Mutation of the Principal Cr Factor Causes Loss of Virulence in a Strain
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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8036-8040, August 1995 Microbiology Mutation of the principal cr factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex (r70/RNA polymerase/transcription factor/Mycobacterium bovis) DESMOND M. COLLINS*t, R. PAMELA KAwAKAMI*, GEOFFREY W. DE LISLE*, LISA PASCOPELLA4§, BARRY R. BLOOMt, AND WILLiAM R. JACOBS, JR.* *AgResearch, Wallaceville Animal Research Centre, P.O. Box 40063, Upper Hutt, New Zealand; and tHoward Hughes Medical Institute, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Contributed by Barry R Bloom, May 4, 1995 ABSTRACT Tuberculosis continues to be responsible for M. tuberculosis H37Rv for mice, two clones with overlapping the deaths ofmillions ofpeople, yet the virulence factors ofthe cosmid inserts had a selective growth advantage in mice causative pathogens remain unknown. Genetic complementa- indicating that these inserts contained one or more factors tion experiments with strains of the Mycobacterium tubercu- contributing to virulence. losis complex have identified a gene from a virulent strain that Here we have used the same in vivo complementation restores virulence to an attenuated strain. The gene, desig- technique to restore virulence to an attenuated strain of M. nated rpoV, has a high degree of homology with principal bovis as assessed by a guinea pig model. A DNA fragment of transcription or r factors from other bacteria, particularly 2.7 kb was eventually shown to confer the virulence phenotype, Mycobacterium smegmatis and Streptomyces griseus. The homol- and this contained a single large open reading frame (ORF) ogous rpoVgene ofthe attenuated strain has a point mutation that encoded a principal a- factor that has been designated causing an arginine -* histidine change in a domain known to rpoV.¶ rpoV was shown to differ from the homologous se- interact with promoters. To our knowledge, association ofloss quence in the avirulent strain by a single point mutation that ofbacterial virulence with a mutation in the principal r factor caused an arginine -> histidine change in a region known to has not been previously reported. The results indicate either interact with promoters. that tuberculosis organisms have an alternative principal or factor that promotes virulence genes or, more probably, that MATERIALS AND METHODS this particular mutant principal cr factor is unable to promote expression of one or more genes required for virulence. Study Bacterial Strains and Growth Conditions. M bovis ATCC- of genes and proteins differentially regulated by the mutant 35721 was obtained from the American Type Culture Collec- transcription factor should facilitate identification of further tion. The strain is synonymous with TMC403, which was virulence factors. originally isolated from cattle, deposited by A. G. Karlson in the Trudeau Culture Collection 45 years ago and described as Tuberculosis caused the death of2.5 million people in 1990 and having low virulence for guinea pigs (6). M bovis WAg200 and is predicted by the World Health Organization to kill 3.5 WAg201 were isolated in New Zealand from tuberculous million by the year 2000 (1). Yet the virulence factors of the animals. The strains were identified as M bovis by their colony causative organisms, Mycobacterium tuberculosis and to a lesser morphology, slow growth, acid-fast staining, susceptibility to extent Mycobacterium africanum and Mycobacterium bovis, thiophene-2-carboxylic acid hydrazide and isoniazid, and remain unknown. These species, referred to as the M. tuber- growth on pyruvate-supplemented but not glycerol-supple- culosis complex, are so closely related genetically that they are mented media. Strains were grown statically at 37°C in Middle- better considered as variants of the same species (2, 3). They brook 7H9 liquid medium (Middlebrook 7H11 for solid me- cause a similar disease in humans and guinea pigs and are likely dium) enriched with albumin/glucose complex (Difco), 0.05% to share similar virulence mechanisms. Although the virulence Tween 80, and 1% sodium pyruvate. For electroporation M of these tuberculosis organisms has been investigated for many bovis ATCC35721 was grown in roller bottles at 1 rpm in liquid years, their slow growth and, until recently, the lack of medium supplemented with 1% glycerol. A library of M applicable molecular genetic techniques have prevented tuberculosis Erdman (V. Balasubramanian, Albert Einstein progress toward convincingly identifying any virulence factors. College of Medicine, Bronx, NY) was also used. E. coli strains The development of mycobacterial shuttle vectors and tech- used were X2764 (7) to prepare the cosmid library, XL1-Blue niques to transfer them from Escherichia coli into pathogenic (Stratagene) and HB101 for general cloning, and DH10B (Life and nonpathogenic mycobacteria enabled molecular genetic Technologies, Grand Island, NY) for cloning Pac I inserts studies of mycobacteria to be performed (4). Recently, an recovered from