridge that are both unprecedented. Sporalactam B (4) shows selective and potent inhibition of Mycobacterium tuberculosis.

Protein tyrosine kinase, PtkA, is required for Mycobacterium tuberculosis growth in macrophages

Protein phosphorylation plays a key role in Mycobacterium tuberculosis (Mtb) physiology and pathogenesis. We have previously shown that a secreted protein tyrosine phosphatase, PtpA, is essential for Mtb inhibition of host macrophage acidification and maturation, and is a substrate of the protein tyrosine kinase, PtkA, encoded in the same operon. In this study, we constructed a ∆ptkA deletion mutant in Mtb and found that the mutant exhibited impaired intracellular survival in the THP-1 macrophage infection model, correlated with the strain’s inability to inhibit macrophage phagosome acidification. By contrast, the mutant displayed increased resistance to oxidative stress in vitro. Proteomic and transcriptional analyses revealed upregulation of ptpA, and increased secretion of TrxB2, in the ΔptkA mutant. Kinase and protein-protein interaction studies demonstrated that TrxB2 is a substrate of PtkA phosphorylation. Taken together these studies establish a central role for the ptkA-ptpA operon in Mtb pathogenesis.