GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK1
London School of Hygiene & Tropical Medicine, Keppel St, London WC1E 7HT, UK2
Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA3
Author for correspondence: Ken Duncan. Tel: +44 1438 763841. Fax: +44 1438 764799. e-mail: kd9430{at}gsk.com
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ABSTRACT |
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Keywords: gene disruption, severe combined immune deficiency (SCID) mouse, attenuation
Abbreviations: BCG, Bacille CalmetteGuérin; SCID, severe combined immune deficiency
b The precise locations of all of the insertions examined in this study can be found as supplementary data in Microbiology Online (http://mic.sgmjournals.org).
a Present address: Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111, USA.
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INTRODUCTION |
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A novel therapeutic agent with the potential to reduce the duration of chemotherapy from the current minimum of six months to a more acceptable period would make a very significant impact on treatment outcomes in the clinic (OBrien & Nunn, 2001 ). However, this strategy relies upon killing a poorly understood population of bacteria that persists for long periods of time despite the presence of highly active drugs. The enzyme isocitrate lyase has been shown to be required for the survival of M. tuberculosis in the persistence phase in a murine model of infection (McKinney et al., 2000
). Identification of more targets within M. tuberculosis with similar properties to isocitrate lyase would increase the likelihood of new drugs against this organism being found.
The only available M. tuberculosis vaccine, the Bacille CalmetteGuérin (BCG), is an attenuated strain of the M. tuberculosis-related species Mycobacterium bovis. Its protective efficacy varies widely (from 0 to 80%) in different parts of the world (Fine, 1995 ) and is thought to be waning with time (Behr & Small, 1997
). A potential route to generating a new vaccine candidate is rational attenuation of M. tuberculosis. This approach requires the identification and characterization of the genes that contribute to the virulence of M. tuberculosis.
Random mutagenesis using mobile genetic elements such as transposons has been a highly successful approach for identifying virulence factors in many bacterial pathogens (Manoil & Beckwith, 1985 ; Bowe et al., 1998
). The transposon inserts into host DNA thus inactivating individual genes and operons. Bacterial strains with mutations in their metabolic pathways can then be identified using selective medium in vitro, and genes which are not essential for growth in vitro can be screened for a role in vivo using virulence assays. The location of the transposon in the chromosome of a mutant may be determined by sequencing the junction of the transposon and chromosomal DNA. After saturating mutagenesis, essential genes or pathways can, in theory, be identified as those without transposon insertions by using genomic footprinting techniques (Akerley et al., 1998
).
In mycobacteria, transposons Tn5367 and Tn5368 (McAdam et al., 1995 ), and Tn5370 (Cox et al., 1999
) derived from IS1096 (Cirillo et al., 1991
; McAdam et al., 1995
) can be efficiently delivered to the bacteria on a temperature-sensitive mycobacteriophage (Bardarov et al., 1997
) or by using a temperature-sensitive, counter-selectable plasmid (Pelicic et al., 1997
). Transposons derived from Mariner can also be used to generate mutants in mycobacteria (Rubin et al., 1999
; Sassetti et al., 2001
). The studies detailed above have been successful in isolating mycobacterial strains with mutations in their metabolic pathways.
To identify virulence factors in M. tuberculosis, the technique of signature-tagged mutagenesis (Hensel et al., 1995 ) has been applied (Camacho et al., 1999
; Cox et al., 1999
). Pools of mutant bacteria generated using a number of transposons, each having different sequence tags, were inoculated into mice and the clearance of individual mutants was monitored by hybridization of the surviving pool to the tags; the identity of mutants that did not grow in the mice was subsequently determined. The advantage of this approach is the direct selection for the virulence phenotype in an animal model, but its success relies on the quality of the mutants in the input pool, the nature of which is not known in advance. Two groups working independently of each other used signature-tagged mutagenesis to screen mutant libraries generated by transposons Tn5367 and Tn5370 in M. tuberculosis; both groups identified mutants in a limited set of genes involved in lipid metabolism (Camacho et al., 1999
; Cox et al., 1999
). Therefore, it is possible that the number of virulence factors that can be detected in M. tuberculosis by this method is low or, alternatively, the libraries created by IS1096-derived transposons are limited in their degree of complexity.
