Mycobacterium: Molecular Microbiology Chapter Abstracts
Chapter 1
DNA Replication and Cell Division
Murty V. Madiraju and Malini Rajagopalan
The eubacterial cell cycle consists of two important aspects; one is the DNA replication cycle and the other is the cell division cycle. Information on how the critical events involved in the Mycobacterium tuberculosis cell cycle are initiated, regulated and coordinated with each other is critical for understanding the state of the bacterium during active and persistent growth in vivo. Although we have a long way to go in understanding the molecular mechanisms responsible for the initiation and the regulation of replication and cell division processes in mycobacteria, we are gaining ground in achieving this goal. Studies on the replication origin(s) and the DnaA initiator protein and their interactions in vitro and in vivo are providing clues to the roles of the initiator protein and the regulation of oriC replication. Epidemiological studies on clinical isolates suggest that M. tuberculosis strains can use alternate origins for replication initiation. The involvement of the inducible DnaE2 polymerase in generating spontaneous mutations following DNA damage has been established. Investigations into activities of DNA chain elongation enzymes and the inducible DNA damage response are actively being pursued. Fluorescent microscopy techniques to visualize the FtsZ protein catalyzed septal rings at the mid-cell location and genetic and biochemical characterization of other cell division protein components that either associate with and or effect the ring FtsZ-ring assembly, are being carried out.
Chapter 2
The Mycobacterial Two-Component Regulatory Systems
Stuart C. G. Rison, Sharon L. Kendall, Farahnaz Movahedzadeh and Neil G. Stoker
The ability to respond to different environments is critical for bacterial survival. Two-component regulatory systems are widely used to sense such changes. These systems usually involve a membrane-bound sensor that detects an environmental change, and activates a cytoplasmic response-regulator that is able to bind to relevant promoters, and to switch gene expression on and off as appropriate. In pathogens, virulence genes are often controlled in this way, and this proves to be the case also for Mycobacterium tuberculosis, which has 11 such paired systems and eight orphan genes (six response regulators and two sensors). In this chapter we review the current state of knowledge of these gene systems in M. tuberculosis (and their orthologues in related bacteria), including functional information from genetic studies, and expression data from microarray studies.
Chapter 3
Protein Secretion and Export in Mycobacterium tuberculosis
Sherry Kurtz and Miriam Braunstein
The secreted and exported proteins of Mycobacterium tuberculosis are protein subsets exposed to the host cell environment, making them attractive candidates for virulence factors and immunogenic antigens. In this chapter, we review the current understanding of the translocation systems responsible for localizing these extracytoplasmic proteins and the available methods for identifying them. Also discussed are examples of secreted and exported M. tuberculosis proteins shown to contribute to virulence and/or the immune response in the host.
Chapter 4
Vaccine Strategies
D.M. Collins, B.M. Buddle and G.W. de Lisle
A wider appreciation in developed countries of the size and intractability of the global tuberculosis problem has resulted in intensive efforts being devoted over the last 15 years towards producing new tuberculosis vaccines. These efforts have been aided by new insights into mycobacterial pathogenesis gained from developments in genomics, molecular genetics and immunology. A wide range of different vaccine candidates have recently been produced, including live attenuated mycobacteria, and protein and DNA subunits. An increasingly advocated concept in the last few years has been the use of different priming and boosting vaccines rather than a single all purpose vaccine, although the single vaccine approach has not been abandoned. Already, a few of the new candidate vaccines tested, have induced protection in animal models that is equivalent or better than that of the current vaccine, bacille Calmette and Guérin (BCG). Several of the candidates that may have moderate advantages over BCG are already in early human trials, but it will be many years before a replacement for BCG is available. Attempts to produce better vaccines than those in the current trials are continuing. It is expected that in some cases these attempts will be successful due to the continual improvements that are occurring in understanding host protection and mycobacterial pathogenesis, and also because some classes of vaccine candidates take longer to optimise than others. Improved animal models are being developed that more closely mimic conditions in human populations, so that a more accurate assessment of candidate vaccines can be made before they enter human trials. There is now almost universal acceptance that much better tuberculosis vaccines than BCG can be made, and considerable attention is being devoted towards designing clinical trials and considering operational issues for their deployment.
Chapter 5
Drug Resistance in Mycobacterium tuberculosis
Rabia Johnson*, Elizabeth M. Streicher*, Gail E. Louw, Robin M. Warren, Paul D. van Helden and Thomas C. Victor
Anti-tuberculosis drugs are a double-edged sword. Whilst they destroy pathogenic Mycobacterium tuberculosis, they also select for drug resistant bacteria against which those drugs are subsequently ineffective. Global surveillance has shown that drug resistant tuberculosis is widespread and is now a threat to tuberculosis control programs in many countries. The application of molecular methods during the last decade has greatly changed our understanding of drug resistance in tuberculosis. Application of molecular epidemiological methods has also been central to the description of outbreaks of drug resistance. We describe the recommendations for tuberculosis treatment according to the WHO guidelines, the drug resistance problem world-wide, mechanisms of resistance to first and second line drugs and the application of molecular methods to detect gene mutations causing resistance. It is envisaged that molecular techniques will be important adjuncts to traditional culture-based procedures for rapid drug resistance screening. Prospective analysis and intervention to prevent transmission may be particularly helpful in areas with ongoing transmission of drug resistant strains as recent mathematical modeling indicate that the burden of multi-drug resistant (MDR) strains cannot be contained in the absence of specific efforts to limit transmission.
