Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.

Isolation and characterization of genetic expression and secretion signals from Enterococcus faecalis through the use of broad-host-range alpha-amylase probe vectors.

We have constructed two broad-host-range Gram+/Gram- probe vectors designed for the cloning of bacterial genetic expression and secretion signals. These vectors make use of a silent reporter gene encoding the mature alpha-amylase from Bacillus licheniformis whose reactivation can easily be monitored on iodine-stained starch plates. Shotgun cloning of Enterococcus faecalis DNA fragments allowed recovery of several cassettes directing transcription, translation of the reporter gene and secretion of alpha-amylase. Sequence analysis revealed, in each case, the presence of a putative promoter, ribosome-binding site and signal peptide similar to those described in other Gram+ bacteria.

A new series of mycobacterial expression vectors for the development of live recombinant vaccines.

Recombinant BCG (bacillus Calmette-Guérin) is a promising candidate as a live vaccine delivery system. Thus far, however, only autoreplicative plasmids carrying the heterologous genes to be expressed in BCG, together with antibiotic-resistance genes, have been successfully used. This could potentially lead to the spreading of antibiotic resistance among other bacteria, and might therefore be unsafe for the environment. In this study, we present a series of three Escherichia coli-Mycobacteria shuttle vectors which enable expression and secretion of antigens without the use of antibiotic-resistance markers. All these plasmids confer mercury resistance to the host bacteria as the only selectable marker and contain a unique restriction site to allow for single-step in-frame cloning of open reading frames downstream from the Mycobacterium tuberculosis 85A antigen promoter and export signal. The system was used to express the free beta-subunit of human chorionic gonadotropin (hCG beta), a potential target of an immunotherapeutic vaccine.

Efficient homologous recombination in fast-growing and slow-growing mycobacteria.

Although homologous recombination is a major mechanism for DNA rearrangement in most living organisms, it has been difficult to detect in slowly growing mycobacteria by a classical suicide vector approach. Among the possible reasons for this are the low levels of transformation efficiency, the relatively high levels of illegitimate recombination, and the peculiar nature of the recA gene in slowly growing mycobacteria. In this report, we present an efficient homologous recombination system for these organisms based on the use of replicative plasmids which facilitates the detection of rare recombination events, because the proportions of recombined molecules increase over time. Intraplasmid homologous recombination in Mycobacterium smegmatis and Mycobacterium bovis BCG was easily selected by the reconstitution of an interrupted kanamycin resistance gene. Chromosomal integration via homologous recombination was selected by the expression of the kanamycin resistance gene under the control of a chromosomal promoter that was not present in the plasmid before recombination. This technique was termed STORE (for selection technique of recombination events). All the clones selected by STORE had undergone homologous recombination, as evidenced by PCR analyses of the kanamycin-resistant clones. This technique should be applicable to all organisms for which homologous recombination has been difficult to achieve, provided the gene of interest is expressed.

Genetic analysis and overexpression of lipolytic activity in Bacillus subtilis.

The previously cloned Bacillus subtilis lipase gene (lip) was mapped on the chromosome and used in the construction of a B. subtilis derivative totally devoid of any lip sequence. Homologous overexpression was performed in this strain by subcloning the lip open reading frame on a multicopy plasmid under the control of a strong gram-positive promoter. A 100-fold overproducing strain was obtained, which should facilitate purification of the secreted protein. Furthermore, the delta lip strain BCL1050 constitutes an ideal host for the cloning of heterologous lipase genes.

Analysis of the Mycobacterium tuberculosis 85A antigen promoter region.

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.

Mercury resistance as a selective marker for recombinant mycobacteria.

The use of antibiotic-resistance markers for the selection of recombinant mycobacteria is widespread but questionable considering the development of live recombinant BCG vaccines. In contrast, vector-encoded resistance to heavy metals such as mercury may represent an interesting alternative for the development of live vaccines compatible with use in humans and in animals. The mercury resistance genes (mer) from Pseudomonas aeruginosa and from Serratia marcescens were cloned into the Escherichia coli-Mycobacterium shuttle vector pRR3. The resulting vectors, designated pMR001 and pVN2, were introduced by electroporation into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. The recombinant mycobacteria were stable in vitro and in vivo, and had high-level mercury resistance, thus indicating that the mer genes can be useful as selective markers in mycobacteria.

Green fluorescent protein as a new expression marker in mycobacteria.

This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM-11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM-11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co-factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free-living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.

Neutralizing antibody responses elicited in mice immunized with recombinant bacillus Calmette-Guérin producing the Schistosoma mansoni glutathione S-transferase.

