Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France1
Tel: +33 1 45688446. Fax: +33 1 40613583. e-mail: stcole{at}pasteur.fr
Keywords: bioinformatics, PE and PPE families, pathogenesis, BCG vaccine, evolution
a This review is based on the 2002 Marjory Stephenson Prize Lecture delivered by the author at the 150th Meeting of the Society for General Microbiology, 9 April 2002.
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Background |
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At the present time, the World Health Organization estimates that eight million new cases of tuberculosis occur every year and that 25 million individuals worldwide will lose their lives to the disease in the coming decade (Dye et al., 1999 ). Although the ultimate solution to the problem of tuberculosis will be socio-economic, many of these deaths could be prevented if better access to treatment were available and if vaccination were more effective. More alarmingly, on the basis of their tuberculin reactivity, a sign of prior infection, it has been calculated that one-third of the worlds population has been infected with Mycobacterium tuberculosis (Dye et al., 1999
), the aetiological agent of the disease. These individuals are thus at risk of presenting with disease later in life as their immunity wanes due to ageing or as a result of HIV infection (Lillebaek et al., 2002
). While immunization with the BCG vaccine prevents tuberculosis, particularly in children in the West, it is of limited efficacy in the developing world where the disease burden is highest (Fine, 1995
).
A highly efficient treatment, known as short course chemotherapy, is available to cure the disease. This involves taking a combination of four drugs for a minimum period of 6 months. The lengthy treatment duration is imposed by the exceptionally slow growth of the tubercle bacillus. While high cure rates can be obtained by means of DOTS (Directly Observed Therapy Short-course) (Espinal et al., 1999 ), this strategy would be even more effective if its duration could be reduced by at least 2 months. Regrettably, despite the efficacy of DOTS, drug resistance is becoming increasingly prevalent for a variety of operational reasons (Dye et al., 2002
). Among the challenges facing mycobacteriologists and biomedical researchers are the development of faster-acting drugs that also act on latent disease, and the creation of a vaccine that is universally efficacious. Genomics, the systematic analysis of the complete genetic material found in an organism by means of DNA sequencing and bio-informatics, is opening new avenues for research in these key areas and catalysing discovery.
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The Mycobacterium tuberculosis complex |
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Evolution of the M. tuberculosis complex |
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Microbiological properties |
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Unlike the other complex members, M. microti and M. bovis require pyruvate as a growth supplement. There are also differences in the natural resistance to certain antibiotics such as pyrazinamide (PZA), due to a missense mutation in the activating enzyme pyrazinamidase (Scorpio & Zhang, 1996 ), and thiophen-2-carboxylic hydrazide (TCH), as well as in the production of niacin (Table 1
). All virulent members of the complex are capable of withstanding phagocytosis and replicating within macrophages and monocytes.
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Genomics of M. tuberculosis |
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The genome contains 4000 genes distributed fairly evenly between the two strands and accounting for >91% of the potential coding capacity. Genes were classified into 11 broad functional groups and, today, precise or putative functions can be attributed to 52%, with the remaining 48% being conserved hypotheticals or unknown (see Camus et al., 2002
). Over 51% of the genes have arisen as a result of gene duplication or domain shuffling events, and 3·4% of the genome is composed of insertion sequences (IS) and prophages (phiRv1, phiRv2). There are 56 copies of IS elements belonging to the well-known IS3, IS5, IS21, IS30, IS110, IS256 and ISL3 families, as well as a new IS family, IS1535, that appears to employ a frameshifting mechanism to produce its transposase (Gordon et al., 1999b
). IS6110, a member of the IS3 family, is the most abundant element and has played an important role in genome plasticity.
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Genomics and biology |
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Whereas the tubercle bacillus appears to employ lipolysis as its principal catabolic pathway, it has no bias or obvious lesions in its anabolic repertoire. While this is fully consistent with our ability to culture M. tuberculosis in defined medium, it is somewhat unusual for an intracellular parasite to have retained such functions as the corresponding metabolites are often scavenged from the host. Although the presence of a complete network of anabolic systems is in agreement with the notion that the tubercle bacillus has only recently emerged as a human pathogen, and thus had insufficient time to adapt to a new host by shedding biosynthetic genes, it may also indicate that the availability of metabolic precursors is limiting within the phagosome. Support for the latter explanation is provided by the finding that genes for anabolic functions have been heavily conserved in the genome of Mycobacterium leprae, a related, obligate intracellular pathogen, in the face of massive reductive evolution that may have eliminated as many as 2600 genes (Cole et al., 2001 ; Eiglmeier et al., 2001
).