We have taken a different approach to using transposon mutagenesis to identify genes essential for the virulence of M. tuberculosis. We generated a library of mutant M. tuberculosis strains by delivery of transposon Tn5370 in pJSC84 to the organism using the temperature-sensitive bacteriophage phAE87 (Cox et al., 1999 ) and subsequently identified the transposon position in 1474 of the mutants. This approach revealed the precise identity of each mutant and provided a choice of candidates to screen in vivo. We found that Tn5370 did not insert into the genome of M. tuberculosis in a completely random fashion, which limited the number of ORFs mutated. A pilot experiment to test the virulence of a random selection of the mutants in severe combined immune deficiency (SCID) mice identified mutants with either significantly increased or decreased virulence, and singled out one ORF of unknown function that contributed strongly to the loss of virulence of M. tuberculosis in the SCID mouse.
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METHODS |
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Transposon mutagenesis.
Lysates of temperature-sensitive phage phAE87 carrying Tn5370 were prepared at 30 °C in Mycobacterium smegmatis mc2155, as described previously (Bardarov et al., 1997 ). M. tuberculosis H37Rv was grown to late exponential phase in 100 ml M-ADC-TW. It was then harvested by centrifugation, washed in 10 ml of a pre-warmed buffer (10 mM Tris/HCl, pH 7·6, 100 mM NaCl, 10 mM MgSO4, 2 mM CaCl2) and resuspended in 10 ml of the same buffer. Cells of M. tuberculosis H37Rv were infected with a high-titre phage lysate (>1x1011 p.f.u. ml-1) at an m.o.i. of 10, and incubated at the non-permissive temperature of 39 °C for 4 h to allow phage attachment. Cells were then plated directly onto selective medium. Transposition frequency, as measured by the number of hygromycin-resistant mutants (total number of input cells)-1, was generally <1x10-6 mutants (input cell)-1. Spontaneous hygromycin resistance was seen in <1x10-9 mutants (input cell)-1.
Ten-thousand colonies were picked from the selective medium and inoculated into individual wells in 96-well microtitre plates containing Middlebrook 7H9 medium, and grown to high cell density at 37 °C. Replica plates were generated and stored at -80 °C.
Genomic DNA extraction.
A protocol based on the method of Pospiech & Neumann (1995) for the extraction of genomic DNA was adapted for a 96-well plate format. For each mutant strain, a 3 ml culture was grown and harvested by centrifugation. The cell pellet was then transferred to a 96-well deep-well plate (Advance Biotech) containing 200 µl lysozyme (5 mg ml-1) in 20 mM Tris/HCl (pH 9·0) and incubated overnight at 37 °C. The cells were then lysed by adding SDS to 2% and proteinase K to 33 µg ml-1 to the wells, followed by further incubation at 37 °C for 3 h. The lysate was transferred to a screw-cap microfuge tube containing 90 µl of 5 M NaCl and 200 µl CHCl3, and the sample was centrifuged at 14000 r.p.m. for 15 min to separate the aqueous and organic phases. The upper aqueous phase was transferred to a 96-well plate; DNA was precipitated from the sample by the addition of 0·6 vol. 2-propanol. The sample was then spun at 2000 r.p.m. for 30 min, and the resulting pellet was resuspended in 50 µl of 5 mM Tris/HCl (pH 8·0) buffer.
Sequencing of the transposon-insertion site.
A restriction-site PCR method adapted from Sarkar et al. (1993) was used for sequencing the transposon-insertion sites. PCRs were performed in 96-well plates (Costar). The first round of PCR used a transposon-specific primer (SP1, 5'-TGCAGCAACGCCAGGTCCACACT-3') and an arbitrarily-degenerate primer (RS6-4, 5'-GTAATACGACTCACTATAGGGCNNNNCATG-3') to amplify the chromosomal sequence flanking the transposon-insertion site. PCRs were carried out in a total volume of 20 µl in 10 mM Tris/HCl (pH 8·3), 50 mM KCl, 1·5 mM MgCl2, 0·01% (w/v) gelatin, 0·25 mM dNTPs, 0·1 µM SP1, 0·5 µM RS6-4 and 0·25 U AmpliTaq (Perkin Elmer). First-round cycling was performed with an initial denaturation step at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 5 min, annealing at 50 °C for 30 s and extension at 72 °C for 90 s, with a final extension step at 72 °C for 7 min.
A second round of PCR was done using 1 µl of the first-round products as template and the nested primers SP2 (5'-CTCTTGCTCTTCCGCTTCTTCTCC-3') and T7 (5'-TAATACGACTCACTATAGGG-3') to generate more specific products for sequencing. Reactions were carried out in a total volume of 20 µl in 10 mM Tris/HCl (pH 8·3), 50 mM KCl, 1·5 mM MgCl2, 0·01% (w/v) gelatin, 0·25 mM dNTPs, 0·5 µM SP2, 0·5 µM T7, 5% (v/v) DMSO and 0·25 U AmpliTaq. Second-round cycling was performed with a denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 15 s and extension at 72 °C for 30 s, with a final extension step at 72 °C for 7 min.
