Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.

The virulence of the mycobacteria that cause tuberculosis depends on their ability to multiply in mammalian hosts. Disruption of the bacterial erp gene, which encodes the exported repetitive protein, impaired multiplication of M. tuberculosis and M. bovis Bacille Calmette-Guérin in cultured macrophages and mice. Reintroduction of erp into the mutants restored their ability to multiply. These results indicate that erp contributes to the virulence of M. tuberculosis.

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Mycobacterium tuberculosis caseinolytic protease ClpP1 (Mt ClpP1) is a self-compartmentalized protease consisting of two heptameric rings stacked on top of each other, thus enclosing a catalytic chamber. Within the chamber, which can be reached through two axial pores, each of the 14 identical monomers possesses a serine protease active site. The unfolding and translocation of substrates into the chamber are mediated by associated hexameric ATPases covering the axial pores. Three crystal structures of Mt ClpP1, determined by molecular replacement, are presented in this study. Two of the models were refined to a resolution of 2.6 A and the third to 3.0 A. It was found that disorder in the handle domain affects the formation and configuration of the tetradecamer and results in condensed structures with larger equatorial pores when compared with ClpPs from other species. Additionally, this disorder accompanies conformational changes of the residues in the catalytic triad. The models also reveal structural differences within the N-terminal hairpin-loop domain, which possibly reflect the significant differences in amino-acid sequence between Mt ClpP1 and other ClpP homologues in this region.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum ␤-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.Mycobacterium bovis and M. tuberculosis, the causative agents of tuberculosis, are facultative intracellular bacteria. In spite of the major health problems linked to these pathogens, little is known about their exported or secreted proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of M. tuberculosis culture filtrates show at least 30 secreted proteins (4,23,44). Some of them have been characterized, with their genes cloned and sequenced (1,8,19,43,45). Other, although immunodominant, antigens of major importance in protective immunity (3, 25) have not been fully identified. In addition, many exported proteins probably stay attached to the cell membrane and are therefore not found in culture supernatants. Proteins compartmentalized on the outer surface of various pathogenic bacteria, such as the 103-kDa invasin of Yersina pseudotuberculosis (16) or the 80-kDa internalin from Listeria monocytogenes (12) have been shown to play an important role in the interactions with the host cells. Thus, membrane-bound proteins might be of significance to the physiopathology of M. tuberculosis infection.In this report, we describe the adaptation to mycobacteria of a genetic method for identifying exported proteins. This methodology is based on translational fusion with the bacterial alkaline phosphatase (PhoA). Such protein fusions must be exported to have PhoA activity (7,15,18). We used a phoA gene from which had been deleted the promoter region, the ribosome binding site, and the complete signal sequence-encoding region including the translational start codon. Thus, alkaline phosphatase activity is dependent upon translational fusion in the correct reading frame with part of an exported protein. We first describe the construction of a phoA plasmid vector for mycobacteria and show that introduction in this vector of the exported M. f...