Signalling inhibitors against Mycobacterium tuberculosis--early days of a new therapeutic concept in tuberculosis.

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

A protein kinase inhibitor as an antimycobacterial agent.

The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.

Monitoring promoter activity and protein localization in Mycobacterium spp. using green fluorescent protein.

Two green fluorescent protein (Gfp) fusion vectors were constructed for use in Mycobacterium spp. The first plasmid facilitates quantification of mycobacterial promoter activity. The second vector permits construction of translational fusions of mycobacterial proteins to Gfp in order to study subcellular localization including protein secretion. Using this translational fusion construct, we verify that a Gfp fusion to the putative secreted M. tuberculosis protein ChoD is translocated to the extracellular milieu when cloned and expressed in Mycobacterium smegmatis.

N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis.

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria. Mycothiol was previously shown to be synthesized from 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside by ligation with cysteine followed by acetylation. A novel mycothiol-dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in Mycobacterium smegmatis and shown to have a homolog, Rv1082, in Mycobacterium tuberculosis. In the present study we found that a protein encoded by the M. tuberculosis open reading frame Rv1170, a homolog of Rv1082, possesses weak mycothiol conjugate amidase activity but shows substantial deacetylation activity with 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic precursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in M. smegmatis at a level about twice that of mycothiol. It was shown that GlcNAc-Ins is absent in mycothiol-deficient mutant strain 49 of M. smegmatis and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol. This demonstrates that GlcNAc-Ins is a key intermediate in the pathway of mycothiol biosynthesis. Assignment of Rv1170 as the gene coding the deacetylase in the M. tuberculosis genome represents the first identification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.

Cholesterol is accumulated by mycobacteria but its degradation is limited to non-pathogenic fast-growing mycobacteria.

In this report we show that fast-growing non-pathogenic mycobacteria degrade cholesterol from liquid media, and are able to grow on cholesterol as a sole carbon source. In contrast, slow-growing mycobacteria, including pathogenic Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), do not degrade and use cholesterol as a carbon source. Nevertheless, pathogenic mycobacteria are able to uptake, modify, and accumulate cholesterol from liquid growth media, and form a zone of clearance around a colony when plated on solid media containing cholesterol. These data suggest that cholesterol may have a role in mycobacterial infection other than its use as carbon source.

DNA-Based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III.

Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V). Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult. The 16S rRNA gene (16S rDNA) and recA gene of these bacteria were examined in order to develop rapid tests for genomovar identification. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B. multivorans and B. vietnamiensis but insufficient to discriminate strains of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recA demonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B. cepacia complex genomovars. Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic tree identified six distinct clusters (recA groups): B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B. Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within the genome of B. cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present. In conclusion, analysis of the recA gene of the B. cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens.

A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase.

Mycothiol, 1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH), is composed of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes. When Mycobacterium smegmatis was treated with the alkylating agent monobromobimane (mBBr), the cellular mycothiol was converted to its bimane derivative (MSmB). The latter was rapidly cleaved to produce GlcN-Ins and the bimane derivative of N-acetylcysteine (AcCySmB), a mercapturic acid that was rapidly exported from the cells into the medium. The other product of cleavage, GlcN-Ins, was retained in the cell and utilized in the resynthesis of mycothiol. The mycothiol S-conjugate amidase (amidase) responsible for cleaving MSmB was purified to homogeneity from M. smegmatis. A value of K(m) = 95 +/- 8 microM and a value of k(cat) = 8 s(-)(1) was determined for the amidase with MSmB as substrate. Activity with 100 microM mycothiol or with the monobromobimane derivative of 1-D-myo-inosityl-2-(L-cysteinyl)amido-2-deoxy-alpha-D-glucopyra nos ide (CySmB-GlcN-Ins) or of 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-(alpha, beta)-D-glucopyranoside (AcCySmB-GlcN) was at least 10(3) lower than with 100 microM MSmB, demonstrating that the amidase is highly specific for S-conjugates of mycothiol. Conjugates of mycothiol with the antibiotic cerulenin, N-ethylmaleimide, 3-(N-maleimidopropionyl)-biocytin, and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin also exhibited significant activity. The sequence of the amino-terminal 20 residues was determined, and an open reading frame (Rv1082) coding for 288 residues having an identical predicted amino-terminal amino acid sequence was identified in the Mycobacterium tuberculosis genome. The Rv1082 gene (mca) from M. tuberculosis was cloned and expressed in Escherichia coli, and the expressed protein was shown to have substrate specificity similar to the amidase from M. smegmatis. These results indicate that mycothiol and mycothiol S-conjugate amidase play an important role in the detoxification of alkylating agents and antibiotics.

The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis.

In bacteria, extracellular signals are generally transduced into cellular responses via a two-component system. However, genome sequence data have now revealed the presence of 'eukaryotic-like' protein kinases and phosphatases. Mycobacterium tuberculosis appears to be unique among bacteria in that its genome contains 11 members of a newly identified protein kinase family. These M. tuberculosis eukaryotic-like protein kinases could be key regulators of metabolic processes, including transcription, cell development and interactions with host cells.