There are, however, two additional arguments in favour of M. tuberculosis recently changing its niche and lifestyle. Firstly, the genome contains numerous genes (>100) encoding regulatory proteins and signal transduction pathways that control gene expression (Cole et al., 1998 ). Secondly, there are 20 enzyme systems that are predicted to use cytochrome P450 as a cofactor and these are often involved in the degradation of xenobiotics, or the modification of organic molecules, such as sterols, by means of their mono-oxygenase activity (Aoyama et al., 1998
). These enzymes are common in soil organisms where they enable diverse organic matter to be degraded to yield metabolizable sources of carbon and energy (Aoyama et al., 1998
; Munro & Lindsay, 1996
). Both the regulatory networks and the P450 systems have been subject to massive gene decay in M. leprae (Cole et al., 2001
; Eiglmeier et al., 2001
).
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The PE and PPE gene families |
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Variability and possible roles of the PE and PPE multigene families |
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There is growing evidence from signature-tagged mutagenesis and micro-array studies that some M. tuberculosis PE-PGRS proteins may be involved in pathogenesis (Camacho et al., 1999 ). In addition, members of the PE-PGRS families have been implicated in the pathogenesis of Mycobacterium marinum (Ramakrishnan et al., 2000
), where at least two genes were shown to be up-regulated strongly following phagocytosis of the bacterium.
Subcellular fractionation studies and immunogold or fluorescent antibody staining localized some PE-PGRS proteins in the cell wall and cell membrane of M. tuberculosis (Banu et al., 2002 ; Brennan & Delogu, 2002
). Disruption of the M. tuberculosis gene encoding the PE-PGRS protein Rv1818c resulted in greatly reduced bacterial clumping, suggesting that this protein may mediate cellcell adhesion, and phagocytosis of the mutant cells by macrophages was also reduced (Brennan et al., 2001
). Another PE-PGRS protein, Rv1759c, that varies between strains, binds fibronectin and could thus mediate bacterial attachment to host cells (Espitia et al., 1999
; Singh et al., 2001
). The PE-PGRS proteins contain no obvious hydrophobic stretch that could act as a trans-membrane anchor and it is difficult to envisage how these proteins cross the cytoplasmic membrane. It has been speculated that a 23-amino-acid sequence that ends the PE domain and precedes the PGRS segment acts in membrane attachment but proof of this is lacking (Brennan et al., 2001
).
The immunogenicity of the PE-PGRS protein Rv1818c has been studied extensively in mice (Delogu & Brennan, 2001 ), where immunization with the PE domain induced Th1-type responses that were not found when the complete PE-PGRS protein was used. Instead, the PGRS part of the protein elicited antibodies and suppressed the Th1 response induced by the PE domain. The PE-PGRS proteins bear some sequence similarity to EBNA, the EpsteinBarr virus nuclear antigens, which block antigen presentation by the MHC class I pathway, through their action as proteasome inhibitors (Cole et al., 1998
). It was speculated that PE-PGRS proteins may also have inhibitory activity and it has recently been shown that the PGRS domain, when fused to GFP, confers increased resistance to proteosomal attack (Brennan & Delogu, 2002
). If these immunological and adhesive properties are shared among other members of the family, it is conceivable that the extensive variation observed at the gene level could bestow very different phenotypes on the different strains.
The PPE proteins of the MPTR class also show variability (Zhang & Young, 1994 ), and the largest predicted PPE-MPTR protein detected contains 3300 amino acids. Extensive sequence variation has been reported for PPE proteins between M. tuberculosis and M. bovis (Gordon et al., 2001a
). Little evidence concerning the possible function of the PPE-MPTR proteins exists but one member of the PPE protein family was recently shown to be cell-wall-associated and surface-exposed (Sampson et al., 2001
). It seems increasingly likely that both the PPE-MPTR and PE-PGRS proteins may correspond to variable surface antigens (Banu et al., 2002
).
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Comparative genomics |
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SNPs do occur in the genomes of members of the M. tuberculosis complex (Table 1) but at a relatively low level for a bacterium of 1 in every 20004000 bp (Sreevatsan et al., 1997
), depending on the species. Some SNPs, like the point mutation in the pncA gene responsible for pyrazinamide resistance (Scorpio & Zhang, 1996
), result in phenotypic change but the majority seem to be silent. Consequently, InDels appear to be the most common means of generating diversity. Most of the insertions result from transposition events, generally involving IS6110, or more rarely from gene duplication. No conclusive evidence in favour of recent horizontal gene transfer occurring in the M. tuberculosis complex is available and the closest example of this is provided by the prophage genomes, phiRv1 or phiRv2, respectively (Brosch et al., 2000
) corresponding to regions of difference (RD) RD3 or RD11.
