Current Molecular Medicine - Volume 7, Issue 3, 2007

It is some twenty years since the first genes were cloned from pathogenic mycobacteria and 8 years since the complete DNA sequence of a Mycobacterium tuberculosis genome was published. When the first genes were cloned TB was commonly regarded as a beaten disease; we had fully effective chemotherapy, an adequate vaccine (BCG) and the remaining problem (people dying elsewhere) was to be solved by field operational and socio-economic developments. The declaration by the World Health Organization in 1993 that tuberculosis had become “A Global Emergency” certainly helped to change the perception that TB was a beaten disease and, although the emphasis remained on the operational issues of improving the application of the existing control measures, particularly DOTS chemotherapy, there was a surge in basic research. This is fortunate, since there has been little sign that the global emergency (2 million deaths a year) is coming under control by application of the existing tools. On the contrary, extremely drug resistant (XDR) TB is disseminating, bringing with it the 40-60% death rates that prevailed in the pre-chemotherapy era. Equally, there is no sign that the synergy between HIV and TB that is devastating some parts of the world, and spreading, is being brought under control either. There is typically a long lag between basic research and applied benefits and the length of that lag is inversely proportional to the financial resources mobilized. The resources have been paltry in relation to the scale of the problem and the benefits to be gained. Yet, already we have attained a dramatically increased understanding of the bacterium and how it goes about its business of growing, causing disease and spreading. Conceptual changes have been brought about and new drugs and new vaccines have been devised. Many of these are reviewed in this issue of the journal. The first four reviews deal with basic studies of the mycobacterium, which may underpin and inform efforts directly aimed at developing new practical tools for disease control. Cox and Cook have drawn together remarkable new insights into the metabolic pathways and growth regulation of the tubercle bacillus and derived mathematical relationships between key variables. Dover et al. provide a comprehensive review of the detailed level at which the synthesis of the complex cell wall is now understood and Bacon and Marsh review the unexpected ways in which the bacteria respond to controlled changes in single environmental variables. Waddell and Butcher discuss the evidence and implications of the findings that the metabolism of the bacterium inside infected cells is dramatically different from its metabolism in conventional laboratory conditions. Whereas the basic studies can be viewed as providing the basis for rational design of new tools, particularly bactericidal drugs aimed at key aspects of bacterial persistence and growth in vivo, the extent to which this is currently being achieved can be assessed in the review of new drug discovery by Williams and Duncan. On the other hand, the molecular typing of strains of tubercle bacillus has developed directly and rapidly out of basic studies and has filled a glaring gap in the TB control toolbox, as reviewed by Behr and Mostowy. The immunology of tuberculosis is proving slow to yield its secrets and undergo paradigm shifts. Accordingly, there is a lack or immune correlates of protection that can be applied to people to predict either the outcome of infection or the effect of vaccination and this unsatisfactory situation is surveyed by Fletcher; clinical evaluation of new vaccines could be severely delayed if adequate correlates are not found soon.Support for a hypothesis relating excessive type 2 (Th2) immune responses to failure of immunity through inadequate bactericidal and cytotoxic T cell responses is drawn together by Rook. In relation to vaccine composition, insight into the role and potential utility of bacterial heat shock proteins as antigens and adjuvants in immunity is reviewed by Walker et al. The thrust for new fully defined and synthetic vaccines to complement or replace the live BCG vaccine is epitomised by the work reviewed by McMurry et al. and efforts to refine and enhance BCG itself by genetic engineering are reviewed by Hernandez-Pando et al. Although the potential of immunotherapy as an adjunct to chemotherapy has been largely ignored until now, encouraging indications of what might be achievable have been obtained in mice and the current position is summarised by Roy et al..........

A framework was developed to provide an integrated view of mycobacterial growth and its regulation. The topics reviewed include the properties of cell cultures and their relation to properties of individual cells, cell sizes and macromolecular compositions, uptake of nutrients through the cell envelope, protein biosynthesis, core metabolic pathways, generation of an electrochemical gradient of protons, ATP synthesis and the control of energy generation.

The replication and growth of Mycobacterium tuberculosis are fundamentally linked to the synthesis and extension of its complex cell wall. Incorporation of new wall material must be tightly regulated so that its deposition does not compromise the extant structure. M. tuberculosis also produces an impressive array of complex bioactive lipids that are intimately involved in pathogenesis and protective immunity. The profiles of these lipids are regulated appropriately to allow the bacterium to respond to the prevailing conditions it faces in vivo. A number of regulatory strategies employed by M. tuberculosis to control cell wall biosynthesis and cell division have now been elucidated. The review highlights the role of alternative sigma factors with extracytoplasmic function in the activation of genes for biosynthesis of complex lipids involved in pathogenicity. RelMtb and CRPMt play roles in cell wall responses to general nutrient deprivation by synthesis and sensing of starvation second messengers, respectively. Recently, the importance of protein phosphorylation networks in cell wall biosynthesis has attracted considerable interest. A plethora of two-component and eukaryotic-like serine/threonine protein kinases systems have been discovered and several are implicated in cell-division, morphogenesis and regulation of the profile of complex bioactive lipids elaborated by the pathogen.

Mycobacterium tuberculosis encounters a range of stimuli in the host. Understanding the environmental cues that initiate the transcriptional response of M. tuberculosis, which enable the bacterium to replicate and/or survive in the host, will provide markers that are specific to different stages of disease, further refining the search for improved treatments and vaccines. Studying M. tuberculosis gene expression in vivo is technically challenging and more amenable in vitro experiments are being used to aid interpretation and to dissect the signals that are responsible for controlling subsets of genes. Key parameters that affect the growth of a pathogen in the host include nutrient status, environmental pH, oxygen availability, and host defences. Studying gene expression, pathogenicity, and physiology of M. tuberculosis that has been exposed to these relevant host conditions in vitro will further increase our understanding of the virulence factors that M. tuberculosis requires to establish disease. Complementary information obtained by metabolic flux analysis, proteomics, and regulatory networks analysis will enable a clearer picture of how transcriptional responses translate to changes in the metabolome and physiology of the organism.

