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Articles by Yossef Av-Gay in JoVE

Other articles by Yossef Av-Gay on PubMed

Expression and Localization of the Mycobacterium Tuberculosis Protein Tyrosine Phosphatase PtpA

Research in Microbiology. May, 2002  |  Pubmed ID: 12066895

The Mycobacterium tuberculosis open reading frame Rv2234 encodes a low molecular weight tyrosine phosphatase named PtpA. Kinetic analyses of PtpA activity reveal that it is capable of dephosphorylation of p-nitrophenyl phosphate, as well as a variety of phosphotyrosine-containing substrates. In contrast, PtpA showed no detectable activity towards substrates containing phosphoserine or -threonine residues. Transcriptional analysis reveals that the M. tuberculosis ptpA promoter is expressed in the slow-growing Mycobacterium species M. bovis BCG, but not in the fast-growing species M. smegmatis. Furthermore, ptpA expression is upregulated upon entry of BCG cultures into stationary phase and increases upon infection of human monocytes. We also show that, despite the lack of a general export pathway signal sequence, the M. tuberculosis PtpA protein can be released from both M. tuberculosis and M. smegmatis during growth.

Evidence That Plant-like Genes in Chlamydia Species Reflect an Ancestral Relationship Between Chlamydiaceae, Cyanobacteria, and the Chloroplast

Genome Research. Aug, 2002  |  Pubmed ID: 12176923

An unusually high proportion of proteins encoded in Chlamydia genomes are most similar to plant proteins, leading to proposals that a Chlamydia ancestor obtained genes from a plant or plant-like host organism by horizontal gene transfer. However, during an analysis of bacterial-eukaryotic protein similarities, we found that the vast majority of plant-like sequences in Chlamydia are most similar to plant proteins that are targeted to the chloroplast, an organelle derived from a cyanobacterium. We present further evidence suggesting that plant-like genes in Chlamydia, and other Chlamydiaceae, are likely a reflection of an unappreciated evolutionary relationship between the Chlamydiaceae and the cyanobacteria-chloroplast lineage. Further analyses of bacterial and eukaryotic genomes indicates the importance of evaluating organellar ancestry of eukaryotic proteins when identifying bacteria-eukaryote homologs or horizontal gene transfer and supports the proposal that Chlamydiaceae, which are obligate intracellular bacterial pathogens of animals, are not likely exchanging DNA with their hosts.

Mycothiol-deficient Mycobacterium Smegmatis Mutants Are Hypersensitive to Alkylating Agents, Free Radicals, and Antibiotics

Antimicrobial Agents and Chemotherapy. Nov, 2002  |  Pubmed ID: 12384335

Mycothiol (MSH; 1D-myo-inosityl 2-[N-acetyl-L-cysteinyl]amido-2-deoxy-alpha-D-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc(2)155 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-alpha-D-glucopyranoside to levels 20- to 25-fold the level found in the parent strain. The cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase (MshC) activities of the three mutant strains were < or =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.

Identification and Characterization of a Diamide Sensitive Mutant of Mycobacterium Smegmatis

FEMS Microbiology Letters. Mar, 2003  |  Pubmed ID: 12670676

A mutant, T7, highly sensitive to oxidative stress as caused by diamide was isolated from a Mycobacterium smegmatis mc(2)155 transposon mutant library. While wild-type M. smegmatis is able to grow well on solid media supplemented with 10 mM diamide, T7 is only able to grow on solid media containing up to 1 mM diamide. This mutant is also sensitive to other thiol modifying agents such as iodoacetamide and chlorodinitrobenzene. By sequencing the genomic DNA flanking the transposon, T7 was found to be mutated in the region upstream of the homolog of M. tuberculosis Rv0274 open reading frame. Sequence analysis revealed that Rv0274 is a member of a superfamily of metalloenzymes comprising enzymes such as extradiol dioxygenases, glyoxalases, and fosfomycin resistant glutathione transferases. Cloning and epichromosomal expression of M. tuberculosis Rv0274 in the mutant resulted in complementation of the sensitivity to diamide.

Inactivation of MshB, a Key Gene in the Mycothiol Biosynthesis Pathway in Mycobacterium Smegmatis

Microbiology (Reading, England). May, 2003  |  Pubmed ID: 12724395

The mshB gene encoding N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. Disruption of mshB in Mycobacterium smegmatis resulted in decreased production of mycothiol (5-10 % of the parent strain mc(2)155) but did not abolish mycothiol synthesis completely. Complementation of the MshB(-) mutants with the mshB gene resulted in increased mycothiol production towards the exponential and stationary phases of the bacterial growth cycle. These results suggest that another enzyme is capable of mycothiol biosynthesis by providing N-acetylglucosaminylinositol deacetylation activity in the absence of MshB. One of the candidate enzymes capable of carrying out such reactions is the MshB orthologue mycothiol amide hydrolase, MCA. However, epichromosomal expression of mca in the MshB(-) mutants did not restore mycothiol levels to the level of the parent strain. Unlike other mutants, which have little or no detectable levels of mycothiol, the MshB(-) mutant did not exhibit increased resistance to isoniazid. However, the MshB(-) mutant was resistant to ethionamide. Phenotypic analysis of other mutants lacking mycothiol revealed that MshA(-) mutants also exhibit ethionamide resistance but that a MshC(-)mutant was sensitive to ethionamide, suggesting that mycothiol or its early intermediates influence ethionamide activation.

The Glycosyltransferase Gene Encoding the Enzyme Catalyzing the First Step of Mycothiol Biosynthesis (mshA)

Journal of Bacteriology. Jun, 2003  |  Pubmed ID: 12754249

Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins). A transposon mutant of Mycobacterium smegmatis shown to be GlcNAc-Ins and mycothiol deficient was sequenced to identify a putative glycosyltransferase gene designated mshA. The ortholog in Mycobacterium tuberculosis, Rv0486, was used to complement the mutant phenotype.

The Crystal Structure of 1-D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside Deacetylase (MshB) from Mycobacterium Tuberculosis Reveals a Zinc Hydrolase with a Lactate Dehydrogenase Fold

The Journal of Biological Chemistry. Nov, 2003  |  Pubmed ID: 12958317

Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two beta-sheets and two alpha-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.

Kinome Analysis of Host Response to Mycobacterial Infection: a Novel Technique in Proteomics

Infection and Immunity. Oct, 2003  |  Pubmed ID: 14500469

An array of mammalian phospho-specific antibodies was used to screen for a host response upon mycobacterial infection, reflected as changes in host protein phosphorylation. Changes in the phosphorylation state of 31 known signaling molecules were tracked after infection with live or heat killed Mycobacterium bovis BCG or after incubation with the mycobacterial cell wall component lipoarabinomannan (LAM). Mycobacterial infection triggers a signaling cascade leading to activation of stress-activated protein kinase and its subsequent downstream target, c-Jun. Mycobacteria were also shown to inhibit the activation of protein kinase C epsilon and to induce phosphorylation of proteins not yet known to be involved in mycobacterial infection, such as the cytoskeletal protein alpha-adducin, glycogen synthase kinase 3beta, and a receptor subunit involved in regulation of intracellular Ca(2+) levels. The mycobacterial cell wall component LAM has been identified as a trigger for some of these modulation events.

Characterization of Mycobacterium Tuberculosis Mycothiol S-conjugate Amidase

Biochemistry. Oct, 2003  |  Pubmed ID: 14556638

Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the predominant thiol found in most actinomycetes. Mycothiol S-conjugate amidase (Mca) cleaves the amide bond of mycothiol S-conjugates of a variety of alkylating agents and xenobiotics, producing GlcN-Ins and a mercapturic acid that can be excreted from the cell. Mca of Mycobacterium tuberculosis (Rv1082) was cloned and expressed as a soluble protein in Escherichia coli. The protein contained 1.4 +/- 0.1 equiv of zinc after purification, indicating that Mca is a metalloprotein with zinc as the native metal. Kinetic studies of Mca activity with 14 substrates demonstrated that Mca is highly specific for the mycothiol moiety of mycothiol S-conjugates and relatively nonspecific for the structure of the sulfur-linked conjugate. The deacetylase activity of Mca with GlcNAc-Ins is small but significant and failed to saturate at up to 2 mM GlcNAc-Ins, indicating that Mca may contribute modestly to the production of GlcN-Ins when GlcNAc-Ins levels are high. The versatility of Mca can be seen in its ability to react with a broad range of mycothiol S-conjugates, including two different classes of antibiotics. The mycothiol S-conjugate of rifamycin S was produced under physiologically relevant conditions and was shown to be a substrate for Mca in both oxidized and reduced forms. Significant activity was also seen with the mycothiol S-conjugate of the antibiotic cerulenin as a substrate for Mca.

