1 Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Department of Medical Microbiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK
3 National Mycobacteria Reference Laboratory, National Institute of Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands
Correspondence
Roger S. Buxton
rbuxton{at}nimr.mrc.ac.uk
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ABSTRACT |
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Present address: Division of Medical Microbiology, Departamento de Patologia, Federal University of Ceara, Rua Monsenhor Furtado, S/N Rodolfo Teofilo, Fortaleza-Ceara, 60441-750, Brazil.
Present address: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
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INTRODUCTION |
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M. microti has also been employed as a vaccine against tuberculosis and was reported to give a high degree of protection, similar to that seen with BCG (Mycobacterium bovis bacille Calmette-Guérin) (Sula & Radkovsky, 1976). An attenuated strain of M. microti was used as a vaccine in Czechoslovakia from 1951 to 1969, and about 500 000 people, mostly newborn, were vaccinated intradermally. In a separate study in the UK between 1950 and 1952, a vaccine was prepared from non-attenuated M. microti and used in vaccination trials organized by the Medical Research Council (Hart & Sutherland, 1977
). The trials in Czechoslovakia and the UK gave similar results, with the M. microti vaccine conferring about 75 % protection. Furthermore, the M. microti vaccine showed low allergenic potency, making it less likely than BCG to compromise the tuberculin test in the vaccinated population. The M. microti vaccine induced fewer than 30 % positive skin test conversions in response to Purified Protein Derivative (PPD) from M. tuberculosis (Brooke, 1941
; Sula & Radkovsky, 1976
; Bloom & Fine, 1994
). A recent comparison of efficacy between the Pasteur substrain of BCG and the M. microti vaccine showed that both could provide protection against tuberculosis in rabbits (Dannenberg et al., 2000
) and mice (Manabe et al., 2002
). Despite conferring good protection against tuberculosis, M. microti can still cause clinical disease in immunocompetent patients, and therefore its use in vaccination may constitute a health hazard (van Soolingen et al., 1998
).
Identification of M. microti by traditional methods is not easy, so the prevalence, geographical distribution and clinical importance of M. microti may have been underestimated (van Soolingen, 2001). M. microti grows slowly on solid media supplemented with pyruvate, and shows similar biochemical properties to M. tuberculosis, giving variable results for pyrazinamidase and urease activity and niacin accumulation (Levy-Frebault & Portaels, 1992
; van Soolingen et al., 1998
). Two separate studies have shown the usefulness of novel genetic markers (IS6110) to characterize M. microti isolates (Kremer et al., 1998
; van Soolingen et al., 1998
). Moreover, cases of M. microti-derived tuberculosis in both immunocompromised and immunocompetent human patients have been identified using molecular methodologies (Foudraine et al., 1998
; Niemann et al., 2000
).
The virulence mechanisms of M. microti in its natural hosts, and its low virulence in humans, are not well understood. Like that of M. tuberculosis, the M. microti cell wall contains 34 % by weight of mycolates. In both species, exponential-phase in vitro cultures and bacteria harvested from mouse lungs contain a high proportion of ketomycolates, whereas in stationary-phase cultures, ketomycolates decrease rapidly to give proportions similar to those of methoxymycolates (Davidson et al., 1982; Watanabe et al., 2001
). Although there is no clear relationship between possession of a particular mycolate and strain pathogenicity, the same mycolate types are present in all M. tuberculosis complex strains, differing only in chain length and other structural features (Daffé & Draper, 1998
; Kremer et al., 2000
).
In this study, comparative genomics, by microarray assay methods, were employed to delineate the genetic differences between M. tuberculosis and M. microti, and to relate these to virulence. DNA microarrays were used to study a group of 12 strains classified as M. microti, including the strain OV254, originally isolated from voles in the 1930s (Wells, 1937). M. microti strains isolated from other mammals, such as pig, llama and hyrax, were also included. Lastly, M. microti strains have more recently been isolated from human tuberculosis infections (van Soolingen et al., 1998
), from both immunocompromised and immunocompetent individuals, and we included these strains in our investigation. These strains were identified as M. microti using the spacer oligonucleotide typing, spoligotyping, DNA fingerprinting method (van Soolingen et al., 1998
).
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METHODS |
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Synthesis of labelled DNA.