The deletions fall into two groups, ancient and recent. The ancient deletions occurred at different stages in the speciation process and are widespread whereas the recent deletions have a more restricted distribution. Examples of the latter are the IS6110-mediated deletion of the 7 kb locus RvD2 in M. tuberculosis H37Rv, still present in the closely related avirulent derivative H37Ra (Brosch et al., 1999 ), or loss of the RD2 region encoding the protein antigen MPB64 from some strains of M. bovis BCG (Mahairas et al., 1996
). The RvD2 region also undergoes great variability in clinical isolates of M. tuberculosis and seems to represent a hot-spot for IS6110 transposition events (Ho et al., 2001
).
In contrast to these recent deletions, the absence of regions RD7, RD8, RD9 and RD10 from M. microti, M. bovis and BCG, which are still present in all M. tuberculosis strains, seems to be a much older event in evolutionary terms (Table 2). From close inspection of the DNA sequences bordering these RD regions it is apparent that deletions occurred within coding regions. Genes that are present in M. tuberculosis in full-length have been disrupted in BCG, M. bovis and M. microti at exactly the same location, whereas these coding sequences are still intact in M. tuberculosis and M. canettii strains. This finding rules out the possibility of the DNA in these regions having been acquired by M. tuberculosis but, instead, argues strongly in favour of loss of the corresponding genetic material by the other species. Based on the presence or absence of such conserved RD regions, a degree of relatedness to the last common ancestor of the M. tuberculosis complex was proposed that shows that the lineages of M. tuberculosis and M. bovis separated before the M. tuberculosis specific deletion TbD1 occurred (Fig. 2
). From this analysis it is clear that M. bovis cannot have been the ancestor of M. tuberculosis but, rather, appears to be descended from M. tuberculosis or to have emerged independently (Brosch et al., 2002
).
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Functional genomics |
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Several of the RD regions described above contain genes that encode potential virulence factors like those characterized in other microbial pathogens (Table 2). These include prophages (RD3, RD11), phospholipases C (RD5), invasins (RD7) and an exopolysaccharide biosynthetic system (RD4). RD1 is the sole region that appears to be missing from the vaccine strains BCG and M. microti but is present in all virulent members of the M. tuberculosis complex. All M. microti strains tested have lost
14 kb of DNA that has removed or inactivated genes Rv3864Rv3876 (Brodin et al., 2002
) and this deletion partially overlaps the RD1 locus of M. bovis BCG (Rv3871Rv3879) (Mahairas et al., 1996
). However, while the proteins encoded by the corresponding genes belong to prominent mycobacterial protein families (Tekaia et al., 1999
), it has not been possible to predict their functions by bio-informatics. Two of them, ESAT-6 and CFP-10 (Berthet et al., 1998
; Harboe et al., 1996
; Sorensen et al., 1995
), are small proteins, belonging to the ESAT-6 family, which might be secreted by early-exponential-phase cultures. They have attracted considerable immunological interest as a result of potent antigenicity for T cells. Interestingly, two other variable regions (RD5, RD8) also encode ESAT-6 family members, suggesting that there may be strong selective pressure imposed by the immune system for variants from which they have been lost (Gordon et al., 1999a
).
To test the biological effect that loss of these regions may have had on the different members of the M. tuberculosis complex, two different approaches are being pursued. On the one hand, the corresponding genes can be knocked-out or removed from the genome of M. tuberculosis using gene replacement technology or, on the other, they could be knocked-into species such as M. bovis BCG from which they are missing. In both cases, the phenotype of the resultant recombinants is assessed using a combination of in vitro and in vivo assay systems. These complementary approaches will almost certainly unravel the basis for phenotypic differences among tubercle bacilli and provide insight into their pathogenesis and the attenuation mechanisms at play. Knowledge of the three-dimensional structures of the corresponding proteins and effectors is being generated by structural genomics programmes in which high-throughput technologies are providing datasets at atomic resolution (Cole, 2002 ). Clearly, all this new information will find rapid application in the development of new diagnostic tests, better drugs and vaccines and, hopefully, help to sway what seems at times a desparately unequal struggle against tuberculosis.
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ACKNOWLEDGEMENTS |
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