The Mycobacterium Tuberculosis Ino1 Gene is Essential for Growth and Virulence

Molecular Microbiology. Feb, 2004  |  Pubmed ID: 14763976

Inositol is utilized by Mycobacterium tuberculosis in the production of its major thiol and of essential cell wall lipoglycans. We have constructed a mutant lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyses the first committed step in inositol synthesis. This mutant is only viable in the presence of extremely high levels of inositol. Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in levels of mycothiol, but phosphatidylinositol mannoside, lipomannan and lipoarabinomannan levels were not altered. The ino1 mutant was attenuated in resting macrophages and in SCID mice. We used site-directed mutagenesis to alter four putative active site residues; all four alterations resulted in a loss of activity, and we demonstrated that a D310N mutation caused loss of the active site Zn2+ ion and a conformational change in the NAD+ cofactor.

Development of a Liposome Formulation of Ethambutol

Antimicrobial Agents and Chemotherapy. May, 2004  |  Pubmed ID: 15105152

A liposomal formulation capable of encapsulating 76 to 92% of the antimycobacterial drug ethambutol and showing prolonged in vitro release kinetics is described. In vitro efficacy is equivalent to that of the free drug, suggesting that encapsulation of ethambutol has the potential to shorten the current regimens for tuberculosis.

The Mycobacterium Tuberculosis Protein Serine/threonine Kinase PknG is Linked to Cellular Glutamate/glutamine Levels and is Important for Growth in Vivo

Molecular Microbiology. Jun, 2004  |  Pubmed ID: 15186418

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 DeltapknG 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.

Targeted Mutagenesis of the Mycobacterium Smegmatis Mca Gene, Encoding a Mycothiol-dependent Detoxification Protein

Journal of Bacteriology. Sep, 2004  |  Pubmed ID: 15342574

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.

Screening of Compounds Toxicity Against Human Monocytic Cell Line-THP-1 by Flow Cytometry

Biological Procedures Online. 2004  |  Pubmed ID: 15472722

The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI) exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death.

Thiol Specific Oxidative Stress Response in Mycobacteria

FEMS Microbiology Letters. Aug, 2005  |  Pubmed ID: 16006064

The cellular response of mycobacteria to thiol specific oxidative stress was studied in Mycobacterium bovis BCG cultures. Two-dimensional gel electrophoresis revealed that upon diamide treatment at least 60 proteins were upregulated. Fourteen of these proteins were identified by MALDI-MS; four proteins, AhpC, Tpx, GroEL2, and GroEL1 are functionally related to oxidative stress response; eight proteins, LeuC, LeuD, Rv0224c, Rv3029c, AsnB, Rv2971, PheA and HisH are classified as part of the bacterial intermediary metabolism and respiration pathways; protein EchA14 belong to lipid metabolism, and NrdE, belongs to the mycobacterial information pathway category. Reverse transcription followed by quantitative real time PCR in response to diamide stress demonstrated that protein expression is directly proportional to the corresponding gene transcription.

Mycobacterial Manipulation of the Host Cell

FEMS Microbiology Reviews. Nov, 2005  |  Pubmed ID: 16040149

Phagosome biogenesis, the process by which macrophages neutralize ingested pathogens and initiate antigen presentation, has entered the field of cellular mycobacteriology research largely owing to the discovery 30 years ago that phagosomes harboring mycobacteria are refractory to fusion with lysosomes. In the past decade, the use of molecular genetics and biology in different model systems to study phagosome biogenesis have made significant advances in understanding subtle mechanisms by which mycobacteria inhibit the maturation of its phagosome. Thus, we are beginning to appreciate the extent to which these pathogens are able to interfere with innate immune responses and manipulate defense mechanisms to enhance their survival within the human host cell. Here, we summarize current knowledge about phagosome maturation arrest in infected macrophages and the subsequent attenuation of the macrophage-initiated adaptive anti-mycobacterial immune defenses.

Deletion of the Mycobacterium Tuberculosis PknH Gene Confers a Higher Bacillary Load During the Chronic Phase of Infection in BALB/c Mice

Journal of Bacteriology. Aug, 2005  |  Pubmed ID: 16077122

The role of the serine/threonine kinase PknH in the physiology and virulence of Mycobacterium tuberculosis was assessed by the construction of a pknH deletion mutant. Deletion of the pknH gene did not affect sensitivity to the antimycobacterial drug ethambutol, although it was previously thought to be involved in regulating expression of emb genes encoding arabinosyl transferases, the targets of ethambutol. Nevertheless, transcription analyses revealed that genes associated with mycobacterial cell wall component synthesis, such as emb and ini operons, are downstream substrates of the PknH signaling cascade. In vitro survival studies revealed that a mutant with a deletion of the pknH gene displayed increased resistance to acidified nitrite stress, suggesting that nitric oxide is one of the potential environmental triggers for PknH activation. The effect of pknH deletion on mycobacterial virulence was investigated in BALB/c mice. In this model, the DeltapknH mutant was found to survive and replicate to a higher bacillary load in mouse organs than its parental strain and the pknH-complemented strain. In contrast, another closely related kinase mutant, the DeltapknE mutant, obtained from the same parental strain, was not affected in its virulence phenotype. Infection of THP-1 cells or in vitro growth studies in 7H9 medium did not reveal a significant in vitro growth advantage phenotype for the DeltapknH mutant. In conclusion, we propose that the serine/threonine kinase PknH plays a role in regulating bacillary load in mouse organs to facilitate adaptation to the host environment, possibly by enabling a regulated chronic infection by M. tuberculosis.

Mycobacterium Bovis BCG Attenuates Surface Expression of Mature Class II Molecules Through IL-10-dependent Inhibition of Cathepsin S

Journal of Immunology (Baltimore, Md. : 1950). Oct, 2005  |  Pubmed ID: 16210638

We have previously shown that macrophage infection with Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) partially inhibits MHC class II surface expression in response to IFN-gamma. The present study examined the nature of class II molecules that do in fact reach the surface of infected cells. Immunostaining with specific Abs that discriminate between mature and immature class II populations showed a predominance of invariant chain (Ii)-associated class II molecules at the surface of BCG-infected cells suggesting that mycobacteria specifically block the surface export of peptide-loaded class II molecules. This phenotype was due to inhibition of IFN-gamma-induced cathepsin S (Cat S) expression in infected cells and the subsequent intracellular accumulation of alphabeta class II dimers associated with the Cat S substrate Ii p10 fragment. In contrast, infection with BCG was shown to induce secretion of IL-10, and addition of blocking anti-IL-10 Abs to cell cultures restored both expression of active Cat S and export of mature class II molecules to the surface of infected cells. Consistent with these findings, expression of mature class II molecules was also restored in cells infected with BCG and transfected with active recombinant Cat S. Thus, M. bovis BCG exploits IL-10 induction to inhibit Cat S-dependent processing of Ii in human macrophages. This effect results in inhibition of peptide loading of class II molecules and in reduced presentation of mycobacterial peptides to CD4(+) T cells. This ability may represent an effective mycobacterial strategy for eluding immune surveillance and persisting in the host.

In Vitro Properties of Antimicrobial Bromotyrosine Alkaloids

Journal of Medical Microbiology. Apr, 2006  |  Pubmed ID: 16533988

A bromotyrosine alkaloid family of antimicrobial agents was synthesized using the known structure of a natural inhibitor of the mycobacterial mycothiol S-conjugate amidase (MCA) as a template. This series of compounds represents a novel class of anti-infective agents against Gram-positive pathogens, including mycobacteria and meticillin- and vancomycin-resistant Staphylococcus aureus. The fact that these compounds are active against mycobacterial strains in which the MCA gene is deleted and against Gram-positive bacteria lacking mycothiol suggests the existence of an alternative target for these compounds. One member of this family, EXEG1706, was identified as the lead compound possessing low MICs (2.5-25 microg ml(-1)) for several clinical isolates, whilst having low toxicity for THP-1 monocytes and macrophages.