Schistosomiasis is a group of severe parasitic diseases, in humans and domestic animals, that are especially of importance in the developing world. No efficacious vaccine is currently available. However, Ab-mediated immune responses against the 28-kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST) appear to be involved in protection. This Ag was produced in recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG). The recombinant protein bound glutathione and expressed enzymatic activity, indicating that the active site of Sm28GST was folded properly. Single i.v., i.p., s.c., or intranasal immunizations with rBCG in BALB/c mice resulted in significant anti-SM28GST Ab responses, which were enhanced by a booster dose. The Ab responses remained high for at least 1 yr after immunization. Analyses of the isotype profiles indicated that i.v. immunized mice produced high titers of anti-Sm28GST IgG2a, and less IgG2b and IgG1. Mice immunized by the s.c. route initially also produced high levels of IgG2a and low titers of IgG1 and IgG2b, but the titers of the latter two isotypes increased gradually thereafter, tending toward a mixed profile. Intraperitoneal immunization provided a mixed profile directly after the first administration. High titers of anti-Sm28GST Abs also corresponded to high levels of neutralization of the enzymatic activity. These results indicate that rBCG induces strong IgG1, IgG2a, and IgG2b, and neutralizing Ab responses against Sm28GST, which has been found to correlate with protection against S. mansoni in humans.

Activation of the pro-drug ethionamide is regulated in mycobacteria.

The anti-tuberculosis drug ethionamide (ETH), which is a structural analog of isoniazid (INH), is known to strongly inhibit mycolic acid synthesis in Mycobacterium tuberculosis. Although several targets have been identified for INH, only speculative information is available concerning ETH. Mutations within the promoter and the coding region of enoyl-acyl carrier protein reductase (InhA) were found to confer resistance to both drugs, thus leading to the impression that INH and ETH may share a common mode of action. However, a notable distinction between the two drugs lies in the lack of cross-resistance in clinical isolates. This may be attributed in part to the fact that the pro-drug INH must be activated via KatG, and no activation step for ETH has yet been described. Here we report the identification of an activator for ETH. The ETH activator (Rv3854c), which we have termed EthA, was found to be homologous to various monooxygenases and induced ETH sensitivity when overexpressed in mycobacteria. Interestingly, the neighboring open reading frame (Rv3855), which was found homologous to transcriptional repressors of the tetR family, led to ETH resistance when overexpressed. In addition, chromosomal inactivation of this gene by transposition led to ETH hypersensitivity. These data strongly suggest that Rv3855, which we have termed EthR, regulates the production of EthA, which subsequently activates the pro-drug ETH. This study opens up new avenues of research relating to ETH activation in mycobacteria, possibly leading to an improved efficacy of ETH and to the generation of new anti-mycobacterial agents.

Antimycobacterial activities of isoxyl and new derivatives through the inhibition of mycolic acid synthesis.

Isoxyl (ISO), a thiourea (thiocarlide; 4, 4'-diisoamyloxythiocarbanilide), demonstrated potent activity against Mycobacterium tuberculosis H37Rv (MIC, 2.5 micrograms/ml), Mycobacterium bovis BCG (MIC, 0.5 microgram/ml), Mycobacterium avium (MIC, 2.0 microgram/ml), and Mycobacterium aurum A+ (MIC, 2.0 microgram/ml), resulting in complete inhibition of mycobacteria grown on solid media. Importantly, a panel of clinical isolates of M. tuberculosis from different geographical areas with various drug resistance patterns were all sensitive to ISO in the range of 1 to 10 microgram/ml. In a murine macrophage model, ISO exhibited bactericidal killing of viable intracellular M. tuberculosis in a dose-dependent manner (0.05 to 2.50 microgram/ml). The selective action of ISO on mycolic acid synthesis was studied through the use of [1, 2-14C]acetate labeling of M. tuberculosis H37Rv, M. bovis BCG, and M. aurum A+. At its MIC for M. tuberculosis, ISO inhibited the synthesis of both fatty acids and mycolic acids (alpha-mycolates by 91.6%, methoxymycolates by 94.3%, and ketomycolates by 91.1%); at its MIC in M. bovis BCG, ISO inhibited the synthesis of alpha-mycolates by 87.2% and that of ketomycolates by 88.5%; and the corresponding inhibitions for M. aurum A+ were 87.1% for alpha-mycolates, 87.2% for ketomycolates, and 86.5% for the wax-ester mycolates. A comparison with isoniazid (INH) and ethionamide (ETH) demonstrated marked similarity in action, i.e., inhibition of the synthesis of all kinds of mycolic acids. However, unlike INH and ETH, ISO also inhibited the synthesis of shorter-chain fatty acids. ISO showed no acute toxicity against primary macrophage cell cultures as demonstrated by diminution of redox activity. A homologous series of ISO derivatives were synthesized. Most derivatives were as effective or more effective than the parent compound in the agar proportion assay. Thus, these thioureas, like INH and ETH, specifically inhibit mycolic acid synthesis and show promise in counteracting a wide variety of drug-sensitive and -resistant strains of M. tuberculosis.

Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis.

Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.