chromosomal DNA. E. coli strains were grown integrating cosmid shuttle vector, pYUB178, was developed with agitation in L broth and X2764 was grown at 30°C. For (5). When this vector is electroporated into strains of the M. selection of clones containing pYUB178, kanamycin was tuberculosis complex it inserts in the genome at a single phage added to culture medium at a concentration of 10 ,ug/ml for integration site in a stable manner without the requirement for M bovis and 50 ,ug/ml for E. coli. continuous antibiotic selection. These properties enabled the Construction and Electroporation of M. bovis WAg2OO entire M. tuberculosis H37Rv genome to be represented in a Library. Genomic DNA was prepared from M bovis WAg200 few hundred clones and for each cosmid to be stably incor- using a scaled-up version of the method described by van porated as a single copy in the avirulent host strain, M. tuberculosis H37Ra (5). While none of the M. tuberculosis Abbreviation: ORF, open reading frame. H37Ra(pYUB178::H37Rv) recombinants was as virulent as tTo whom reprint requests should be addressed. §Present address: Rocky Mountain Laboratories, 903 South 4th Street, Hamilton, MT 59840. The publication costs of this article were defrayed in part by page charge 1The sequences reported in this paper have been deposited in the payment. This article must therefore be hereby marked "advertisement" in GenBank data base [accession nos. U21130 (WAg200), U21131 accordance with 18 U.S.C. §1734 solely to indicate this fact. (WAg201), U21132 (ATCC35721), and U21134 (Erdman)]. 8036 Microbiology: Collins et aL Proc. Natl. Acad. Sci. USA 92 (1995) 8037 Soolingen et al. (8) and partially digested with Sau3AI. Frag- A Recombinant M. bovis clones inoculated ments of 30-50 kb were prepared using sucrose gradient centrifugation (9), ligated to Bcl I-digested pYUB178, pack- aged into A heads (GigaPack II Gold, Stratagene), and trans- 1781 1178 duced into E. coli X2764 (5). Kanamycin-resistant clones were pooled and cosmid DNA was prepared by alkaline lysis (10). Washed M. bovis ATCC35721 cells were prepared and an aliquot (0.4 ml) was electroporated with 4 jig of pooled cosmid DNA using previously described methods (11). Guinea Pig Infection. The virulence of M bovis strains was tested in female Duncan-Hartley guinea pigs using a variation of a method described previously (6). Each strain was tested in three guinea pigs that were housed in a level 3 containment ,_4. facility. Guinea pigs were inoculated subcutaneously in the flank with 106 colony-forming units (cfu), except that 107 cfu were used in the case of the pool of M bovis ATCC- 35721(pYUB178::WAg200) clones. Animals were sacrificed after 12 weeks for the initial experiment with library and control animals and after 8 weeks for subsequent experiments. .:...... Animals were examined for the presence of gross lesions of -_ _ -1.2 tuberculosis. Formalin-fixed tissues from the spleen, liver, kidney, and lung were embedded in paraffin, sectioned at 3-5 ,um, stained either with hematoxylin/eosin or by the Ziehl- Neelsen method, and examined for histological evidence of tuberculosis. Representative samples from the injection site, preferred lymph nodes, and spleen were cultured for M bovis. B Recombinant M. bovis clones from lesions Characterization ofM. bovis Recombinants. Recombinants containing integrated cosmids were characterized by junction xI 175 fragment analysis (5). DNA was extracted (8) from M bovis ATCC35721(pYUB178::WAg200) clones, digested with Pst I, and subjected to agarose gel electrophoresis. DNA was trans- ferred to nylon membranes by Southern blotting and the membranes were hybridized with probes of pYUB178 labeled by nick-translation with 32p (10) to reveal the size of fragments - 5.7 at the junction sites where the integrated vector arms join the cosmid insert and host chromosomal DNA. - fto --- 9 .0 0 - 3.8 Cosmid Subcloning. Vector pUHA8 was constructed by .... incorporating aPac I site on either side of the Bcl I cloning site of pYUB178. A cosmid that conferred virulence on M bovis ATCC35721 was partially digested with Sau3AI and fragments of 2-4 kb were prepared by sucrose gradient fractionation (9) and ligated into the Bcl I cloning site of pUHA8. DNA Sequencing and Analysis. Ordered deletions of the 2.7-kb fragment in a pBluescript derivative that contained a - 1.2 Pac I cloning site were generated using the Erase-a-base system (Promega). Complete sequencing in both directions was performed using a cycle sequencing kit (Life Technolo- gies) with 33P-labeled ATP. Homologous fragments from other M tuberculosis complex strains were sequenced in both FIG. 1. Autoradiographs of Southern blots of DNA from recom- directions using oligonucleotide primers designed from the binant M. bovis ATCC35721(pYUB178::WAg200) clones. DNA was sequence of the 2.7-kb fragment. Sequencing of the relevant digested with Pst I and hybridized with a probe of the vector pYUB178 genes from wild-type M bovis and M tuberculosis Erdman to reveal the size of fragments at the junction sites between the strain was performed by the same methods.