Purification and Characterization of Mycobacterium Tuberculosis 1D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside Deacetylase, MshB, a Mycothiol Biosynthetic Enzyme

Protein Expression and Purification. Jun, 2006  |  Pubmed ID: 16630724

Mycothiol (MSH, AcCys-GlcN-Ins) is the major low molecular weight thiol in actinomycetes and is essential for growth of Mycobacterium tuberculosis. MshB, the GlcNAc-Ins deacetylase, is a key enzyme in MSH biosynthesis. MshB from M. tuberculosis was cloned, expressed, purified, and its properties characterized. Values of k(cat) and K(m) for MshB were determined for the biological substrate, GlcNAc-Ins, and several other good substrates. The substrate specificity of MshB was compared to that of M. tuberculosis mycothiol S-conjugate amidase (Mca), a homologous enzyme having weak GlcNAc-Ins deacetylase activity. Both enzymes are metalloamidases with overlapping amidase activity toward mycothiol S-conjugates (AcCySR-GlcN-Ins). The Ins residue and hydrophobic R groups enhance the activity with both MshB and Mca, but changes in the acyl group attached to GlcN have opposite effects on the two enzymes.

Mycothiol-dependent Mycobacterial Response to Oxidative Stress

FEBS Letters. May, 2006  |  Pubmed ID: 16643903

The effect of exogenous oxidative stress on mycothiol (MSH) levels and redox balance was investigated in mycobacteria. Both the thiol-specific oxidant diamide and hydrogen peroxide induced up to 75% depletion of MSH to form the disulfide form, mycothione (MSSM), in Mycobacterium bovis BCG. In comparison, Mycobacterium smegmatis, a saprophytic mycobacterium, displays a greater tolerance towards these oxidants, reflected by the lack of fluxes in MSH levels and redox ratios upon oxidative stress treatments. The basal ratio of MSH to MSSM was established to be 50:1 in M. bovis BCG and 200:1 in M. smegmatis.

Mycobacterium Tuberculosis Transporter MmpL7 is a Potential Substrate for Kinase PknD

Biochemical and Biophysical Research Communications. Sep, 2006  |  Pubmed ID: 16879801

The Mycobacterium tuberculosis serine/threonine protein kinases are attractive potential drug targets, and protein kinase D (PknD) is particularly interesting, as it is autophosphorylated on 11 residues, binds proteins containing forkhead associated domains, and contains a beta-propeller motif that likely functions as an anchoring sensor domain. We created a pknD knockout of a clinical M. tuberculosis isolate, and found that on in vitro phosphorylation of cell wall fractions it lacked a family of phosphorylated polypeptides seen in the WT. Mass spectrometry identified the phosphorylated polypeptides as MmpL7, a transporter of the RND family. MmpL7 is essential for virulence, presumably because it transports polyketide virulence factors such as phthiocerol dimycocerosate (PDIM) to the cell wall. Phosphorylation of the MmpL family of transporters has not been previously described, but these results suggest that PknD, and perhaps other serine/threonine kinases, could regulate their critical role in the formation of the M. tuberculosis envelope.

Mycobacterium Avium Subsp. Paratuberculosis PtpA is an Endogenous Tyrosine Phosphatase Secreted During Infection

Infection and Immunity. Dec, 2006  |  Pubmed ID: 16982836

Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like protein kinase systems. The genome sequence of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of Mycobacterium avium subsp. paratuberculosis by dephosphorylating essential macrophage signaling and/or adaptor molecules.

Mycothiol-dependent Proteins in Actinomycetes

FEMS Microbiology Reviews. Apr, 2007  |  Pubmed ID: 17286835

The pseudodisaccharide mycothiol is present in millimolar levels as the dominant thiol in most species of Actinomycetales. The primary role of mycothiol is to maintain the intracellular redox homeostasis. As such, it acts as an electron acceptor/donor and serves as a cofactor in detoxification reactions for alkylating agents, free radicals and xenobiotics. In addition, like glutathione, mycothiol may be involved in catabolic processes with an essential role for growth on recalcitrant chemicals such as aromatic compounds. Following a little over a decade of research since the discovery of mycothiol in 1994, we summarize the current knowledge about the role of mycothiol as an enzyme cofactor and consider possible mycothiol-dependent enzymes.

Novel Substrates of Mycobacterium Tuberculosis PknH Ser/Thr Kinase

Biochemical and Biophysical Research Communications. Mar, 2007  |  Pubmed ID: 17286964

PknH Ser/Thr protein kinase of Mycobacterium tuberculosis controls the expression of a variety of cell wall related enzymes and regulates the in vivo growth in mice. Therefore, we predicted that the PknH kinase could phosphorylate several substrates controlling different metabolic and physiological pathways. Using a bioinformatic approach, we identified 40 potential substrates. Two substrates were shown to be phosphorylated by recombinant PknH kinase in vitro. Point mutation studies verified that substrates are phosphorylated at the in silico-predicted sites. Kinetic studies revealed a similar relative-phosphorylation rate (V(max)) of PknH towards two new substrates and the only previously known substrate, EmbR. Unlike the EmbR protein, the Rv0681 and DacB1 proteins do not contain an FHA domain and are possible participants of new signaling pathways mediated by the PknH kinase in M. tuberculosis.

Innate Protection of Mycobacterium Smegmatis Against the Antimicrobial Activity of Nitric Oxide is Provided by Mycothiol

Antimicrobial Agents and Chemotherapy. Sep, 2007  |  Pubmed ID: 17638697

Nitric oxide (NO) is an efficient antimicrobial agent. A role for mycothiol in protecting mycobacteria from nitrosative damage was revealed by showing that a Mycobacterium smegmatis mutant is sensitive to NO. A direct correlation between NO and mycothiol levels confirmed that mycothiol is important for protecting mycobacteria from NO attack.

Lipoamide Dehydrogenase Mediates Retention of Coronin-1 on BCG Vacuoles, Leading to Arrest in Phagosome Maturation

Journal of Cell Science. Aug, 2007  |  Pubmed ID: 17652161

Mycobacterium tuberculosis evades the innate antimicrobial defenses of macrophages by inhibiting the maturation of its phagosome to a bactericidal phagolysosome. Despite intense studies of the mycobacterial phagosome, the mechanism of mycobacterial persistence dependent on prolonged phagosomal retention of the coat protein coronin-1 is still unclear. The present study demonstrated that several mycobacterial proteins traffic intracellularly in M. bovis BCG-infected cells and that one of them, with an apparent subunit size of M(r) 50,000, actively retains coronin-1 on the phagosomal membrane. This protein was initially termed coronin-interacting protein (CIP)50 and was shown to be also expressed by M. tuberculosis but not by the non-pathogenic species M. smegmatis. Cell-free system experiments using a GST-coronin-1 construct showed that binding of CIP50 to coronin-1 required cholesterol. Thereafter, mass spectrometry sequencing identified mycobacterial lipoamide dehydrogenase C (LpdC) as a coronin-1 binding protein. M. smegmatis over-expressing Mtb LpdC protein acquired the capacity to maintain coronin-1 on the phagosomal membrane and this prolonged its survival within the macrophage. Importantly, IFNgamma-induced phagolysosome fusion in cells infected with BCG resulted in the dissociation of the LpdC-coronin-1 complex by a mechanism dependent, at least in part, on IFNgamma-induced LRG-47 expression. These findings provide further support for the relevance of the LpdC-coronin-1 interaction in phagosome maturation arrest.

Comparative Analysis of Mutants in the Mycothiol Biosynthesis Pathway in Mycobacterium Smegmatis

Biochemical and Biophysical Research Communications. Nov, 2007  |  Pubmed ID: 17826740

The role of mycothiol in mycobacteria was examined by comparative analysis of mutants disrupted in the four known genes encoding the protein machinery needed for mycothiol biosynthesis. These mutants were sensitive to acid stress, antibiotic stress, alkylating stress, and oxidative stress indicating that mycothiol and mycothiol-dependent enzymes protect the mycobacterial cell against attack from various different types of stresses and toxic agents.