Sequence determination.
PCR products were treated with Exonuclease I (USB) and shrimp alkaline phosphatase (USB) for 30 min at 37 °C, to degrade residual primers. This treatment was followed by heat inactivation at 65 °C for 20 min. A further nested primer (SP3, 5'-GCACACCCAAGCCAACCAGACC-3') was used as the sequencing primer. PCR products were sequenced on an ABI 3700 sequencer (PE Biosystems) using the BigDye terminator chemistry. Cycle sequencing was performed with 30 cycles of denaturation at 95 °C for 5 min, annealing at 55 °C for 15 s and extension at 64 °C for 4 min.
Location of the transposon-insertion site.
Customized PERL scripts that made extensive use of BioPerl modules (http://bioperl.org) were used to analyse the sequencing results. To identify the precise genome insertion position of the transposon in each mutant, the transposon sequence was trimmed and a BLASTN search of the sequence against the complete H37Rv genome sequence (obtained from http://www.sanger.ac.uk/Projects/M_tuberculosis/) was used to align the two sequences. Only alignments in which at least 20 bp of the chromosomal sequence completely matched the H37Rv genome sequence were retained for further analysis; sequences without transposon sequences were discarded.
Infection of mice and tissue analysis.
For the first in vivo screens, CB-17/Icr SCID mice obtained from Dr C. M. Hetherington (National Institute for Medical Research, Mill Hill, London) were bred under aseptic conditions at the London School of Hygiene & Tropical Medicine. Female mice of between 8 and 10 weeks of age were used. Mice were infected with 1x106 viable mycobacteria in 200 µl of pyrogen-free saline via a lateral tail vein. Where appropriate, infected mice were killed by cervical dislocation in accordance with the humane end-point protocols covered under the Animals Scientific Procedures Act, 1986 (UK).
In a parallel set of in vivo screens, 6-week-old female SCID mice were obtained from the Albert Einstein College of Medicine Animal Institute and infected by the intravenous route with 1x103 viable mycobacteria.
Median survival times were calculated for each group of mice; statistical analyses were performed using KaplanMeier plots and log rank tests of survival.
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RESULTS |
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In total, 1474 insertion sites were mapped. Unique insertion sites (1329 in total) were identified, 116 insertions appeared in the exact same position twice, 13 appeared in the exact same position three times and one appeared in the exact same position four times. These identical mutants could have arisen from either sibling colonies or cross-contamination. On 20 occasions mutants with the same insertion position were located on the same sequencing plate; on five occasions mutants with the same insertion position were found in adjacent wells. The precise locations of all of the insertions examined in this study can be found as supplementary data in Microbiology Online (http://mic.sgmjournals.org). Eighty-one percent (1189) of the insertions were located within ORFs and 19% (285) were intergenic; the proportion of insertions into ORFs (81%) was slightly less than the proportion of the M. tuberculosis genome that encodes proteins (90%; Cole et al., 1998 ). In total, 351 ORFs were mutated; the characteristics of these ORFs are shown in Table 1
. The distribution of the transposon mutants amongst the TubercuList (http://genolist.pasteur.fr/TubercuList/) functional classes of M. tuberculosis is shown in Table 2
; the percentage of mutated ORFs within each functional class was between 5 and 12%, with the exception of the PE/PPE class [acidic, glycine-rich proteins of the PE (ProGlu) and PPE (ProProGlu) protein families], which was overrepresented. Sequencing efforts were terminated at this stage, as the number of insertions into ORFs that had not previously been interrupted had dropped to less than 10% on each microtitre plate.
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We compared the insertion positions of Tn5370 in our library to literature reports of IS6110 insertion loci and deletions occurring in clinical strains of M. tuberculosis. Of the 45 ORFs in the clinical strains of M. tuberculosis described as being disrupted by IS6110 (Sampson et al., 2001 ), 14 matched ORFs containing insertions obtained in this study. In addition, three of the 22 intergenic insertion regions were common to both sets of data. In a study to detect small-scale genomic deletions in 19 clinical isolates of M. tuberculosis, Kato-Maeda et al. (2001
) found that 11 of the 25 regions deleted within these isolates contained transposon insertions. These insertion regions include three of the five regions described as being associated with IS6110. Six of the 23 regions deleted in the M. tuberculosis clinical strain CDC 1551 compared to H37Rv (Betts et al., 2000
) were disrupted by Tn5370, and of the 19 regions deleted from M. bovis BCG (Salamon et al., 2000
; Domenech et al., 2001
), 11 were found to contain transposon insertions. It is of note that a high proportion of these previously reported unstable regions of the genome are susceptible to Tn5370 insertion. However, only four of the regions that were found to have high transposon-insertion rates in our library (Rv1917c, Rv1500Rv1507c, Rv2351cRv2353c and Rv3343c) contained IS6110 insertion loci or were deleted from M. tuberculosis clinical strains or M. bovis BCG, suggesting that transposition was not biased to these regions.
