Drift of the HIV-1 envelope glycoprotein gp120 toward increased neutralization resistance over the course of the epidemic: a comprehensive study using the most potent and broadly neutralizing monoclonal antibodies.

Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.

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 cell surface associated phosphatase activity of Mycobacterium bovis BCG is not regulated by environmental inorganic phosphate.

Non-specific phosphomonoesterase activities (alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2)) were examined at the cell surface of Mycobacterium bovis BCG. Using p-nitrophenylphosphate as the substrate, peaks of phosphatase activity were detected at pH 6.0, pH 10.0 and pH 12.0, suggesting the presence of one acid phosphatase and two alkaline phosphatases with distinct optimum pH values. Contrary to the situation observed in several other microorganisms, the expression of these enzymes is not regulated by the environmental inorganic phosphate concentration.

The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis.

We have undertaken the inventory and assembly of the typical subunits of the ABC transporters encoded by the complete genome of Mycobacterium tuberculosis. These subunits, i.e. the nucleotide binding domains (NBDs), the membrane-spanning domains (MSDs) and the substrate binding proteins (SBPs), were identified on the basis of their characteristic stretches of amino acids and/or conserved structure. A total of 45 NBDs present in 38 proteins, of 47 MSDs present in 44 proteins and of 15 SBPs were found to be encoded by M. tuberculosis. Analysis of transcriptional clusters and searches of homology between the identified subunits of the transporters and proteins characterized in other organisms allowed the reconstitution of at least 26 complete (including at least one NBD and one MSD) and 11 incomplete ABC transporters. Sixteen of them were unambiguously classified as importers whereas 21 were presumed to be exporters. By searches of homology with already known transporters from other organisms, potential substrates (peptides, macrolides, carbohydrates, multidrugs, antibiotics, iron, anions) could be attributed to 30 of the ABC transporters identified in M. tuberculosis. The ABC transporters have been further classified in nine different sub-families according to a tree obtained from the clustering of their NBDs. Contrary to Escherichia coli and similarly to Bacillus subtilis, there is an equal representation of extruders and importers. Many exporters were found to be potentially implicated in the transport of drugs, probably contributing to the resistance of M. tuberculosis to many antibiotics. Interestingly, a transporter (absent in E. coli and in B. subtilis) potentially implicated in the export of a factor required for the bacterial attachment to the eukaryotic host cells was also identified. In comparison to E. coli and B. subtilis, there is an under-representation of the importers (with the exception of the phosphate importers) in M. tuberculosis. This may reflect the capacity of this bacterium to synthesize many essential compounds and to grow in the presence of few external nutrients. The genes encoding the ABC transporters occupy about 2.5% of the genome of M. tuberculosis.

Characterization of a Mycobacterium bovis BCG insertion sequence related to the IS21 family.

The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group.

Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors.

Using culture filtrate Ag-specific mAbs generated from mycobacteria-infected H-2b haplotype mice, we have previously identified three genes in the Mycobacterium tuberculosis genome, encoding proteins homologous to the periplasmic ATP-binding cassette phosphate-binding receptor PstS of the phosphate-specific transport system of E. coli. To define the potential vaccinal properties of these phosphate-binding proteins, female C57BL/6 mice were injected i.m. with plasmid DNA encoding PstS-1, PstS-2, or PstS-3 proteins from M. tuberculosis and immunogenicity and protective efficacy against i.v. challenge with M. tuberculosis H37Rv was analyzed. Significant levels of highly Ag-specific Abs and Th1-type cytokines IL-2 and IFN-gamma could be detected following vaccination with each of the three genes. However, only mice vaccinated with PstS-3 DNA demonstrated significant and sustained reduction in bacterial CFU numbers in spleen and lungs for 3 mo after M. tuberculosis challenge, as compared with CFU counts in mice vaccinated with control DNA. Vaccination with PstS-2 DNA induced a modest reduction in CFU counts in spleen only, whereas vaccination with PstS-1 DNA was completely ineffective in reducing bacterial multiplication. In conclusion, our results indicate that DNA vaccination is a powerful and easy method for comparative screening of potentially protective Ags from M. tuberculosis and that the PstS-3 protein is a promising new subunit vaccine candidate.

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.