Mycobacterium Bovis Bacillus Calmette-Guérin Secreting Active Cathepsin S Stimulates Expression of Mature MHC Class II Molecules and Antigen Presentation in Human Macrophages

Journal of Immunology (Baltimore, Md. : 1950). Oct, 2007  |  Pubmed ID: 17911599

A successful Th cell response to bacterial infections is induced by mature MHC class II molecules presenting specific Ag peptides on the surface of macrophages. In recent studies, we demonstrated that infection with the conventional vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) specifically blocks the surface export of mature class II molecules in human macrophages by a mechanism dependent on inhibition of cathepsin S (Cat S) expression. The present study examined class II expression in macrophages infected with a rBCG strain engineered to express and secrete biologically active human Cat S (rBCG-hcs). Cat S activity was completely restored in cells ingesting rBCG-hcs, which secreted substantial levels of Cat S intracellularly. Thus, infection with rBCG-hcs, but not parental BCG, restored surface expression of mature MHC class II molecules in response to IFN-gamma, presumably as result of MHC class II invariant chain degradation dependent on active Cat S secreted by the bacterium. These events correlated with increased class II-directed presentation of mycobacterial Ag85B to a specific CD4(+) T cell hybridoma by rBCG-hcs-infected macrophages. Consistent with these findings, rBCG-hcs was found to accelerate the fusion of its phagosome with lysosomes, a process that optimizes Ag processing in infected macrophages. These data demonstrated that intracellular restoration of Cat S activity improves the capacity of BCG-infected macrophages to stimulate CD4(+) Th cells. Given that Th cells play a major role in protection against tuberculosis, rBCG-hcs would be a valuable tuberculosis vaccine candidate.

Mycobacterium Smegmatis Biofilm Formation and Sliding Motility Are Affected by the Serine/threonine Protein Kinase PknF

FEMS Microbiology Letters. Jan, 2008  |  Pubmed ID: 18031532

Eighteen 'eukaryotic-like' serine/threonine kinases are present in the Mycobacterium smegmatis genome. One of them encoded by the ORF 3677 demonstrates high similarity to the Mycobacterium tuberculosis protein kinase PknF. A merodiploid strain was generated, which showed reduced growth associated with irregular cell structure. The merodiploid strain displayed altered colony morphology, defective sliding motility and biofilm formation. These data indicate a role for PknF in biofilm formation, possibly associated with alterations in glycopeptidolipid composition.

Molecular Cloning and Biochemical Characterization of a Serine Threonine Protein Kinase, PknL, from Mycobacterium Tuberculosis

Protein Expression and Purification. Apr, 2008  |  Pubmed ID: 18276158

PknL, a eukaryotic like serine threonine protein kinase from Mycobacterium tuberculosis, is predicted to be involved in transcriptional regulation and cell division. Attempts to clone and over-express the protein in Escherichia coli using pET43.1c as the vector were unsuccessful. The fusion protein was expressed as a truncated product and showed feeble autokinase activity. To overcome this technical glitch, the pknL ORF was cloned into a mycobacterial expression vector, pALACE and the purified His-tagged protein was evaluated for autokinase activity. Phosphorylation experiments with exogenous substrates like myelin basic protein (MBP) were performed. For the fast identification of protein phosphorylation sites, chromatographic methods of separating the [gamma-32P]phosphate radio labeled amino acids using thin-layer chromatography (TLC) on cellulose sheets was carried out. Thus, the activity of PknL was demonstrated using autophosphorylation and substrate phosphorylation experiments. Phospho amino acid determinations revealed that PknL was phosphorylated predominantly on serine and also on threonine residues. A single amino acid substitution of lysine to methionine in the active site completely abolished enzymatic action, thereby confirming the authenticity of the kinase function of the expressed protein.

Mycobacterium Tuberculosis Virulence is Mediated by PtpA Dephosphorylation of Human Vacuolar Protein Sorting 33B

Cell Host & Microbe. May, 2008  |  Pubmed ID: 18474358

Entry into host macrophages and evasion of intracellular destruction mechanisms, including phagosome-lysosome fusion, are critical elements of Mycobacterium tuberculosis (Mtb) pathogenesis. To achieve this, the Mtb genome encodes several proteins that modify host signaling pathways. PtpA, a low-molecular weight tyrosine phosphatase, is a secreted Mtb protein of unknown function. The lack of tyrosine kinases in the Mtb genome suggests that PtpA may modulate host tyrosine phosphorylated protein(s). We report that a genetic deletion of ptpA attenuates Mtb growth in human macrophages, and expression of PtpA-neutralizing antibodies simulated this effect. We identify VPS33B, a regulator of membrane fusion, as a PtpA substrate. VPS33B and PtpA colocalize in Mtb-infected human macrophages. PtpA secretion combined with active-phosphorylated VPS33B inhibited phagosome-lysosome fusion, a process arrested in Mtb infections. These results demonstrate that PtpA is essential for Mtb intracellular persistence and identify a key host regulatory pathway that is inactivated by Mtb.

Mycothiol Biosynthesis is Essential for Ethionamide Susceptibility in Mycobacterium Tuberculosis

Molecular Microbiology. Sep, 2008  |  Pubmed ID: 18651841

Spontaneous mutants of Mycobacterium tuberculosis that were resistant to the anti-tuberculosis drugs ethionamide and isoniazid were isolated and found to map to mshA, a gene encoding the first enzyme involved in the biosynthesis of mycothiol, a major low-molecular-weight thiol in M. tuberculosis. Seven independent missense or frameshift mutations within mshA were identified and characterized. Precise null deletion mutations of the mshA gene were generated by specialized transduction in three different strains of M. tuberculosis. The mshA deletion mutants were defective in mycothiol biosynthesis, were only ethionamide-resistant and required catalase to grow. Biochemical studies suggested that the mechanism of ethionamide resistance in mshA mutants was likely due to a defect in ethionamide activation. In vivo, a mycothiol-deficient strain grew normally in immunodeficient mice, but was slightly defective for growth in immunocompetent mice. Mutations in mshA demonstrate the non-essentiality of mycothiol for growth in vitro and in vivo, and provide a novel mechanism of ethionamide resistance in M. tuberculosis.

Gaseous Nitric Oxide Bactericidal Activity Retained During Intermittent High-dose Short Duration Exposure

Nitric Oxide : Biology and Chemistry. Feb, 2009  |  Pubmed ID: 18789393

Previously, we have shown that gaseous Nitric oxide (gNO) has great potential as an effective topical anti-infective agent for non-healing wounds due to its non-specific antimicrobial properties. These same antimicrobial attributes may be useful for pulmonary infections. However, gNO would have limited usefulness as an inhaled antimicrobial agent as continuous exposure to the concentration required for a bactericidal effect (160-200 ppm) leads to methemoglobinemia. To overcome this problem, we investigated whether a thirty minute exposure of 160 ppm every four hours would retain the same antimicrobial effect as continuous delivery. In vitro, exposure of clinical multi-drug resistant Staphylococcus aureus and Escherichia coli strains isolated from the lungs of nosocomial pneumonia patients and a lethal antibiotic-resistant strain of Pseudomonas aeruginosa, isolated from a deceased cystic fibrosis patient resulted in over a 5 log(10) reduction in bacterial load after multiple thirty minute treatments (4 cycles) every four hours to 160 ppm gNO. The intermittent regimen required 320 (SD=0)ppm h for 100% lethality whereas the continuous exposure required 800 (SD=160)ppm h. We have also shown that selection for a gNO resistant phenotype did not lead to decrease sensitivity to gNO therapy (p>0.05). In addition, no host cellular toxicity was observed in human THP-1 monocytes and macrophages following intermittent delivery of a high concentration of gNO, and the proliferation and migration of pulmonary epithelial cells was not adversely affected by the administration of intermittent high-dose gNO. These results justify further studies that should focus on whether intermittent delivery of 160 ppm of gNO every four hours can technically be administered while keeping inhaled NO(2) levels less than 2 ppm and methemoglobin saturation less than 2.5 percent.