Approximately 173 (49%) of the disrupted ORFs could lie within operons. Fifty-eight of these were within the last gene of the operon and were, therefore, unlikely to affect expression of other genes. In total, an estimated 90 operons were disrupted. This includes seven insertions in the phosphophthiocerol operon, which has been identified by others as a virulence gene cluster using signature-tagged transposon mutagenesis (Camacho et al., 1999 ; Cox et al., 1999
).
Virulence of mutants in SCID mice
Since strains were obtained with mutations in ORFs covering a variety of functional classes, the first screen for virulence in vivo was designed to encompass mutations in a broad range of metabolic pathways to determine whether any played an essential role in vivo. Strains with mutations in ORFs from functional classes including DNA repair, DNA transcription, cell division, cell processes, regulators, degradation of macromolecules, small-molecule degradation, energy metabolism and lipid metabolism were chosen (Table 4). A random selection of mutants was made, to avoid biasing the outcome by testing those mutants that we predicted would be attenuated because of some pre-existing knowledge. None of the mutants chosen displayed any obvious phenotype in vitro. The SCID mouse has previously proven very sensitive in revealing changes in M. tuberculosis virulence in the absence of acquired immunity (Smith et al., 2001
). Mice were infected intravenously with either mutants or wild-type M. tuberculosis H37Rv and their survival was monitored (Table 4
). Fig. 3
shows the statistical analysis of median survival times for the transposon mutants compared to the wild-type strain. This experiment was conducted in two groups of mice. Mice infected with M. tuberculosis H37Rv died with a median survival time of 32·5 (Fig. 3a
) and 32 days (Fig. 3b
). Of the 11 mutants tested, five were unchanged in their virulence when compared to the wild-type strain. One mutant was marginally attenuated in its virulence, as defined by statistical analysis (P<0·05); this strain had a mutation in pvdS, which encodes a putative regulator. The strain with a mutation in Rv1773c, encoding a transcriptional regulator belonging to the IclR family, was marginally more virulent (P<0·05) than the pvdS mutant. Two further mutant strains were more virulent at the P<0·01 level than the pvdS or Rv1773c mutants; the increased virulence of these strains was due to mutations in Rv3252c, encoding a possible monooxygenase, and in Rv3213c, encoding a protein with similarity to Soj, a sporulation inhibitor from Bacillus subtilis, respectively. Interestingly, two of the mutants tested, Rv2467 and Rv0469, were significantly more virulent (P<0·001) than the other mutants, and had disruptions in pepD (encoding an aminopeptidase) and umaA1 (encoding an unknown mycolic acid methyltransferase), respectively.
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DISCUSSION |
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The advance in homologous recombination techniques for M. tuberculosis now means that it is possible to target any gene of choice. However, the process of selecting and inactivating individual genes on a large scale is an awesome one. Transposon mutagenesis offers a convenient way of generating large numbers of mutant bacteria. Isolation of a mutant strain implies that disruption of the gene into which the transposon has inserted has not had a lethal effect on survival of the bacterium in culture. This technique is, therefore, unsuitable for generating mutations in genes essential for growth. Thus, we set out to saturate the chromosome of M. tuberculosis with insertions of transposon Tn5370 by generating a library of mutants, and then to identify the insertion points, and thus the genes disrupted by these insertions, by sequencing the junction between the transposon and chromosomal DNA. It was hoped that this genome-scale approach would suggest which genes were essential for growth of M. tuberculosis by identifying those that did not have a transposon insertion. This approach was limited by the randomness of transposition if there was any insertion bias and by polar effects caused when the insertion was within an operon that had an essential gene or genes distal to the site of disruption. Our use of the mini-transposon Tn5370 ensured that the resulting M. tuberculosis mutants were stable. The presence of the res sites in the reshygres gene cassette facilitated removal of the hygromycin-resistance marker, by transient expression of the Tn resolvase gene in the mutated strain. This would allow us to further analyse phenotypically the unmarked mutant strains as well as to generate multiple mutations in isogenic backgrounds.