Analysis of the changing mRNA expression profile of Mycobacterium tuberculosis though the course of infection promises to advance our understanding of how mycobacteria are able to survive the host immune response. The difficulties of sample extraction from distinct mycobacterial populations, and of measuring mRNA expression profiles of multiple genes has limited the impact of gene expression studies on our interpretation of this dynamic infection process. The development of whole genome microarray technology together with advances in sample collection have allowed the expression pattern of the whole M. tuberculosis genome to be compared across a number of different in vitro conditions, murine and human tissue culture models and in vivo infection samples. This review attempts to produce a summative model of the M. tuberculosis response to infection derived from or reflected in these gene expression datasets. The mycobacterial response to the intracellular environment is characterised by the utilisation of lipids as a carbon source and the switch from aerobic/microaerophilic to anaerobic respiratory pathways. Other genes induced in the macrophage phagosome include those likely to be involved in the maintenance of the cell wall and genes related to DNA damage, heat shock, iron sequestration and nutrient limitation. The comparison of transcriptional data from in vitro models of infection with complex in vivo samples, together with the use of bacterial RNA amplification strategies to sample defined populations of bacilli, should allow us to make conclusions about M. tuberculosis physiology and host microenvironments during natural infection.

There is an urgent need for new drugs to treat tuberculosis. During the last forty years the only drugs to have been developed are variations on existing ones, but new drug candidates must offer improvements over existing agents. In particular, we require new drugs having novel mechanisms of action that are active against drug-resistant strains and also kill persistent bacilli, thus shortening the length of chemotherapy. Recent advances in our understanding of the biology of Mycobacterium tuberculosis, in particularly the availability of the genome sequence coupled with development of new genetic tools, have greatly contributed to the discovery of potential drug targets for new antituberculars. However, although many potential new drug targets have been identified, greater effort is required in target validation to show properly that they are essential for bacterial growth and survival. In this review, the current drug development pipeline and the strategies employed to identify and validate novel tuberculosis drug targets are presented.

During the past two decades, a number of variable genetic sequences have been uncovered that permit molecular typing of Mycobacterium tuberculosis complex (MTC) organisms. Since the determination of the M. tuberculosis, and later M. bovis, genome sequences, the nature of these variable genetic sequences has become more evident, permitting a clearer recognition of which molecular tools lend themselves best to certain applications. In this review, ‘classical’ genotyping methods for molecular epidemiologic uses are briefly discussed, followed by a more detailed description of post-genomic typing methods, including large sequence polymorphisms otherwise referred to as genomic deletions. Because genomic deletions represent unique event polymorphisms not prone to reversion, these mutations effectively ‘brand’ bacterial lineages, including species/subspecies of the MTC and specific clades of M. tuberculosis sensu stricto. Genomic deletions therefore provide a new opportunity to accurately classify organisms for diagnostic and epidemiologic purposes, serving as the basis for further study of the natural variability across MTC organisms.

Due to the failure of chemotherapy and the only available vaccine, BCG, to control tuberculosis (TB) disease, there is an urgent need to develop new vaccines and therapeutics. The identification of correlates of immune protection or “biomarkers” will facilitate the rational design of vaccines and drugs for the prevention and clearance of TB infection. Although it is known that IFN-γ is essential for protective immunity, animal and human studies have found that IFN-γ alone is not sufficient for the prevention of TB disease. There is evidence that IL-23, a recently described member of the IL-12 family of cytokines, is important in the immuno-pathogenesis of TB. There is also evidence that regulatory T cells (Treg) are present in TB disease and that Treg may suppress effector T cell responses. In the last five years, clinical studies have been able to use Mycobacterium tuberculosis specific antigens, such as ESAT-6, to focus on recently infected, healthy contacts of TB patients in endemic countries. Advances in techniques such as multi-parameter flow cytometry and DNA microarray analysis will enable us to study these cohorts in great detail and facilitate the identification of immune correlates for the rational design of drugs and vaccines for the treatment and prevention of TB.

We need to understand what is different about susceptibility to tuberculosis (TB) in developing countries where most TB occurs, and where the current vaccine, Bacillus Calmette et Guerin (BCG) usually fails to protect. The presence of a background mixed IFN-γ and Th2 response to mycobacterial antigens before infection with M. tuberculosis (Mtb), and the development of a large IL-4 response during progressive TB, are characteristics of individuals in the locations where BCG fails, which are also seen in animal models in the same countries. Recent data suggest that the background Th1 component in developing countries protects from low dose challenge with Mtb in mouse and man, but that following high dose challenge the pre-existing IL-4 component increases and blocks immunity unless the individual's immune system releases IL-4δ2, an antagonist of IL-4, which is raised in the blood of donors with stable latent TB. We outline how IL-4 (and IL-13) can undermine Th1-mediated immunity and drive inappropriate alternative activation of macrophages. The mechanisms of the effects of IL-4 include impaired antimicrobial activity due to reduced TNF-α-mediated apoptosis of infected cells, reduced activity of iNOS, increased availability of iron to intracellular Mtb, and increased proliferation of antigen-specific FOXP-3+ regulatory T cells. IL-4 also increases the toxicity of TNF-α and drives pulmonary fibrosis, thus enhancing immunopathology. The conclusion is that a vaccine that will work in developing countries might need to do more than enhance the existing Th1 response. In these environments it might be more important to block the Th2 component.