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Genotype and phenotype plasticity, slow growth, the unique and complex cell envelope, dormancy, intracellular pathogenesis, persistence, drug-tolerance (survival despite drug treatment), resistance (genetic mutations that result in heritable loss of susceptibility to antibiotics) and production of a range of virulence factors, are some of the biological features displayed by M. tuberculosis during infection. Understanding the basic biology of M. tuberculosis should aid in designing new vaccines and chemotherapy.
Classification and Taxonomy.
The current classification of mycobacteria is now based in chemotaxonomy markers, morphological and physiological characteristics, as well as molecular analysis, such as analyses of DNA-DNA re-association and determination of the 16S rRNA and rDNA sequence similarities. All these approaches have contributed to re-define the actinomycetes and therefore the genus Mycobacterium (Stackebrandt et al., 1997; Tortoli, 2003; Ventura et al., 2007). Though, the defined Mycobacterium genus is highly diverse and comprises around 85 different species; the members share certain characteristics, especially the ability to form mycelia. Due to the diversity of the genus it is not easy to propose a simple definition, but in general mycobacteria are aerobic, acid-alcohol fast actinomycetes having a genome rich in guanidine and cytosine nucleotides. They are aerobic, acid-alcohol fast actinomycetes that usually produce slightly curved or straight rods and coccoid elements, of around 0.2-0.7 X 1.0-10.0 μm in size (Wayne & Kubica, 1994).
Stackebrandt, E., F.A. Rainey, and N.L. Ward-Rainey (1997). "Proposal for a New Hierarchic Classification System, Actinobacteria classis nov." International Journal of Systematic Bacteriology, 47(2), 479-491.Tortoli, E. (2003). "Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s." Clin Microbiol Rev, 16(2), 319-354.Ventura, M., et al (2007). "Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum." Microbiol Mol Biol Rev, 71(3), 495-548.Wayne, L.G. and G.P. Kubica (1994). "The mycobacteria". In: Bergey’s manual of determinative bacteriology. J.G. Holt, N.R. Krieg, P.H.A. Sneath, J.T. Staley and S.T.Williams (eds). 9th ed. Baltimore, Md. Williams & Wilkins.
Life Style of Mycobacteria.
The diversity of biological life style is immense in mycobacteria. Generally, mycobacteria are free-living saprophytes and are well adapted to different habitats, such as soil and aquatic environments, whereas other species are obligate intracellular pathogens that infect humans, birds and other animals (Goodfellow & Williams, 1983). Although, M. ulcerans has been isolated as a soil inhabitant in symbiosis with roots of certain plants present in tropical rain forests or similar environments (Hayman, 1991), this species is the third most common in mycobacterial disease mediating a chronic disease leading to expanding skin ulcers, the so-called Buruli ulcer (van der Werf et al., 2005).
In general, mycobacteria are the causative agents of a broad epidemiological, clinical, and pathological spectrum of diseases in humans. Mycobacterial diseases are very often associated with immunocompromised individuals, especially HIV-AIDS patients. Some pathogenic mycobacteria prefer a single host, such as M. tuberculosis and M. leprae being human pathogens, whereas others can infect different hosts, for instance M. bovis infecting cattle or humans, M. microti being able to infect wood mice, shrews, llamas, cats and very occasionally humans (Brosch et al., 2001).
Mycobacterial genomes: insight to the bacilli.
The clinical course and pattern of TB is highly variable, ranging from life long asymptomatic infection to rapidly progressive pulmonary or disseminated disease. This variability was assumed to be result of differences in individual responses to the disease. However, modern molecular biology techniques are revealing key biological features of M. tuberculosis at the genomic level, which are partially explaining the phenotype and genotype plasticity found in pathogenic mycobacteria (Brosch et al., 2001).
The architecture, size, and function of the genome within mycobacterial species are diverse. The genome of the M. tuberculosis H37Rv strain is 4.4 Mb and GC content about 65.6%, with a 91% coding capacity. More than 20% of the M. tuberculosis genome is devoted to genes encoding two different classes of proteins: enzymes involved in fatty acid metabolism and acidic, glycine-rich polypeptides of unknown function, the PE and PPE proteins (Cole et al., 1998). The remarkable number of genes encoding for fatty acid biosynthesis maybe related to the ability of the bacillus to grow in the host’s tissue, where fatty acids maybe the major carbon source (Cole et al., 1998). Genome comparisons of the M. tuberculosis H37Rv and CDC1551 strains, the latter being a highly virulent strain, revealed interesting polymorphisms. The CDC1551 strain contains an extra gene that shares homology to the early secreted antigen of 6 kDa (ESAT-6), a major T-cell antigen (Betts et al., 2000).
The M. tuberculosis complex comprises M. tuberculosis together with M. africanum, M. bovis, M. canettii, M. pinnipedii and M. microti, which are closely related organism sharing >99% identity at the nucleotide level. They share 99.9% similarity at the nucleotide level and identical 16S RNA sequences, but differ in terms of their host tropisms, phenotypes and pathogenicity (Brosch et al., 2001). It is intriguing that some members are exclusively human pathogens, M. tuberculosis, M. africanum, M. canettii, whereas others have a wide host spectrum, such as M. bovis.