Our sequencing effort has so far revealed 1474 Tn5370 transposon-insertion sites within the M. tuberculosis H37Rv genome, disrupting 351 genes. As expected, the proportion of insertions into ORFs (81%) is less than the proportion of the M. tuberculosis genome that is protein-coding (90%). Analysis of clusters of insertions showed that Tn5370 insertion generally favours regions of the genome that have a lower-than-average G+C content. This limits our ability to identify those rare insertions into regions of the chromosome with an above-average G+C content. That 25% of the regions containing IS6110 loci in M. tuberculosis, and 42% of the regions deleted from M. tuberculosis clinical strains or M. bovis BCG, were found to have Tn5370 insertions suggests that these regions of the genome are susceptible to transposon insertion. It is thought that recombination between adjacent insertion elements is a common mechanism through which genomic regions become deleted (Brosch et al., 2001 ). Insertions into these regions, however, were not the most frequently represented insertions in the Tn5370 library and did not bias the distribution of the disrupted genes. With the exceptions of the plcABC cluster and pks2 (Parish & Stoker, 2000
; Sirakova et al., 2001
), none of the insertions we generated were into genes that have already been disrupted by homologous recombination methods; therefore, we expect that very many more genes can be disrupted in the M. tuberculosis genome. It should, however, be noted that if a mutant in a particular gene of interest is required homologous recombination methods are more appropriate than transposon mutagenesis.
We randomly selected strains from our library that had mutations in a variety of metabolic pathways and in some ORFs of unknown function and tested their virulence in SCID mice, as we were interested to discover whether any of our mutants had an altered phenotype in vivo. We, and others, have used the SCID mouse model successfully to define the strong attenuation in virulence seen in a number of auxotrophic mutants of M. tuberculosis unable to synthesize tryptophan (trpD), proline (proC) or arginine (argF) (Smith et al., 2001 ; Gordhan et al., 2002
) these mutants also exhibited a very strong attenuation in virulence, reflected in a reduction in c.f.u. values and survival in immunocompetent mice. In the screening of the transposon mutants generated in this study, we found only marginal attenuation of virulence in some of the strains and 39% of the mutants screened showed virulence equal to that of wild-type M. tuberculosis. The latter included M. tuberculosis strains with mutations in fpg, responsible for the repair endonuclease protein, and in lsr2, which encodes a protein that is a putative cell-wall antigen recognized by T-cells from leprosy patients (Laal et al., 1991
). Surprisingly, a significant proportion of the mutants were hypervirulent these comprised strains with mutations in genes encoding members of several of the functional classification groups studied and included strains with mutations in pepD, an aminopeptidase or metalloprotease gene, and umaA1, a gene encoding a mycolic acid methyltransferase involved in cell-wall biosynthesis.
At present, we are unable to explain the mechanism for the strong attenuation of virulence observed with the Rv1290c mutant because of the unknown function of the protein that this gene encodes. The fact that this gene is present in M. tuberculosis and M. bovis strains but is missing from M. leprae, M. smegmatis and M. avium clearly shows that the Rv1290c gene product does not play a role in determining the cell-division rate. Its insertional inactivation, however, shows that it is not essential for the in vitro growth of virulent M. tuberculosis. The strong attenuation of virulence of the Rv1290c mutant in the SCID mouse model clearly shows that this gene has an important in vivo function in determining the extent of the virulence of M. tuberculosis. This makes this mutant strain a good candidate for further study of the in vivo growth kinetics of M. tuberculosis and any effect on the immunopathology of infection by this organism in an immunocompetent mouse model.
In conclusion, we have demonstrated that it is possible to generate and identify large numbers of M. tuberculosis mutants using transposon mutagenesis. We have also shown that transposon Tn5370 favours insertion into low-G+C regions of the M. tuberculosis chromosome. In a pilot experiment in a highly immunocompromised mouse model, we have shown that several of the mutants we generated have altered phenotypes in vivo. This suggests to us that M. tuberculosis is a pathogen well-adapted to its niche, and that inactivation of individual genes within this organism can slightly increase or decrease its virulence. However, inactivation of some genes of M. tuberculosis, notably Rv1290c, can have a significant impact on the virulence of this bacterium in the absence of acquired immunity. Hence, the Rv1290c gene is a candidate for further investigation of its role in M. tuberculosis virulence. Further studies using immunocompetent mice will identify genes within M. tuberculosis which regulate its survival in the presence of adaptive immunity.
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ACKNOWLEDGEMENTS |
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Received 26 April 2002;
revised 5 June 2002;
accepted 25 June 2002.