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Molecular Basis of Pathogenicity

M. tuberculosis uses the respiratory tract as the principal portal of entry, where it travels deep into the alveoli and is engulfed by alveolar macrophages. While these immune cells have the capability to destroy most potential invaders, M. tuberculosis can persist and even replicate in this extremely hostile intracellular environment (Russell, 2001).

The outcome of infection ultimately depends on the induced T-cell response (Kaufmann, 2001). This immune response is usually only capable of containing the infection and rarely sterilizes the lungs, allowing the bacteria to establish what is known as a "latent" infection. The physiological and anatomical nature of latent or dormant infection is currently unknown. Most latent infections are thought to be perpetual, though in at least 5% of cases (much greater in the presence of HIV coinfection) the apparently dormant bacteria eventually exploit lapses in host immunity to multiply and establish overt disease. The resulting proliferation of bacteria in the lungs produces aerosol transmission and infection of new cases. Thus, although M. tuberculosis is highly pathogenic with the capacity to cause the severe pulmonary disease required for transmission, it can also exist in a quasi-commensal state with its host, permitting the establishment of a large reservoir of dormantly infected individuals. These varied clinical states suggest that the molecular basis of pathogenicity of M. tuberculosis involves a complex interplay between bacterial and human factors. A key interface between M. tuberculosis and its host is the macrophage. One of the major features of M. tuberculosis in this respect is its capacity to block the acidification of the phagosome and consequently to disable or retard phagosome-lysosome fusion in phagocytic cells (Goren et al. 1976; Anes et al. 2003). Many aspects of this phenomenon remain unclear, but studies have started to shed some light on the molecular mechanisms involved. Complex lipids from the mycobacterial cell wall are thought to be important for blocking the normal biogenesis of the phagolysosome. Recently the SapM lipid phosphatase of M. tuberculosis, via hydrolysis of phosphatidylinositol 3-phosphate, has been proposed as a key protein in this process (Vergne et al. 2005). A eukaryotic-like serine-threonine protein kinase (PknG) also appears essential for the manipulation of membrane trafficking and intracellular survival (Walburger et al. 2004).

Several approaches for gene inactivation have been developed to enable the identification of the genetic basis of other aspects of pathogenicity. In one of them, a vector with a thermosensitive origin of replication derived from plasmid pAL5000 of Mycobacterium fortuitum has been used together with the gene sacB to select the rare genetic event of double cross-over recombination in M. tuberculosis (Pelicic et al. 1997). For example, this approach was used to inactivate gene phoP, part of a two-component system of M. tuberculosis that did not affect intracellular survival but impaired growth in mouse lungs (Perez et al. 2001). The same vector (ts/sacB) together with marked transposons also allowed signature-tagged mutagenesis (STM) to be applied to M. tuberculosis, thereby identifying transposon mutants of M. tuberculosis that could not survive an in vivo mouse passage. In one study four transposon insertions were identified in a 50-kbp region, which groups 13 genes required for the synthesis or transport of phthiocerol dimycoserosates (Camacho et al. 1999). The absence of these complex lipids affects the permeability and structure of the mycobacterial cell wall as well as the virulence of the bacteria (Camacho et al. 1999). A similar approach based on mycobacteriophages (Braunstein et al. 2002) confirmed the importance of phthiocerol dimycoserosate synthesis and transport for the virulence of M. tuberculosis (Cox et al. 1999).

The use of these genetic techniques in conjunction with animal models of tuberculosis (usually the mouse) has produced a lengthening list of diverse genes implicated in pathogenesis. However, in many cases the precise mechanism by which these genes act has yet to be defined. Hingley-Wilson and colleagues have tried to categorize various M. tuberculosis mutants generated by different research groups by their in vivo growth characteristics, which are shown in Table 10.1 (Hingley-Wilson et al. 2003). The strains listed there include (a) severe growth in vivo (sgiv) mutants, which show a very severe reduction in colony-forming units with time; (b) growth in vivo (giv) mutants, which do not grow as robustly as wildtype M. tuberculosis in the lungs of immunocompetent mice, yet still grow better than sgiv mutants; (c) persistence (per) mutants, which fail to grow or persist after the onset of acquired immunity; and (d) mutants that have the same growth characteristics as per mutants, but show altered pathology (pat) compared with that of wild-type M. tuberculosis. Whilst this may be a useful framework for understanding the mouse model of tuberculosis, it is unclear how these phenotypes relate to human tuberculosis.

A different method for identifying genes implicated in the infection process was developed by Sassetti et al., who adapted the mariner transposon for use in M. tuberculosis and named the method "transposon site hybridization" or TraSH (Sassetti et al. 2001). The use of high-density mutagenesis allowed them to mutate virtually every gene of M. tuberculosis and determine the effect of gene disruption on the growth rate during infection. Microarray hybridization served as the readout for the presence or absence of individual transposon mutants in the pool of bacteria after mouse passage. Comparing the results of mutant pools passaged in mice with pools grown in artificial media, they identified 194 genes that seemed to be specifically required for mycobacterial growth in vivo (Sassetti and Rubin, 2003). According to their data, approximately 5% of the genes present in the genome of M. tuberculosis code for proteins that are required for virulence in the mouse. Interestingly, a high proportion of these proteins are only found in closely related species, suggesting mycobacteria have acquired a specific repertoire of virulence determinants distinct from those identified in other bacterial pathogens.

Table 10.1 Classification ofthe described M. tuberculosis mutants (after Hingley-Wilson et al. 2003).

Class M. tuberculosis mutant

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