The Serine/threonine Protein Kinase PknI Controls the Growth of Mycobacterium Tuberculosis Upon Infection

FEMS Microbiology Letters. Jun, 2009  |  Pubmed ID: 19341393

The protein kinase PknI is one of 11 functional serine/threonine protein kinases in Mycobacterium tuberculosis. Specialized transduction was performed to create a null mutant in the pknI gene. The resulting mutant was used to determine the role of PknI in M. tuberculosis growth and infectivity. The pknI mutant grows better under acidic pH and limited oxygen availability. We observed a modest increased growth of pknI mutant within macrophages during an in vitro infection and a hypervirulence phenotype in severe combined immunodeficiency mice. The internal signals used to activate PknI are most likely the host-associated signals such as low pH associated with limited oxygen availability. Thus, we have shown that PknI plays a role in sensing the host macrophage's environment and translating it to slow the growth of M. tuberculosis within the infected host.

Mycobacterium Tuberculosis PtkA is a Novel Protein Tyrosine Kinase Whose Substrate is PtpA

The Biochemical Journal. May, 2009  |  Pubmed ID: 19366344

In Mycobacterium tuberculosis, signal transduction is mediated by 11 serine/threonine kinases, but no tyrosine kinases have been identified thus far. The protein encoded by the ORF (open reading frame) Rv2232 has been annotated as a member of the HAD (haloacid dehydrogenase-like hydrolase) superfamily, which includes phosphatases, phosphomanno- and phosphogluco-mutases, and haloacid dehydrogenases. In the present paper, we report, on the basis of biochemical and mutational analyses, that the Rv2232-encoded protein, named protein tyrosine kinase A (PtkA) is a bona fide protein tyrosine kinase. The cognate substrate of PtkA is the secreted protein tyrosine phosphatase A (PtpA).

Glutathione Disulfide and S-nitrosoglutathione Detoxification by Mycobacterium Tuberculosis Thioredoxin System

FEBS Letters. Oct, 2009  |  Pubmed ID: 19737561

Mycobacterium tuberculosis resides within alveolar macrophages. These phagocytes produce reactive nitrogen and oxygen intermediates to combat the invading pathogens. The macrophage glutathione (GSH) pool reduces nitric oxide (NO) to S-nitrosoglutathione (GSNO). Both glutathione disulfide (GSSG) and GSNO possess mycobactericidal activities in vitro. In this study we demonstrate that M. tuberculosis thioredoxin system, comprises of thioredoxin reductase B2 and thioredoxin C reduces the oxidized form of the intracellular mycothiol (MSSM) and is able to efficiently reduce GSSG and GSNO in vitro. Our study suggests that the thioredoxin system provide a general reduction mechanism to cope with oxidative stress associated with the microbe's metabolism as well as to detoxify xenobiotics produced by the host.

Protein Kinase and Phosphatase Signaling in Mycobacterium Tuberculosis Physiology and Pathogenesis

Biochimica Et Biophysica Acta. Mar, 2010  |  Pubmed ID: 19766738

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), evades the antimicrobial defenses of the host and survives within the infected individual through a complex set of strategies. These include active prevention of host cellular killing processes as well as overwhelming adaptive gene expression. In the past decade, we have gained an increased understanding of how mycobacteria not only have the ability to adapt to a changing host environment but also actively interfere with the signaling machinery within the host cell to counteract or inhibit parts of the killing apparatus employed by the macrophage. Mtb is able to sense its environment via a set of phospho-signaling proteins which mediate its response and interaction with the host in a coordinated manner. In this review, we summarize the current knowledge about selected Mtb serine, threonine, and tyrosine kinase and phosphatase signaling proteins, focusing on the protein kinases, PknG and PtkA, and the protein phosphatase, PtpA.

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

Antimicrobial Agents and Chemotherapy. Jan, 2010  |  Pubmed ID: 19884372

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 10(4) CFU/ml.

Inositol Monophosphate Phosphatase Genes of Mycobacterium Tuberculosis

BMC Microbiology. 2010  |  Pubmed ID: 20167072

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.

Synthesis, Characterization, and Evaluation of Antimicrobial and Cytotoxic Effect of Silver and Titanium Nanoparticles

Nanomedicine : Nanotechnology, Biology, and Medicine. Oct, 2010  |  Pubmed ID: 20215045

Microbial resistance represents a challenge for the scientific community to develop new bioactive compounds. Nosocomial infections represent an enormous emerging problem, especially in patients with ambulatory treatment, which requires that they wear medical devices for an extended period of time. In this work, an evaluation of the antimicrobial activity of both silver and titanium nanoparticles was carried out against a panel of selected pathogenic and opportunistic microorganisms, some of them commonly associated with device-associated infections. Cytotoxicity assays monitoring DNA damage and cell viability were evaluated using human-derived monocyte cell lines. We show that silver-coated nanoparticles having a size of 20-25 nm were the most effective among all the nanoparticles assayed against the tested microorganisms. In addition, these nanoparticles showed no significant cytotoxicity, suggesting their use as antimicrobial additives in the process of fabrication of ambulatory and nonambulatory medical devices.

Inhibition of Mycobacterium Tuberculosis Tyrosine Phosphatase PtpA by Synthetic Chalcones: Kinetics, Molecular Modeling, Toxicity and Effect on Growth

Bioorganic & Medicinal Chemistry. Jun, 2010  |  Pubmed ID: 20462762

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes pi stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents.

Convergence of Ser/Thr and Two-component Signaling to Coordinate Expression of the Dormancy Regulon in Mycobacterium Tuberculosis

The Journal of Biological Chemistry. Sep, 2010  |  Pubmed ID: 20630871

Signal transduction in Mycobacterium tuberculosis is mediated primarily by the Ser/Thr protein kinases and the two-component systems. The Ser/Thr kinase PknH has been shown to regulate growth of M. tuberculosis in a mouse model and in response to NO stress in vitro. Comparison of a pknH deletion mutant (ΔpknH) with its parental M. tuberculosis H37Rv strain using iTRAQ enabled us to quantify >700 mycobacterial proteins. Among these, members of the hypoxia- and NO-inducible dormancy (DosR) regulon were disregulated in the ΔpknH mutant. Using kinase assays, protein-protein interactions, and mass spectrometry analysis, we demonstrated that the two-component response regulator DosR is a substrate of PknH. PknH phosphorylation of DosR mapped to Thr(198) and Thr(205) on the key regulatory helix α10 involved in activation and dimerization of DosR. PknH Thr phosphorylation and DosS Asp phosphorylation of DosR cooperatively enhanced DosR binding to cognate DNA sequences. Transcriptional analysis comparing ΔpknH and parental M. tuberculosis revealed that induction of the DosR regulon was subdued in the ΔpknH mutant in response to NO. Together, these results indicate that PknH phosphorylation of DosR is required for full induction of the DosR regulon and demonstrate convergence of the two major signal transduction systems for the first time in M. tuberculosis.

Antimycobacterial Activity of UDP-galactopyranose Mutase Inhibitors

International Journal of Antimicrobial Agents. Oct, 2010  |  Pubmed ID: 20678902

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.

MmpS4 Promotes Glycopeptidolipids Biosynthesis and Export in Mycobacterium Smegmatis

Molecular Microbiology. Nov, 2010  |  Pubmed ID: 21062372

The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram-negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single-chain antibodies against the surface-exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C-terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery.

Antibacterial and Cytotoxic Activities of the Sesquiterpene Lactones Cnicin and Onopordopicrin

Natural Product Communications. Feb, 2011  |  Pubmed ID: 21425665

The antimicrobial and cytotoxic activities of chloroform extracts from the weeds Centaurea tweediei and C. diffusa, and the main sesquiterpene lactones isolated from these species, onopordopicrin and cnicin, respectively, were assayed. Results show that the chloroform extracts from both Centaurea species possess antibacterial activities against a panel of Gram-positive and Gram-negative bacteria. Remarkable antibacterial activity against methicillin-resistant Staphylococcus aureus was also measured. Both the extracts and the purified sesquiterpene lactones show high cytotoxicity against human-derived macrophages. Despite this cytotoxicity, C. diffusa chloroform extract and cnicin are attractive candidates for evaluation as antibiotics in topical preparations against skin-associated pathogens.