Expression and characterization of the Mycobacterium tuberculosis serine/threonine protein kinase PknB.

PknB is a member of the newly discovered eukaryotic-like protein serine/threonine kinase (PSTK) family of proteins. The pknB gene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and Creb. In vitro kinase assays demonstrated that PknB is a functional kinase that is autophosphorylated on serine/threonine residues and is also able to phosphorylate the peptide substrate myelin basic protein. Analysis of pknB expression in Mycobacterium tuberculosis indicates the presence of pknB mRNA in (i) organisms grown in vitro in bacteriological media, (ii) a murine macrophage in vitro infection model, and (iii) in vivo alveolar macrophages from a patient with tuberculosis.

Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus.

The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.

Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis.

Mycothiol (MSH) is the major low molecular weight thiol in mycobacteria. Two chemical mutants with low MSH and one with no MSH (strain 49) were produced in Mycobacterium smegmatis mc2155 to assess the role of MSH in mycobacteria. Strain 49 was shown to not produce 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside (GlcN-Ins), an intermediate in MSH biosynthesis. Relative to the parent strain, mutant 49 formed colonies more slowly on solid media and was more sensitive to H2O2 and rifampin, but less sensitive to isoniazid. Complementation of mutant 49 with DNA from M. tuberculosis H37Rv partially restored production of GlcN-Ins and MSH, and resistance to H2O2, but largely restored colony growth rate and sensitivity to rifampin and isoniazid. The results indicate that MSH and GlcN-Ins are not essential for in vitro survival of mycobacteria but may play significant roles in determining the sensitivity of mycobacteria to environmental toxins.

Gene organization in the trxA/B-oriC region of the Streptomyces coelicolor chromosome and comparison with other eubacteria.

The gene organization was determined in the trxA/B-rnpA region of the Streptomyces coelocolor chromosome, near to the origin of replication, oriC. Previously, we showed that the trxA and trxB genes, coding for thioredoxin and thioredoxin reductase, respectively, occur in S. coelicolor as a gene cluster and are contained on a cosmid H24 that carries oriC and several genes involved in DNA replication. Here we show that the trxA/B locus is positioned approx. 9.4kb from oriC, present the nucleotide sequence of the trxA/B-rnpA region and use sequence analysis to identify the nature of the intervening genes. Seven open reading frames were found, all oriented in the same direction, five of which were identified as the S. coelicolor homologs of SpoIIIJ, Jag, GidB, Soj and SpoOJ in Bacillus subtilis and which have been ascribed different functions in this and other bacteria for either DNA replication, chromosomal partitioning or morphological development. The arrangement of the genes coding for the above five proteins in the trxA/B-rnpA region in S. coelicolor resembles that in Mycobacterium leprae, Mycobacterium tuberculosis, B. subtilis and Pseudomonas putida, and supports the view that many of the genes necessary for development and cell division in bacteria are organized in a similar fashion. In B. subtilis and P. putida, however, the trxA/B genes are not present in the above gene arrangement.

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.

Characterization of a broad-range disulfide reductase from Streptomyces clavuligerus and its possible role in beta-lactam antibiotic biosynthesis.

Streptomyces clavuligerus is a potent producer of penicillin and cephalosporin antibiotics. A key step in the biosynthesis of these beta-lactam compounds is the cyclization of the thiol tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). However, bis-ACV, the oxidized disulfide form of the tripeptide, is not a substrate for IPNS. We show here that S. clavuligerus possesses an NADPH-dependent disulfide reductase of broad substrate specificity that efficiently catalyzes the reduction of disulfide bonds in bis-ACV and in other low-molecular-weight disulfide containing compounds and proteins. The disulfide reductase comprises two protein components, a 70-kDa reductase consisting of two identical subunits, and a 12-kDa heat-stable protein reductant. The structural and functional properties of the disulfide reductase resemble those of the thioredoxin class of oxidoreductases. When the disulfide reductase system is coupled with IPNS, it quantitatively converts bis-ACV to isopenicillin N. These findings suggest that the disulfide reductase may play a role in the biosynthesis of penicillins and cephalosporins in streptomycetes. We also show here that S. clavuligerus lacks glutathione reductase and have previously reported that Streptomyces species do not contain glutathione. This disulfide reductase may therefore be important in determining the thiol-disulfide redox balance in streptomycetes.

High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies.

A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp. 12,154 and Streptomyces jumonjinensis in Escherichia coli. Most of the IPNS synthesized at 37 degrees C, and representing some 22% and 51% of the total cell protein respectively, occurred in an insoluble, enzymatically inactive form. Active IPNS was recovered in a rapid and simple two-step procedure in which the insoluble material was first denatured in 5 M urea and then refolded by passing the solubilized IPNS through a G-25 Sephadex sizing column. Further chromatography on DEAE-Sepharose resulted in highly active IPNS preparations. This procedure was found to be well suited for scaling up to produce large amounts of IPNS.