Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene

Science

Volumn 282, Issue 5389, 1998, Pages 759-762

  Berthet, François Xavier ^a   Lagranderie, Micheline ^a   Gounon, Pierre ^a   Laurent Winter, Christine ^a   Ensergueix, Danielle ^a   Chavarot, Pierre ^a   Thouron, Françoise ^a   Maranghi, Eddie ^a   Pelicic, Vladimir ^a   Portnoï, Denis ^a   Marchal, Gilles ^a   Gicquel, Brigitte ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL PROTEIN;

ANIMAL CELL; ANIMAL EXPERIMENT; ANIMAL MODEL; ARTICLE; BACTERIAL GENE; BACTERIAL VIRULENCE; BACTERIUM MUTANT; COLONY FORMING UNIT; CONTROLLED STUDY; GENE DISRUPTION; MACROPHAGE; MOUSE; MYCOBACTERIUM BOVIS; MYCOBACTERIUM TUBERCULOSIS; NONHUMAN; PRIORITY JOURNAL; TUBERCULOSIS;

ANIMALS; BACTERIAL PROTEINS; BCG VACCINE; CELL LINE; GENES, BACTERIAL; GENETIC COMPLEMENTATION TEST; IMMUNOHISTOCHEMISTRY; LUNG; MACROPHAGES; MEMBRANE PROTEINS; MICE; MICE, INBRED BALB C; MUTATION; MYCOBACTERIUM BOVIS; MYCOBACTERIUM TUBERCULOSIS; PHAGOSOMES; RECOMBINANT FUSION PROTEINS; TUBERCULOSIS; VACCINES, ATTENUATED; VIRULENCE;

ANIMALIA; BACTERIA (MICROORGANISMS); CORYNEBACTERINEAE; MAMMALIA; MYCOBACTERIUM; MYCOBACTERIUM BOVIS; MYCOBACTERIUM TUBERCULOSIS;

EID: 0032561394     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5389.759     Document Type: Article

References (30)

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16. The ERP coding region, deleted of its signal sequence, was amplified by polymerase chain reaction (PCR) using His-2 (5′-AAGGAGATCTTGTGCATATTTTCTTGTCTAC-3′) and His-3 (5′-AAGGAGATCTGGCGACCGGCACGGTGATTGG-3′) oligonucleotide primers, digested with Bgl II and cloned into the Bam HI site of the expression plasmid pQE70 (QIAGEN, Hilden, Germany). The resulting plasmid, designated pHis233, was electroporated into E. coli strain M15.
17. Liquid cultures of E. coli strain M15 [pHis233] were grown in Luria-Bertani broth, induced with isopropyl-β-D-thiogalactoside, and processed for protein purification under denaturing conditions using nickel-nitrilotriacetic acid agarose resin (QIAGEN). Eluted ERP-6His was dialyzed twice for 12 hours against phosphate-buffered saline (PBS) and stored frozen at -20°C. Two rabbits (strain New Zealand) were injected with 100 μg of protein and then injected every 15 days with 150, 200, and 250 μg of ERP-6His emulsified in incomplete Freund's adjuvant. Hyperimmune anti-ERP serum was obtained by bleeding animals 6 weeks after immunization.
18. Cells were fixed with 1% paraformaldehyde in 0.1 M phosphate buffer, washed in the same buffer, and then applied to Formvar - carbon-coated nickel grids that had previously been made hydrophylic by glow discharge. Grids were then processed for immunocytochemistry (76), rinsed with distilled water, and negatively stained with 1% ammonium molybdate in water.
19. 4Cl (50 mM) in PBS for 10 min, bovine serum albumin (BSA) (1% w/v) in PBS for 5 min, antiserum to ERP diluted 1/100 in PBS-BSA for 60 min, and PBS-BSA (three washes, 2 to 5 min each). Samples were then labeled with goat anti-rabbit immunoglobulin G (H and L chains); conjugated to gold grains (5 or 10 nm in size; British Biocell International, UK); diluted 1/20 in PBS plus 0.1% skin fish gelatin (Sigma) for 30 to 45 min; and washed in PBS (one wash, 1 min) and distilled water (three washes, 1 min each). Finally, samples were fixed with 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 min.
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21. V. Pelicic, J.-M. Reyrat, B. Gicquel, FEMS Microbiol. Lett. 144, 61 (1996).
22. A 3.9-kb DNA fragment encompassing the full-length erp gene was excised from pIPX412 by Pst I digestion and cloned into the corresponding site of pACYC177. The resulting plasmid (pPB1) was linearized by Eco RI, which cuts at a unique site within erp. In parallel, an aph cassette conferring resistance to kanamycin was excised by Pst I digestion from the plasmid pUC-4K. Both pPB1 and the aph fragment were treated with T4 DNA polymerase (Boehringer Mannheim) to create blunt ends and were ligated to produce pPB2. A 5.2-kb DNA fragment containing erp::aph was excised from pPB2 by Pst I digestion and cloned into the nonreplicative plasmid vector pJQ200, resulting in the vector pPB3. Five micrograms of pPB3 was introduced by electroporation into M. bovis BCG, which was subsequently plated onto 7H11 Middelbrook plates supplemented with kanamycin (20 μg/ml). Colonies were screened by PCR, with oligonucleotides flanking the Eco RI site used for insertion of aph and by replica-spotting on plates containing kanamycin (20 μg/ml) and 2% sucrose. One clone that contained an insertion of 1.3 kb and that was no longer sensitive to sucrose was analyzed by Southern blotting.
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30. We thank J. Monis for the bacteriophage Ms6 integrative cassette (pIPX70); M. Gheorghiu for helpful discussions; and J. A. Triccas, D. Ojcius, and C. Guilhot for critical reading of the manuscript. F.X.B. is the recipient of a Bourse de la Fondation Roux doctoral grant. This work was supported by grants from the European Economic Community BIOMED 2 (program number BMH4-CT97-2134), the United Nation Development Program/World Bank/WHO Special Program for Research and Training in Tropical Diseases, and NIH (grant number AI 35207).