Precise Null Deletion Mutations of the Mycothiol Synthesis Genes Reveal Their Role in Isoniazid and Ethionamide Resistance in Mycobacterium Smegmatis

Antimicrobial Agents and Chemotherapy. Jul, 2011  |  Pubmed ID: 21502624

Mycothiol (MSH; AcCys-GlcN-Ins) is the glutathione analogue for mycobacteria. Mutations in MSH biosynthetic genes have been associated with resistance to isoniazid (INH) and ethionamide (ETH) in mycobacteria, but rigorous genetic studies are lacking, and those that have been conducted have yielded different results. In this study, we constructed independent null deletion mutants for all four genes involved in the MSH biosynthesis pathway (mshA, mshB, mshC, and mshD) in Mycobacterium smegmatis and made complementing constructs in integrating plasmids. The resulting set of strains was analyzed for levels of MSH, INH resistance, and ETH resistance. The mshA and mshC single deletion mutants were devoid of MSH production and resistant to INH, whereas the mshB deletion mutant produced decreased levels of MSH yet was sensitive to INH, suggesting that MSH biosynthesis is essential for INH susceptibility in M. smegmatis. Further evidence supporting this conclusion was generated by deleting the gene encoding the MSH S-conjugate amidase (mca) from the ΔmshB null mutant. This double mutant, ΔmshB Δmca, completely abolished MSH production and was resistant to INH. The mshA, mshC, and mshB single deletion mutants were also resistant to ETH, indicating that ETH resistance is modulated by the level of MSH in M. smegmatis. Surprisingly, the mshD deletion mutant lacked MSH production but was sensitive to both INH and ETH. The drug sensitivity was likely mediated by the compensated synthesis of N-formyl-Cys-GlcN-Ins, previously demonstrated to substitute for MSH in an mshD mutant of M. smegmatis. We conclude that MSH or N-formyl-Cys-GlcN-Ins is required for susceptibility to INH or ETH in M. smegmatis.

Synergistic Drug Combinations for Tuberculosis Therapy Identified by a Novel High-throughput Screen

Antimicrobial Agents and Chemotherapy. Aug, 2011  |  Pubmed ID: 21576426

Therapeutic options for tuberculosis (TB) are limited and notoriously ineffective despite the wide variety of potent antibiotics available for treating other bacterial infections. We investigated an approach that enables an expansion of TB therapeutic strategies by using synergistic combinations of drugs. To achieve this, we devised a high-throughput synergy screen (HTSS) of chemical libraries having known pharmaceutical properties, including thousands that are clinically approved. Spectinomycin was used to test the concept that clinically available antibiotics with limited efficacy against Mycobacterium tuberculosis might be used for TB treatment when coadministered with a synergistic partner compound used as a sensitizer. Screens using Mycobacterium smegmatis revealed many compounds in our libraries that acted synergistically with spectinomycin. Among them, several families of antimicrobial compounds, including macrolides and azoles, were also synergistic against M. tuberculosis in vitro and in a macrophage model of M. tuberculosis infection. Strikingly, each sensitizer identified for synergy with spectinomycin uniquely enhanced the activities of other clinically used antibiotics, revealing a remarkable number of unexplored synergistic drug combinations. HTSS also revealed a novel activity for bromperidol, a butyrophenone used as an antipsychotic drug, which was discovered to be bactericidal and greatly enhanced the activities of several antibiotics and drug combinations against M. tuberculosis. Our results suggest that many compounds in the currently available pharmacopoeia could be readily mobilized for TB treatment, including disease caused by multi- and extensively drug-resistant strains for which there are no effective therapies.

Antimicrobial, Anti-inflammatory, Antiparasitic, and Cytotoxic Activities of Galium Mexicanum

Journal of Ethnopharmacology. Sep, 2011  |  Pubmed ID: 21586319

To study the potential benefit of the traditional Mexican medicinal plant Galium mexicanum Kunth (Rubiaceae). Hexane, chloroform, and methanol extracts as well as various fractions from these extracts were tested to determine antibacterial, antifungal, antiparasitic or anti-inflammatory activities in vitro.

Coresistance to Isoniazid and Ethionamide Maps to Mycothiol Biosynthetic Genes in Mycobacterium Bovis

Antimicrobial Agents and Chemotherapy. Sep, 2011  |  Pubmed ID: 21709101

A search to identify new mechanisms of isoniazid resistance in Mycobacterium bovis led to the isolation of mutants defective in mycothiol biosynthesis due to mutations in genes coding for the glycosyltransferase (mshA) or the cysteine ligase (mshC). These mutants showed low-level resistance to isoniazid but were highly resistant to ethionamide. This study further illustrates that mutations in mycothiol biosynthesis genes may contribute to isoniazid or ethionamide resistance across mycobacterial species.

Comparative Efficacy of Commercially Available and Emerging Antimicrobial Urinary Catheters Against Bacteriuria Caused by E. Coli in Vitro

Urology. Aug, 2011  |  Pubmed ID: 21820571

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.

Antimicrobial Activities of Sesquiterpene Lactones and Inositol Derivatives from Hymenoxys Robusta

Phytochemistry. Dec, 2011  |  Pubmed ID: 21963130

Six compounds from the aerial parts of the Argentinean plant Hymenoxys robusta (Rusby) Parker were isolated and their structures elucidated using extensive spectroscopic analyses. These compounds comprise two inositol derivatives and four 3,4-seco-pseudoguaianolides, including vermeerin. Bioactivity assays of these compounds against bacterial and fungal pathogens showed that only vermeerin possessed antimicrobial activity specific against Staphylococcus aureus, and showed no toxicity when exposed to human-derived macrophages.

Mycobacterium Tuberculosis Protein Tyrosine Phosphatase (PtpA) Excludes Host Vacuolar-H+-ATPase to Inhibit Phagosome Acidification

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2011  |  Pubmed ID: 22087003

Mycobacterium tuberculosis (Mtb) pathogenicity depends on its ability to inhibit phagosome acidification and maturation processes after engulfment by macrophages. Here, we show that the secreted Mtb protein tyrosine phosphatase (PtpA) binds to subunit H of the macrophage vacuolar-H(+)-ATPase (V-ATPase) machinery, a multisubunit protein complex in the phagosome membrane that drives luminal acidification. Furthermore, we show that the macrophage class C vacuolar protein sorting complex, a key regulator of endosomal membrane fusion, associates with V-ATPase in phagosome maturation, suggesting a unique role for V-ATPase in coordinating phagosome-lysosome fusion. PtpA interaction with host V-ATPase is required for the previously reported dephosphorylation of VPS33B and subsequent exclusion of V-ATPase from the phagosome during Mtb infection. These findings show that inhibition of phagosome acidification in the mycobacterial phagosome is directly attributed to PtpA, a key protein needed for Mtb survival and pathogenicity within host macrophages.

Antibacterial Activity, Inflammatory Response, Coagulation and Cytotoxicity Effects of Silver Nanoparticles

Nanomedicine : Nanotechnology, Biology, and Medicine. Apr, 2012  |  Pubmed ID: 21718674

The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.

The Mycobacterial Transcriptional Regulator WhiB7 Gene Links Redox Homeostasis and Intrinsic Antibiotic Resistance

The Journal of Biological Chemistry. Jan, 2012  |  Pubmed ID: 22069311

Intrinsic drug resistance in Mycobacterium tuberculosis limits therapeutic options for treating tuberculosis. The mycobacterial transcriptional regulator whiB7 contributes to intrinsic resistance by activating its own expression and many drug resistance genes in response to antibiotics. To investigate whiB7 activation, we constructed a GFP reporter to monitor its expression, and we used it to investigate the whiB7 promoter and to screen our custom library of almost 600 bioactive compounds, including the majority of clinical antibiotics. Results showed whiB7 was transcribed from a promoter that was conserved across mycobacteria and other actinomycetes, including an AT-rich sequence that was likely targeted by WhiB7. Expression was induced by compounds having diverse structures and targets, independent of the ability of whiB7 to mediate resistance, and was dependent on media composition. Pretreatment with whiB7 activators resulted in clinically relevant increases in intrinsic drug resistance. Antibiotic-induced transcription was synergistically increased by the reductant dithiothreitol, an effect mirrored by a whiB7-dependent shift to a highly reduced cytoplasm reflected by the ratio of reduced/oxidized mycothiol. These data provided evidence that intrinsic resistance resulting from whiB7 activation is linked to fundamental changes in cell metabolism.

