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Current Molecular Medicine
ISSN: 1566-5240
Current Molecular Medicine
Volume 7, Number 3, May 2007
Contents
Tuberculosis (Part 1)
Guest Editor: Douglas B. Lowrie
Editorial Pp. 229-230
Growth Regulation in the Mycobacterial Cell
Pp. 231-245
Robert A. Cox and Gregory M. Cook
[Abstract]
Regulation of Cell Wall Synthesis and Growth
Pp. 247-276
Lynn G. Dover, Like J. Alderwick, Alistair K. Brown, Klaus
Futterer and Gurdyal S. Besra
[Abstract]
Transcriptional Responses of Mycobacterium tuberculosis
Exposed to Adverse Conditions In Vitro Pp.
277-286
Joanna Bacon and Philip D. Marsh
[Abstract]
Microarray Analysis of Whole Genome Expression of
Intracellular Mycobacterium tuberculosis
Pp. 287-296
Simon J. Waddell and Philip D. Butcher
[Abstract]
Current Strategies for Identifying and Validating
Targets for New Treatment-Shortening Drugs for TB
Pp. 297-307
Kerstin J. Williams and Ken Duncan
[Abstract]
Molecular Tools for Typing and Branding the Tubercle
Bacillus Pp. 309-317
Marcel A. Behr and Serge Mostowy
[Abstract]
Correlates of Immune Protection from Tuberculosis
Pp. 319-325
Helen A. Fletcher
[Abstract]
Th2 Cytokines in Susceptibility to Tuberculosis
Pp. 327-337
Graham A.W. Rook
[Abstract]
Abstracts
[Back to top]
Editorial
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. The
emerging threat of XDR TB may well drive clinical reappraisals
before long.
Overall it is evident that the increased funding of studies
of the molecular biology of both the tubercle bacillus and
of tuberculosis disease has begun to yield new clinical tools,
but we are still a long way from seeing much practical benefit.
We have come to the hard part, clinical testing of new drugs,
immunotherapies and vaccines. It is not clear that adequate
political or commercial will has been generated to fund clinical
assessment of a representative range of lead candidates for
any of these applications. A five-fold increase in resources
for new tools research has been calculated necessary, from
$206 million to $1.05 billion per year, which would still
be only about a third of the current AIDS research budget
(Treatment Action Group (2006) Tuberculosis Research and Development.
A Critical Analysis, 2nd edition; http://www.aidsinfonyc.org/tag/tbhiv/tbrabdd2.html.
As it stands, best-guess and biased choices to test a limited
selection of new tools based on our still-inadequate understanding
seem unlikely to meet the objectives of The Global Plan to
Stop TB.
That said, we can be pleased with the progress that has been
made and I am very appreciative of the time and effort that
the authors of these reviews have put in to bring us up to
date in their respective fields. It has been a pleasure and
privilege to edit this special issue.
Douglas B. Lowrie
Department of Medical Microbiology
Cardiff University
Heath Park
Cardiff
CF14 4XN
UK
E-mail: lowried@cardiff.ac.uk
[Back to top]
Growth Regulation in the Mycobacterial Cell
Robert A. Cox and Gregory M. Cook
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.
[Back to top]
Regulation of Cell Wall Synthesis and Growth
Lynn G. Dover, Like J. Alderwick, Alistair K. Brown, Klaus
Futterer and Gurdyal S. Besra
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.
[Back to top]
Transcriptional Responses of Mycobacterium tuberculosis
Exposed to Adverse Conditions In Vitro
Joanna Bacon and Philip D. Marsh
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.
[Back to top]
Microarray Analysis of Whole Genome Expression of
Intracellular Mycobacterium tuberculosis
Simon J. Waddell and Philip D. Butcher
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.
[Back to top]
Current Strategies for Identifying and Validating
Targets for New Treatment-Shortening Drugs for TB
Kerstin J. Williams and Ken Duncan
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.
[Back to top]
Molecular Tools for Typing and Branding the Tubercle
Bacillus
Marcel A. Behr and Serge Mostowy
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/sub-species 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.
[Back to top]
Correlates of Immune Protection from Tuberculosis
Helen A. Fletcher
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.
[Back to top]
Th2 Cytokines in Susceptibility to Tuberculosis
Graham A.W. Rook
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
Guérin (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.
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