Chapter 6
Virulence Factors of Nontuberculosis Mycobacteria
P.L.C. Small
Nontuberculosis mycobacteria are environmental mycobacteria which are widespread in nature in soil, water, animals and invertebrates. Although almost one third of these species have been associated with human disease, fewer than 15 species are important human pathogens. Several factors have led to increased interest in environmental mycobacterial pathogens: 1) The spread of HIV has resulted in a large population of immunosuppressed people leading to the identification of several new mycobacterial pathogens (such as Mycobacterium genovese), as well as increasing the incidence of well known organisms such as Mycobacterium avium 2) The spread of Mycobacterium ulcerans which causes serious disease in immunocompetent hosts and contains unique virulence determinants and 3) The growing awareness that some environmental pathogens such as Mycobacterium marinum can provide important insight into the pathogenesis of Mycobacterium tuberculosis and 4) The fact that genome sequences for M. avium (http://www.ebi.ac.uk/genomes/AE016958.html), M. marinum (www.sanger.ac.uk/Projects/M_marinum), and M. ulcerans (http://genopole.pasteur.fr/Mulc/BuruList.html) are available. Although virulence determinants have not been identified in the vast majority of environmental mycobacterial pathogens, there is a growing body of literature on M. avium subspecies avium, Mycobacterium kansasii, M. marinum and M. ulcerans that will be explored in this chapter.
Chapter 7
The Stress Response
G. R Stewart, I. Papatheodorou and D.B. Young
Heat shock provides a convenient and well-characterised model to study the response of cells exposed to environmental stress. The major stress proteins induced by heat shock are molecular chaperones which play an essential role in the protein-folding activities of the cell under all conditions, but are particularly important when the protein complement of the cell begins to denature and aggregate during exposure to stress. The dramatic increase in the expression of chaperones during infection identifies the stress response as an important element of mycobacterial pathogenesis. Recent genome-wide analyses allow us to view the regulation and function of the major stress proteins in the context of the whole cell response to infection. The prominence of conserved stress proteins in the microbial response to infection presents a convenient target for recognition by the host immune system, triggering innate immune signalling as well as providing antigens for adaptive immunity. In this review, we describe advances in understanding the biology of mycobacterial stress proteins from the perspective both of the microbe and of the infected host.
Chapter 8
Mycobacterial Dormancy and Its Relation to Persistence
Michael Young, Galina V. Mukamolova and Arseny S. Kaprelyants
Latent tuberculosis is a long-term, asymptomatic infection caused by the persistence of Mycobacterium tuberculosis within the human body. The question of how bacteria survive for decades in immunologically educated hosts without causing disease has puzzled microbiologists for a century (Stewart et al., 2003). Several models have been established to mimic presumed or deduced features of the persistent state. Many, though not all of them, are associated with reduced bacterial culturability. Since our understanding of the precise nature of the organisms that persist during TB latency is so rudimentary, we do not know which models are most relevant. In this chapter we consider the proposition that persistence of M. tuberculosis during TB latency is associated with the adoption of a dormant state, in which the bacteria lose culturability, sometimes for protracted periods. We suggest that the persisting organisms may be a dynamic and mixed population, in which individual bacteria are able to adopt or cycle between different physiological states, some of which may be characterised by impaired culturability.
Chapter 9
Mycobacterial Resistance to Reactive Oxygen and Nitrogen Intermediates: Recent Views and Progress in M. tuberculosis
Kyu Y. Rhee
Resistance to the antimicrobial actions of reactive oxygen and reactive nitrogen intermediates (ROI/RNI) is a common property of pathogenic mycobacteria. However, the molecular basis of mycobacterial resistance to host-derived ROI/RNI in vivo remains poorly understood. This gap in our understanding of mycobacterial resistance to ROI/RNI is explained by three factors: (1) the historical absence of a facile system to genetically manipulate mycobacteria, (2) our limited understanding of the free radical stresses encountered by mycobacteria in vivo, and (3) apparent redundancies in the ROI/RNI defense mechanisms utilized by mycobacteria. This chapter will review mycobacterial ROI/RNI resistance placed in the framework of our current understanding of the antimicrobial biology of immune-derived ROI/RNI. Reflecting the weight of the published literature, studies of Mtb and murine models of tuberculosis will be emphasized.
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