A Phase I Clinical Study of Inhaled Nitric Oxide in Healthy Adults

Journal of Cystic Fibrosis : Official Journal of the European Cystic Fibrosis Society. Jul, 2012  |  Pubmed ID: 22520076

Nitric oxide (NO) is an approved pulmonary vasodilator for neonates and full term infants up to a dose of 80 ppm. At 100 ppm to 200 ppm, NO has potent antimicrobial activities in vitro and in animal studies which suggest its therapeutic use for infectious diseases in humans. However, whether inhaled NO is safe at 160 ppm in healthy human adults is unknown. The aim of the phase I study was to assess the safety of delivery and the physiologic effects of intermittent 160 ppm NO in healthy human adults.

Nitazoxanide Stimulates Autophagy and Inhibits MTORC1 Signaling and Intracellular Proliferation of Mycobacterium Tuberculosis

PLoS Pathogens. 2012  |  Pubmed ID: 22589723

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

Antimicrobial, Anti-inflammatory, Antiparasitic, and Cytotoxic Activities of Laennecia Confusa

TheScientificWorldJournal. 2012  |  Pubmed ID: 22623891

The current paper investigated the potential benefit of the traditional Mexican medicinal plant Laennecia confusa (Cronquist) G. L. Nesom (Asteraceae). Fractions from the hexane, chloroform, methanol, and aqueous extracts were analyzed for antibacterial, antifungal, anti-inflammatory, and antiparasitic activities. The antimicrobial activity of the extracts and fractions was assessed on bacterial and fungal strains, in addition to the protozoa Leishmania donovani, using a microdilution assay. The propensity of the plant's compounds to produce adverse effects on human health was also evaluated using propidium iodine to identify damage to human macrophages. The anti-inflammatory activity of the extracts and fractions was investigated by measuring the secretion of interleukin-6. Chemical analyses demonstrated the presence of flavonoids, cyanogenic and cardiotonic glycosides, saponins, sesquiterpene lactones, and triterpenes in the chloroform extract. A number of extracts and fractions show antibacterial activity. Of particular interest is antibacterial activity against Staphylococcus aureus and its relative methicillin-resistant strain, MRSA. Hexanic and chloroformic fractions also exhibit antifungal activity and two extracts and the fraction CE 2 antiparasitic activity against Leishmania donovani. All bioactive extracts and fractions assayed were also found to be cytotoxic to macrophages. In addition, the hexane and methane extracts show anti-inflammatory activity by suppressing the secretion of interleukine-6.

Mycobacterium Tuberculosis Modulators of the Macrophage's Cellular Events

Microbes and Infection. Nov, 2012  |  Pubmed ID: 22841679

A number of mycobacterial macromolecules have been shown to target biological processes within host macrophages; however, the exact mechanism for the majority of these host-pathogen interactions is poorly understood. The following review summarizes current knowledge and expands on a subset of mycobacterial effectors for which a cognate substrate, cellular partner or signaling pathway have been experimentally identified within the human host.

Chemical Constituents, Anti-inflammatory and Antioxidant Activities of Bark Extracts from Prunus Tucumanensis Lillo

Natural Product Research. 2013  |  Pubmed ID: 22889184

The anti-inflammatory, antioxidant, antimicrobial and cytotoxic activities of the hexane (HE), chloroform (CE) and methanol (ME) extracts obtained from the bark of Prunus tucumanensis Lillo were investigated. Both ME and CE extracts displayed a significant in vitro anti-inflammatory activity similar to dexamethasone and to a commercial formulation (Pygeum) used for the treatment of benign prostatic hyperplasia (BPH). ME exhibited powerful antioxidant (67.6% relative to BHT) and free radical scavenging (RC50 = 5 ppm) activities, antimicrobial activities against Staphylococcus aureus and Mycobacterium smegmatis and did not show any cytotoxic effect on human-derived macrophage cells. Chemical analyses showed that (2 R,3 R)-3,5,7,3',5'-pentahydroxyflavan, β-sitosterol and β-sitosterol-3-O-β-D-glucopyranoside (daucosterol) are relevant components of ME.

Mycobacterium Tuberculosis-secreted Phosphatases: from Pathogenesis to Targets for TB Drug Development

Trends in Microbiology. Feb, 2013  |  Pubmed ID: 23084287

Mycobacterium tuberculosis (Mtb) infects human alveolar macrophages and relies on the inhibition of phagosome acidification and maturation. This is, in part, dependent on the disruption of host signaling networks within the macrophage. In recent years, Mtb-secreted protein- and lipid-phosphatases protein-tyrosine phosphatase A (PtpA), PtpB, and secreted acid phosphatase M (SapM) have been shown to contribute to Mtb pathogenicity. Here, we review the current knowledge on PtpA, PtpB, and SapM focusing on their ability to interfere with host functions. We further explore how these phosphatase-dependent host-pathogen interactions can be targeted for novel tuberculosis (TB) drug discovery and examine the ongoing development of inhibitors against these phosphatases.

Disruption of the Serine/threonine Protein Kinase H Affects Phthiocerol Dimycocerosates Synthesis in Mycobacterium Tuberculosis

Microbiology (Reading, England). Apr, 2013  |  Pubmed ID: 23412844

Mycobacterium tuberculosis possesses a complex cell wall that is unique and essential for interaction of the pathogen with its human host. Emerging evidence suggests that the biosynthesis of complex cell-wall lipids is mediated by serine/threonine protein kinases (STPKs). Herein, we show, using in vivo radiolabelling, MS and immunostaining analyses, that targeted deletion of one of the STPKs, pknH, attenuates the production of phthiocerol dimycocerosates (PDIMs), a major M. tuberculosis virulence lipid. Comparative protein expression analysis revealed that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted pknH strain. Furthermore, we analysed the composition of the major lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), and found a twofold higher LAM/LM ratio in the mutant strain. Thus, we provide experimental evidence that PknH contributes to the production and synthesis of M. tuberculosis cell-wall components.

Antimicrobial Activity, Cytotoxicity and Inflammatory Response of Novel Plastics Embedded with Silver Nanoparticles

Future Microbiology. Mar, 2013  |  Pubmed ID: 23464375

Infections associated with medical devices are an important cause of morbidity and mortality. Microorganisms are responsible for catheter infections that may then result in the local or systemic dissemination of the microorganism into the bloodstream. The aim of this study was to evaluate the antimicrobial activity of silver nanoparticles (AgNPs) embedded in polyurethane plastics, commonly used for catheter fabrication.

Gaseous Nitric Oxide Reduces Influenza Infectivity in Vitro

Nitric Oxide : Biology and Chemistry. May, 2013  |  Pubmed ID: 23562771

Gaseous nitric oxide (gNO) is an approved vasodilator drug for inhalation up to a maximum dose of 80 ppm. While gNO has been shown, in vitro, to be an effective antibacterial agent (at 160 ppm), NO-donor compounds have been shown to inhibit a variety of viruses at varying stages of replication. This research was done in order to determine whether gNO at 80 or 160 ppm possesses an antiviral effect on influenza viruses. Three strains of influenza (A and B) were exposed to gNO for up to 180 min, before and after infection of MDCK cells. In search for possible mechanism of antiviral action, Neuraminidase (NA) inhibition assay of H1N1 that was exposed to gNO was performed. Results show that when virions were exposed to gNO prior to infection a complete inhibition of infectivity was achieved for all three strains. Post infection exposure of influenza with gNO resulted in about 30% inhibition of infectivity. Further testing showed that when eliminating the pH effect by exposing a dried virus to gNO, 90% inhibition was found after 2h exposure. NA activity, of whole dried H1N1 virus, was found to be inhibited by gNO (80%). These results suggest that 80 and 160 ppm gNO have a time dependent antiviral effect on influenza strains of viruses during various stages of cellular infection, which are not due to concomitant changes in pH in the surrounding milieu. Viral NA inhibition by gNO was shown and may be responsible for this antiviral effect.