Genomic DNA was labelled with dCTP coupled to Cy3 or Cy5 dyes (Amersham Pharmacia Biotech). DNA (3 µg) was mixed with 3 µl random hexamer primers (1 µg ml1, Invitrogen) in a total volume of 41·5 µl, and the primers were annealed to the DNA by incubating the mix at 95 °C for 5 min, followed by rapid cooling on ice. The tubes were centrifuged briefly, and the following were added: 5 µl 10x Klenow polymerase buffer (Promega), 1 µl dNTP mix (5 mM dA/G/TTP and 2 mM dCTP, Amersham Pharmacia Biotech), 1·5 µl Cy3- or Cy5-dCTP (25 nM, Amersham Pharmacia Biotech) and 1 µl Klenow DNA polymerase (5 U µl1, Promega). The reaction mixture was incubated at 37 °C for 90 min, and labelled cDNA was eluted in 13 µl distilled water after extraction in a MiniElute column (Qiagen). The probe was mixed with 3·2 µl 20x SSC (0·15 M NaCl, 0·015 M sodium citrate), and 0·3 µl 20 % (w/v) SDS, and then denatured by heating at 95 °C for 2 min. The mix was allowed to cool briefly before being added to the array.
Slide processing.
Spotted microarray slides, representing 100 % of the genes of the M. tuberculosis H37Rv genome, were prepared at the Bacterial Microarray Group, St George's Hospital Medical School, London. The rehydration and blocking steps were carried out as described by Eisen & Brown (1999).
Hybridization and washing.
The processed array slides were placed in prehybridization solution (3·5x SSC, 0·1 %, w/v, SDS and 0·1 % fraction V BSA [Sigma]) at 60 °C for 20 min, to block non-specific probe-binding sites. The slides were then rinsed sequentially for 1 min in distilled water and 1 min in 2-propanol (Merck), and then centrifuged in 50 ml centrifuge tubes at 433 g for 5 min. Samples of denatured, labelled probe (16 µl) were applied to a dried, prehybridized slide. Post-hybridization and slide-washing steps were performed as described by Eisen & Brown (1999).
Data collection and analysis.
Slides were scanned in a dual-laser microarray scanner (Genepix 4000A, Axon Instruments) and the images obtained were analysed using GenePix Pro software (Axon Instruments). The software calculated the mean signal intensity and local background for each spot on the array and subtracted the local background from the signal intensity of each spot. Spots showing a high background or poor hybridization were eliminated from the analysis. Data obtained from six slides, including dye swaps, were analysed with GeneSpring software, version 4.1.5 (Silicon Genetics). To account for dye swap, the signal channel and control channel measurements for dye swap samples were reversed. Each gene's measured intensity was divided by its control channel value in each sample; if the control channel was below 10, then 10 was used instead. If the control channel and the signal channel were both below 10 then no data were reported. Each measurement was divided by the 50·0th percentile of all measurements in that sample. The percentile was calculated using only genes marked present.
PCR amplification and DNA sequencing of deletions.
Primers were designed to flank known deletion sites (Table 2). DNA was amplified using HotStar Taq polymerase (Qiagen), and the products purified using a Qiaquick PCR purification kit (Qiagen). DNA sequencing was carried out on an ABI377 DNA sequencer using a Big Dye terminator kit (ABI).
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RESULTS |
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Table 3 shows genes deleted in M. microti and demonstrates that, apart from three strains isolated from humans, the pattern of deleted sequences was different in every strain examined. In comparison with M. tuberculosis H37Rv, each M. microti strain had an average of 9·4 deleted regions. Among the 12 strains, a total of 13 different deleted sequences were detected.
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Presence of esat6 within the RD1 deletion in M. microti
esat6 (Rv3875) appears in a cluster region along with lhp (Rv3874) in M. tuberculosis and other GC-rich bacteria, including M. bovis and Mycobacterium leprae (Gey van Pittius et al., 2001). Recent results analysing the function of the ESAT-6 and CFP-10 proteins, known to form a tight 1 : 1 complex (Renshaw et al., 2002
), have shown them to be required for invasion of lung interstitial tissue (Hsu et al., 2003
). The extent of the RD1 deletion, where esat6 is located, was found to be highly variable in M. microti (Fig. 2
). Besides the deletion of genes Rv3871Rv3876, most strains had the additional, contiguous RD1
deletion, which meant that the entire deleted region extended from Rv3864 through to Rv3876, compared with Rv3871 to Rv3879c for BCG (Gordon et al., 1999
). However, one strain, vole scab15, had the RD1
deletion in the absence of RD1, and in two strains RD1 resulted in the deletion of a single gene only: Rv3871 in strain vole 15498, and esat6 (Rv3875) in strain human 97-0770.