Although, human and bovine tubercle bacilli can be differentiated by host range, virulence and physiological features, the genetic basis for these differences are still unknown. The M. bovis AF2122/97 genome has been recently deciphered (Garnier et al., 2003). The genome sequence is 4.3 Mb in length, with an average GC content of 65.63%, which potentially encodes 3952 proteins (Garnier et al., 2003). The bovine bacillus genome shares >99.95% identity at the nucleotide level to that of M. tuberculosis. However, deletion of genetic information is the dominant trend in the M. bovis genome, which has many pseudogenes. These genes resemble intact genes that are involved in molecular transport, structure and biosynthesis of the cell surface, detoxification, intermediary metabolism, fatty acid metabolism and cofactor biosynthesis (Garnier et al., 2003).
Likewise, the obligate intracellular pathogen, M. leprae, has the smallest genome sequenced among the mycobacteria. The complete genome of M. leprae TN strain contains 3.2 Mb, roughly 1.4 Mb smaller than that of M. tuberculosis, and has an average GC content of 57.8%. The M. leprae genome only encodes 49.5% of its coding capacity, whereas 27% contains pseudogenes, randomly distributed, and the remaining 23.5% of the genome does not appear to be coding (Cole et al., 2001a). The extensive genome downsizing and rearrangement must have occurred during evolution of the leprosy bacillus and this might be associated with the obligate intracellular habitat of the bacteria. Supporting this hypothesis, the prolyl-tRNA synthase, encoded by proS, is more similar to the enzymes of Borrelia burdorferi and eukaryotes. The M. leprae proS gene is both displaced and inverted with respect to the M. tuberculosis counterpart, consistent with a recent acquisition through horizontal gene transfer, from host to pathogen. Other interesting findings are the deletion of some polyketide synthases (PKS), involved in lipid biosynthesis, and the mbt operon required for the production of mycobactins (Cole et al., 2001a; b).
The cell wall.
M. tuberculosis possesses an unusual cell wall that provides resistance to many antibiotics and the components of the killing mechanisms of macrophages. Historically, the inhibition of the biosynthesis of this structure has proven useful in TB chemotherapy, with the frontline agents ethambutol (arabinan) and isonazid (mycolic acids) and second-line agents like D-cycloserine (peptidoglycan) and ethionamide (mycolic acids) inhibiting the production of its various components. A more complete understanding of its biosynthesis and structure has been a major research focus in order to define potential targets for the development of new drugs (Boshoff et al., 2004; Scherman et al., 2003).
The cell envelope of M. tuberculosis consists of three main structural components. The plasma membrane, cell wall and the capsule (Rastogi et al., 1986). The plasma membrane appears to be a typical bacterial membrane and perhaps contributes very little towards the pathology of the bacilli.
Beyond the membrane is peptidoglycan (PG) in covalent attachment to arabinogalactan (AG), which in turn is linked to the mycolic acids. All these molecules form the cell wall core, the mAGP complex (Brennan, 2003; Minnikin et al., 2002). Brennan and Minnikin proposed a model of mycobacteria cell wall.
The distinguishing characteristic of all Mycobacterium species is that the cell wall is thicker than in many other bacteria, which is hydrophobic, waxy, and rich in mycolic acids. M. tuberculosis and M. leprae possess a complex cell wall and its extensive hydrophobic domains contribute to poor permeability which, in turn, leads to an inherent resistance against many drugs (Minnikin et al., 2002).
The mycolic acids are key lipid components in the cell wall of M. tuberculosis. These lipids are high molecular weight α-alkyl-b-hydroxy fatty acids, which are predominantly linked to AG, but some are found as extractable glycolipids such as trehalose monomycolates (TMM) and trehalose 6,6’-dimycolates (TDM). The main part of the branched chain is called “meromycolic acid” and the other part the a-branch (Minnikin et al., 2002). Mycolic acids from mycobacteria contain about 70-90 carbon atoms and 20-25 carbon atoms in the α-branch, while in Corynebacterium diphtheriae, corynomycolates have 30-36 C atoms in total (Dover et al., 2004a). The molecular organisation of mycolic acids plays an important role in nutrient uptake into the bacterium as well as conferring resistance to a wide range of antibacterial drugs (Takayama et al., 2005). Moreover, the mycolic acids also play a role during cellular and innate immune responses during M. tuberculosis infection (Korf et al., 2005).
Other components of the cell envelope of M. tuberculosis are the lipoarabinomannan (LAM) and related lipomannan (LM); both lipoglycans are based on phosphatidylinositol mannoside (PIM) anchors, which may locate into the plasma membrane (Minnikin et al., 2002). It is speculated that LAM is linked to the interaction of the pathogen with the host, possibly leading to resistance mechanisms associated with macrophage killing (Chatterjee, 1997; Karakousis et al., 2004). On the other hand, a family of sulfated acyl trehaloses (SL) (Goren, 1970), and phenolic glycolipids (PGL) have been characterised from M. tuberculosis and M. canettii, respectively (Daffé et al., 1987; Watanabe et al., 1994). The sulfolipids of M. tuberculosis are five structurally related sulfatides, from which the sulfolipid-I (SL-I), the most abundant sulfatide, has been identified as a 2,3,6,6’-tetraacyl-a,a’-D-trehalose 2’-sulfate (Goren, 1970). The PGL consist of a lipid core formed by a long-chain b-diol, occurring naturally as diester of the polymethyl branched fatty acids (Malaga et al., 2008). Since both SLs and PGLs are probably located in the outer leaflet of the envelope, their location has prompted speculation that they may be virulence factors involved in host-pathogen interactions (Reed et al., 2004; Zhang et al., 1991).
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