Mycobacterium Tuberculosis Nucleoside Diphosphate Kinase Inactivates Small GTPases Leading to Evasion of Innate Immunity

PLoS Pathogens. 2013  |  Pubmed ID: 23874203

Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.

Microbial Protein-tyrosine Kinases

The Journal of Biological Chemistry. Apr, 2014  |  Pubmed ID: 24554699

Microbial ester kinases identified in the past 3 decades came as a surprise, as protein phosphorylation on Ser, Thr, and Tyr amino acids was thought to be unique to eukaryotes. Current analysis of available microbial genomes reveals that "eukaryote-like" protein kinases are prevalent in prokaryotes and can converge in the same signaling pathway with the classical microbial "two-component" systems. Most microbial tyrosine kinases lack the "eukaryotic" Hanks domain signature and are designated tyrosine kinases based upon their biochemical activity. These include the tyrosine kinases termed bacterial tyrosine kinases (BY-kinases), which are responsible for the majority of known bacterial tyrosine phosphorylation events. Although termed generally as bacterial tyrosine kinases, BY-kinases can be considered as one family belonging to the superfamily of prokaryotic protein-tyrosine kinases in bacteria. Other members of this superfamily include atypical "odd" tyrosine kinases with diverse mechanisms of protein phosphorylation and the "eukaryote-like" Hanks-type tyrosine kinases. Here, we discuss the distribution, phylogeny, and function of the various prokaryotic protein-tyrosine kinases, focusing on the recently discovered Mycobacterium tuberculosis PtkA and its relationship with other members of this diverse family of proteins.

Mycobacterium Tuberculosis Promotes Anti-apoptotic Activity of the Macrophage by PtpA Protein-dependent Dephosphorylation of Host GSK3α

The Journal of Biological Chemistry. Oct, 2014  |  Pubmed ID: 25187516

Mycobacterium tuberculosis tyrosine phosphatase PtpA inhibits two key cellular events in macrophages required for the elimination of invading organisms, phagosome acidification, and maturation. Kinome analysis revealed multiple PtpA-dependent changes to the phosphorylation status of macrophage proteins upon M. tuberculosis infection. Among those proteins we show that PtpA dephosphorylates GSK3α on amino acid Tyr(279), which leads to modulation of GSK3α anti-apoptotic activity, promoting pathogen survival early during infection.

Phosphorylation Control of Protein Tyrosine Phosphatase A Activity in Mycobacterium Tuberculosis

FEBS Letters. Jan, 2015  |  Pubmed ID: 25535696

Protein tyrosine phosphatase A (PtpA) has been shown to play a key role in human macrophage infection by Mycobacterium tuberculosis (Mtb). Protein tyrosine kinase A (PtkA) was the first protein tyrosine kinase shown to phosphorylate PtpA. Here, we found that PtkA-mediated phosphorylation of PtPA on Tyr-128 and Tyr-129 enhances the PtPA phosphatase activity. Moreover, ex-vivo protein-protein interaction assays showed that PtpA can be phosphorylated by several eukaryotic-like Ser/Thr protein kinases, such as protein kinase A (PknA). PknA was found to regulate PtpA phosphatase activity through Thr-45 phosphorylation. These results indicate that members of two independent families of protein kinases tune PtpA activity in Mtb.

Immunoevasion and Immunosuppression of the Macrophage by Mycobacterium Tuberculosis

Immunological Reviews. Mar, 2015  |  Pubmed ID: 25703562

By virtue of their position at the crossroads between the innate and adaptive immune response, macrophages play an essential role in the control of bacterial infections. Paradoxically, macrophages serve as the natural habitat to Mycobacterium tuberculosis (Mtb). Mtb subverts the macrophage's mechanisms of intracellular killing and antigen presentation, leading ultimately to the development of tuberculosis (TB) disease. Here, we describe mechanisms of Mtb uptake by the macrophage and address key macrophage functions that are targeted by Mtb-specific effector molecules enabling this pathogen to circumvent host immune response. The macrophage functions described in this review include fusion between phagosomes and lysosomes, production of reactive oxygen and nitrogen species, antigen presentation and major histocompatibility complex class II expression and trafficking, as well as autophagy and apoptosis. All these are Mtb-targeted key cellular pathways, normally working in concert in the macrophage to recognize, respond, and activate 'proper' immune responses. We further analyze and discuss major molecular interactions between Mtb virulence factors and key macrophage proteins and provide implications for vaccine and drug development.

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium Tuberculosis Growth and Infectivity

The Journal of Biological Chemistry. Sep, 2015  |  Pubmed ID: 26229105

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr(213). The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages.

Phosphorylation of Mycobacterium Tuberculosis Protein Tyrosine Kinase A PtkA by Ser/Thr Protein Kinases

Biochemical and Biophysical Research Communications. Nov, 2015  |  Pubmed ID: 26417687

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has inflicted about one third of mankind and claims millions of deaths worldwide annually. Signalling plays an important role in Mtb pathogenesis and persistence, and thus represents attractive resource for drug target candidates. Here, we show that protein tyrosine kinase A (PtkA) can be phosphorylated by Mtb endogenous eukaryotic-like Ser/Thr protein kinases (eSTPKs). Kinase assays showed that PknA, PknD, PknF, and PknK can phosphorylate PtkA in dose- and time-dependent manner. Enzyme kinetics suggests that PknA has the highest affinity and enzymatic efficiency towards PtkA. Furthermore, protein-protein interaction assay in surrogate host showed that PtkA interacts with multi-eSTPKs in vivo, including PknA. Lastly, we show that PtkA phosphorylation by eSTPKs occurs on threonine residues and may effect tyrosine phosphorylation levels and thus PtkA activity in vitro. These results demonstrate that PtkA can serve as a substrate to many eSTPKs and suggests that's its activity can be regulated.

Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium Tuberculosis Growth in Human Macrophages

Antimicrobial Agents and Chemotherapy. Oct, 2015  |  Pubmed ID: 26503663

Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.

Intracellular Growth of Bacterial Pathogens: The Role of Secreted Effector Proteins in the Control of Phagocytosed Microorganisms

Microbiology Spectrum. Dec, 2015  |  Pubmed ID: 27337278

The ability of intracellular pathogens to subvert the host response, to facilitate invasion and subsequent infection, is the hallmark of microbial pathogenesis. Bacterial pathogens produce and secrete a variety of effector proteins, which are the primary means by which they exert control over the host cell. Secreted effectors work independently, yet in concert with each other, to facilitate microbial invasion, replication, and intracellular survival in host cells. In this review we focus on defined host cell processes targeted by bacterial pathogens. These include phagosome maturation and its subprocesses: phagosome-endosome and phagosome-lysosome fusion events, as well as phagosomal acidification, cytoskeleton remodeling, and lysis of the phagosomal membrane. We further describe the mode of action for selected effectors from six pathogens: the Gram-negative Legionella, Salmonella, Shigella, and Yersinia, the Gram-positive Listeria, and the acid-fast actinomycete Mycobacterium.

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium Tuberculosis

Cell Reports. Jan, 2016  |  Pubmed ID: 26774486

The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping but distinct functions of EGT and MSH. Last, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.

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

Organic Letters. Feb, 2017  |  Pubmed ID: 28164711

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.

Epigenetic Phosphorylation Control of Mycobacterium Tuberculosis Infection and Persistence

Microbiology Spectrum. Mar, 2017  |  Pubmed ID: 28281439

Reversible protein phosphorylation is the most common type of epigenetic posttranslational modification in living cells used as a major regulation mechanism of biological processes. The Mycobacterium tuberculosis genome encodes for 11 serine/threonine protein kinases that are responsible for sensing environmental signals to coordinate a cellular response to ensure the pathogen's infectivity, survival, and growth. To overcome killing mechanisms generated within the host during infection, M. tuberculosis enters a state of nonreplicating persistence that is characterized by arrested growth, limited metabolic activity, and phenotypic resistance to antimycobacterial drugs. In this article we focus our attention on the role of M. tuberculosis serine/threonine protein kinases in sensing the host environment to coordinate the bacilli's physiology, including growth, cell wall components, and central metabolism, to establish a persistent infection.

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