Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions

Abstract

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...

RecombinantMycobacterium bovisBCG Expressing the Sm14 Antigen ofSchistosoma mansoniProtects Mice from Cercarial Challenge

“…pGEMEX-sm14 (containing the sm14 gene (31), pGEM-T Easy (Promega), and the mycobacterial expression vectors pLA71, pLA73 (22), and pMIP12 (21) were used. The mycobacterial expression vectors contain the E. coli and mycobacterial origins of replication, a kanamycin resistance gene, the up-regulated Mycobacterium fortuitum promoter pBlaF*, its ATG initiation codon, and a multicloning site which places the heterologous gene in fusion with the signal sequence or the whole ␤-lactamase-encoding gene in pLA71 and pLA73, respectively.…”

β-Lactamase can function as a reporter of bacterial protein export during Mycobacterium tuberculosis infection of host cells

“…The genetic methods rely on reporter enzymes that are fused to M. tuberculosis protein sequences and report on the subcellular location of the fusion proteins (Braunstein et al, 2000;Chubb et al, 1998;Downing et al, 1999;Lim et al, 1995;Wiker et al, 2000). Surrogate hosts such as non-pathogenic Mycobacterium smegmatis or Escherichia coli have been used in most of these studies, often because endogenous enzyme activities in M. tuberculosis precluded their use directly in the pathogen.…”

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

“…Based on this observation, a comparative study of the transformability of M. vaccae (ATCC 15483) and M. smegmatis mc#155 was performed. We employed previously characterized vectors including pJEM15 (Timm et al, 1994), pJEM11 (Lim et al, 1995) pMV261 (Stover et al, 1991) and pBEN, a derivative of pMV262 expressing a mutated form of green fluorescent protein (Cormack et al, 1996). In addition, a novel series of plasmids (pUS series) was developed and evaluated.…”

“…When necessary, media were supplemented with hygromycin B at 50 µg ml − " (for mycobacteria) or 200 µg ml − " (for E. coli), or 25 µg kanamycin ml − " for plasmid selection and 40 µg X-Gal ml − " for identification of recombinant clones with β-galactosidase activity. The E. coli\mycobacteria shuttle vectors pJEM11 (Lim et al, 1995) and pJEM15 (Timm et al, 1994) were kindly provided by Dr Brigitte Gicquel, Institute Pasteur, Paris, France. The vector pMV261 (Stover et al, 1991) was a gift from Dr A. Cataldi (Instituto de Biotecnologia, INTA, Hurlingham, Argentina) and pBEN, a derivative of pMV262 expressing a mutated form of green fluorescent protein (Cormack et al, 1996) under control of the hsp60 promoter, was generously supplied by Dr L. Ramakrishnan (Stanford University, USA).…”