Antimycobacterial activity of synthetic pamamycins.

OBJECTIVES: Early studies have indicated that pamamycins, a group of macrodiolides first isolated from Streptomyces alboniger, have potent antimicrobial activity against Gram-positive bacteria, fungi and mycobacteria but not against Gram-negative bacteria. The recent availability of highly purified and reasonable quantities of several pamamycins through their total syntheses has rendered possible more extensive studies on their effects on mycobacteria. METHODS: Bioluminescent strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis, expressing the luxA and luxB genes from Vibrio harveyi were used for the comparison of the antimycobacterial activity of the two synthetic macrodiolides pamamycin-607 and pamamycin-621A and a non-naturally occurring cyclic dimer of pamamycin-607, i.e. yukomycin. RESULTS: Pamamycin-607 was the most active of the three macrocycles and was more active against M. tuberculosis than against M. smegmatis. Twenty-five clinical isolates of M. tuberculosis were susceptible to pamamycin-607 in a narrow MIC range of 1.5-2.0 mg/L. The new assay was also validated by comparison with the BACTEC radiometric test. CONCLUSION: Rapid screening of a new class of macrocyclic antimycobacterials using bioluminescent mycobacteria identified pamamycin-607 as a potential antituberculous agent. The latter was active against clinical isolates of M. tuberculosis within a narrow MIC range of 1.5-2.0 mg/L irrespective of their resistance to isoniazid or rifampicin. Our findings warrant further investigations.

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated.

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.

Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG.

A gene encoding a protein homologous to the periplasmic ABC phosphate binding receptor PstS from Escherichia coli was cloned and sequenced from a lambda gt11 library of Mycobacterium tuberculosis by screening with monoclonal antibody 2A1-2. Its degree of similarity to the E. coli PstS is comparable to those of the previously described M. tuberculosis phosphate binding protein pab (Ag78, Ag5, or 38-kDa protein) and another M. tuberculosis protein which we identified recently. We suggest that the three M. tuberculosis proteins share a similar function and could be named PstS-1, PstS-2, and PstS-3, respectively. Molecular modeling of their three-dimensional structures using the structure of the E. coli PstS as a template and their inducibility by phosphate starvation support this view. Recombinant PstS-2 and PstS-3 were produced and purified by affinity chromatography. With PstS-1, these proteins were used to demonstrate the specificity of three groups of monoclonal antibodies. Using these antibodies in flow cytometry and immunoblotting analyses, we demonstrate that the three genes are expressed and their protein products are present and accessible at the mycobacterial surface as well as in its culture filtrate. Together with the M. tuberculosis genes encoding homologs of the PstA, PstB, and PstC components we cloned before, the present data suggest that at least one, and possibly several, related and functional ABC phosphate transporters exist in mycobacteria. It is hypothesized that the mycobacterial gene duplications presented here may be a subtle adaptation of intracellular pathogens to phosphate starvation in their alternating growth environments.

A serine/threonine protein kinase from Mycobacterium tuberculosis.

Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein. The size of the protein was confirmed by in vitro coupled transcription/translation. Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring. The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase. Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis. Its expression was detected in cultures of M. bovis BCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.

A Mycobacterium tuberculosis gene cluster encoding proteins of a phosphate transporter homologous to the Escherichia coli Pst system.

We report the cloning and sequencing of three M. tuberculosis genes encoding proteins homologous to E. coli PstA, PstC and PstB. They are tentatively called pstA-2, pstC-1 and pstB. They encode proteins of 302, 336 and 275 amino acids, respectively. In E. coli, PstB is the ATP binding component and PstA/PstC are the two hydrophobic subunits of a phosphate permease belonging to the family of ABC (ATP-binding cassette) transporters. In mycobacteria, PstS-1, the phosphate binding subunit (Andersen and Hansen, 1989), is encoded by a gene directly surrounded by pstB, pstC-1 and pstA-2 within a potential operon (pstB, pstS-1, pstC-1, pstA-2). Phosphate uptake by whole, suspension grown, M. bovis BCG cells was measured and could be inhibited by a monoclonal antibody directed against the PstS-1 subunit, suggesting that these genes encode subunits of a functional mycobacterial phosphate permease.

Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter.

Following the identification of a M. tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS-3 and pstC-2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively. Together with the previously isolated M. tuberculosis gene similar to the E. coli pstA, these are included in a cluster encoding a second putative phosphate transport system. We demonstrate that pstS-3 encodes the previously described Ag 88, a 40 kDa M. bovis BCG culture filtrate antigen (immunodominant in H-2b haplotype type mice). Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding-dependent permeases is proposed.

Structure of the Mycobacterium tuberculosis antigen 88, a protein related to the Escherichia coli PstA periplasmic phosphate permease subunit.

We report the cloning and sequencing of the gene coding for antigen 88 from Mycobacterium tuberculosis by using monoclonal antibodies to screen an expression library in lambda gt11. The gene encodes a 403-amino-acid-residue protein with a calculated molecular mass of 43,790 Da which contains seven putative transmembrane alpha-helical domains and presents a significant homology to the PstA protein of Escherichia coli. In its N-terminal region, it contains a 61-amino-acid region highly homologous to the fifth transmembrane helix of E. coli PstC. PstA and PstC are the two hydrophobic subunits of an E. coli periplasmic phosphate permease. Since the phosphate-binding subunit of this putative permease in M. tuberculosis has previously been characterized, i.e., the 38-kDa mycobacterial protein (also called protein antigen b, Ag 5, and Ag 78) homologous to PstS of E. coli, it seems likely that functional permeases analogous to the periplasmic permeases of gram-negative bacteria also exist in mycobacteria.