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The plcABC gene cluster
The RD5 region, which includes the phospholipase C operon, was variable among the M. microti strains, with Rv2350c (plcB) being the most frequently deleted gene (Fig. 3). In BCG, RD5 deletion was different, deleting from Rv2344 to Rv2354. Interestingly, plcD appeared to be deleted in two M. microti strains (hyrax and human 97-0770). In M. tuberculosis, plcD is not located close to plcA, B or C ; at the time of writing the precise location of plcD in M. microti is not known.
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RD6 and RD8 deletions
The RD6 region, which encodes PE/PPE proteins, was described by Brodin et al. (2002) as not being deleted in M. microti strains. In the present study, the situation was variable, with the region being deleted in three of the twelve strains. The RD8 region was deleted in the majority of the strains, but in each case the deletion was not continuous, with two genes (Rv3619c and Rv3620c, encoding proteins of unknown function) present in all the analysed strains, as evidenced by hybridization to the Rv3619c and Rv3620c microarray probes. However when the microarray probes for these two genes were analysed for potential cross-hybridization with other M. tuberculosis genes, each gave very high BLAST homology scores with two other genes (data not shown). Thus the presence of Rv3619c and Rv3620c within the M. microti genome could be an artefact due to cross-hybridization. We believe, therefore, that the RD8 deletion is probably continuous in M. microti. The RD8 deletion in BCG extends from Rv3617 to Rv3622c.
Newly identified deletions: MiD1, MiD2 and MiD3
Two of the newly identified deletions, MiD1 and MiD3, were deleted in the majority of the M. microti strains; MiD1 encodes conserved hypothetical proteins and MiD3 encodes insertion sequences and PE/PPE proteins. The MiD2 region was deleted only in two vole isolates (OV254 and vole 15498), both being strains originally isolated from voles in the 1960s. Therefore, this deletion could have occurred during multiple passages of these strains in culture media. The Rv3345c, Rv3346c and Rv3348 microarray probes from the variable MiD3 region also show cross-hybridization with other genes encoding PE/PPE and PGRS proteins; nevertheless the deleted region from PE PGRS50 (Rv3345c) to Rv3349c was confirmed by PCR amplification and sequencing (see Table 2).
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DISCUSSION |
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With regard to deleted regions and virulence, this study shows that it is difficult to ascribe virulence to any particular pattern of deletion. Although RD1 of BCG and M. microti is thought to be crucial for attenuation (Mahairas et al., 1996; Brodin et al., 2002
; Pym et al., 2002
), in this study three of the four M. microti strains from immunocompetent patients had the RD1 region deleted. It is also noticeable that of the 12 strains studied, only three were identical in deletion pattern; these strains were all isolated from immunocompetent humans, suggesting that they may have arisen from a single source.
The genomic comparison of M. tuberculosis with M. microti identified 13 deletion regions in M. microti, compared to M. tuberculosis. Nine deleted regions (RD1 to RD10) had already been reported in BCG (Behr et al., 1999; Gordon et al., 1999
), although five of these (RD1, RD4, RD5, RD6 and RD8) were not identical to those described for BCG. Although M. microti mainly causes disease in small rodents, it can also, rarely, cause disease in man and other large mammals, indicating that the deleted regions do not totally eliminate virulence (van Soolingen et al., 1998
; Horstkotte et al., 2001
). RD1 is thought to be the principal deletion resulting in attenuation of BCG and M. microti (Mahairas et al., 1996
; Brodin et al., 2002
; Pym et al., 2002
); even so, one of the M. microti strains investigated here did not have an RD1 deletion and two more had only a single gene deleted within this region. For this reason, although RD1 deletions undoubtedly contribute to attenuation of M. microti (Pym et al., 2002
, 2003
), they are not the only mechanism of attenuation. In fact, only deletion RD3 was present in all of the strains examined, while RD7, RD8 and MiD1 were found in almost all M. microti, and may therefore have some relation to the host range of M. microti. Interestingly, the human isolate 97-0770 has an exceptional and divergent deletion pattern, and since this isolate caused pulmonary tuberculosis in an immunocompetent 34 year old male (van Soolingen et al., 1998
), it may have unknown extra genes enabling it to cause disease in immunocompetent individuals.
In general, although the results support the evolutionary scenario proposed for the M. tuberculosis complex by Brosch et al. (2002), in that most isolates of M. microti had the RD7, RD8, RD9 and RD10 deletions, some isolates did not appear to fit into this neat picture. In particular, one isolate did not have the RD7, RD8, RD9 or RD10 deletions, another did not have the RD9 deletion and a third lacked both the RD9 and RD10 deletions an apparent discrepancy for which at present there is no explanation.
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ACKNOWLEDGEMENTS |
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Received 18 September 2003;
revised 23 December 2003;
accepted 7 January 2004.
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