Current
Volume 4, Number 6, 2004
Contents
Biology
and Biochemistry of the Kinetoplastid Parasites
Executive
Editor: Hemanta K. Majumder
The Determinants of Chagas Disease:
Connecting Parasite and Host Genetics Pp. 549-562
David
A. Campbell, Scott J. Westenberger and Nancy R. Sturm
The Molecular Control of Antigenic Variation
in Trypanosoma brucei Pp. 563-576
David
Horn
Genetic Regulation of Protein Synthesis in
Trypanosomes Pp. 577-584
Anish
Das and Vivian Bellofatto
cAMP Signalling in the Kinetoplastid Protozoa
Pp. 100%-599
T.
Seebeck, R. Schaub and A. Johner
New Insights into the Developmental Biology
and Transmission Mechanisms of Leishmania Pp. 601-609
P.A.
Bates and M.E. Rogers
The Application of Gene Expression Microarray
Technology to Kinetoplastid Research Pp. 611-621
Robert C. Duncan, Poonam Salotra, Neena Goyal, Natalia S. Akopyants, Stephen M. Beverley and Hira L. Nakhasi
The Structure and Replication of Kinetoplast
DNA Pp. 623-647
Joseph Shlomai
Surface Determinants of Leishmania
Parasites and their Role in Infectivity in the Mammalian Host Pp.
649-665
Thomas Naderer, James E. Vince and Malcolm J. McConville
Leishmaniasis: Current Status of Vaccine
Development Pp. 667-679
Bindu Sukumaran and Rentala Madhubala
Macrophage Specific Drug Delivery in
Experimental Leishmaniasis Pp. 681-689
Mukul
Kumar Basu and Sanchaita Lala
Role of Chemokines in Leishmania
Infection Pp. 691-696
Kaushik Roychoudhury and Syamal Roy
Progress in Vaccine Research and Possible
Effector Mechanisms in Visceral Leishmaniasis Pp. 697-709
Rajesh Ravindran and Nahid Ali
DNA Topoisomerases in Life and Death:
Implications in Kinetoplastid Protozoa Pp. 711-722
Arnab
Roy Chowdhury and Hemanta K. Majumder
Abstracts
[Back to top] The Determinants of Chagas Disease:
Connecting Parasite and Host Genetics
David
A. Campbell, Scott J. Westenberger and Nancy R. Sturm
As a consequence
of infection by Trypanosoma cruzi, 30% of victims may develop chronic
Chagas disease, which presents a spectrum of pathology including
cardiomyopathy, megacolon and megaesophagus. The outcome of infection in a
particular individual is the result of a set of complex interactions among the
host genetic background, environmental and social factors, and the genetic
composition of the parasite, all of which can be complicated by mixed
infections and re-infections. Initially we consider what is known about the
genetic structure and biological properties of the protozoan. Currently, six
distinct subgroups have been characterized by different combinations of four distinct
genotypic classes. The recent demonstration of genetic exchange via non-meiotic
cell fusion illustrates a mechanism by which maintained heterogeneous
polyploidy may have been generated in these parasites. Subsequently, we
consider factors in humans and in experimental mouse-infection and tissue
culture models that have contributed to our understanding of the host's
susceptibility or resistance to disease. Identification of the direct players
in host-pathogen interactions at the establishment and chronic phases of the
disease is perhaps the best hope of a clinical handle for treatment. At some
point in the future, these disparate areas of study will have to come together.
It is to be hoped that this scientific fusion will result in better prognosis
and treatment of Chagas disease.
[Back to top] The Molecular Control of Antigenic Variation
in Trypanosoma brucei
David
Horn
The African
trypanosome, Trypanosoma brucei, is a protozoan that causes sleeping
sickness in humans and N’gana in livestock. These flagellated parasites are
directly exposed to immune defences as they circulate in the mammalian host
bloodstream but they maintain persistent infections by undergoing antigenic
variation. Central to this process is mono-allelic transcription and switching
of the expressed variant-surface glycoprotein (VSG) gene which encodes the vast
majority of their dense surface coat. The active telomeric VSG is transcribed
by RNA polymerase I in an ‘expression site body’ (ESB) while transcription
attenuation occurs at ‘inactive’ telomeres. Here, I review what is known about
the molecular mechanisms involved in achieving antigenic variation and outline
how we intend to exploit genome sequence and new tools, particularly RNA
interference, to identify and characterise factors required for VSG regulation.
[Back to top] Genetic Regulation of Protein Synthesis in
Trypanosomes
Anish
Das and Vivian Bellofatto
It is becoming increasingly
clear that parasitic protozoa remain a scourge to humans in the 21st
century. The trypanosomes are a diverse group of insect-transmitted parasites
that wiggle their way through multiple life cycle stages as they destroy human
lives. Exquisitely detailed studies of these organisms reveal basic differences
in gene expression that separate these single celled eukaryotes from
multicellular eukaryotic organisms and have suggested numerous potential drug
targets.
[Back to top] cAMP Signalling in the Kinetoplastid
Protozoa
T.
Seebeck, R. Schaub and A. Johner
Several species of
kinetoplastid protozoa cause major human infectious diseases. Trypanosoma
cruzi is responsible for the fatal Chagas disease in large parts of South
America, the various species of Leishmania cause a number of different
human diseases with millions of patients world-wide, and the African
trypanosome Trypanosoma brucei is the agent of human sleeping sickness,
a disastrously re-emerging epidemic of fatal infections in Sub-Saharan Africa.
Chemotherapy of all of these infections is in a very unsatisfactory state. cAMP
signalling pathways in humans have provided interesting drug targets for a
number of clinical conditions, from asthma to impotency. Similarly, cAMP
signalling in kinetoplastids might offer useful targets for the development of
novel antiparasitic drugs, which makes their exploration an urgent need.
Current knowledge
suggests that cAMP signalling proceeds along very similar pathways in all
kinetoplastid pathogens (T. cruzi, the Leishmanias and T. brucei). Their
adenylyl cyclases are structurally very different from the human enzymes and
appear to function as enzyme-linked cell surface receptors. They might
represent the major sensory apparatus of the kinetoplastids, guiding much of
their environmental sensing and host/parasite interaction. The cAMP-specific
phosphodiesterases of the kinetoplastids are rather similar to those of human
cells and might function in similar ways. Essentially nothing is known on
downstream effectors of cAMP in the kinetoplastids. Homologues of protein
kinase A and its regulatory subunits have been identified, but their
biochemical properties seem to be disctinct from that of mammalian protein
kinase A.
[Back to top] New Insights into the Developmental Biology
and Transmission Mechanisms of Leishmania
P.A. Bates and M.E. Rogers
Leishmania alternates between two main morphological
forms in its life cycle: intracellular amastigotes in the mammalian host and
motile promastigotes in the sandfly vector. Several different forms of
promastigote can be recognised in sandfly infections. The first promastigote
forms, which are found in the sandfly in the bloodmeal phase, are
multiplicative procyclic promastigotes. These differentiate into nectomonad
promastigotes, which are a non-dividing migratory stage moving from the
posterior to the anterior midgut. When nectomonad promastigotes arrive at the
anterior midgut they differentiate into leptomonad forms, a newly named life
cycle stage, which resume replication. Leptomonad promastigotes, which are
found in the anterior midgut, are the developmental precursors of the
metacyclic promastigotes, the mammal-infective stages. Leptomonad forms also
produce promastigote secretory gel, a substance that plays a key role in
transmission by forming a physical obstruction in the gut, forcing the sandfly
to regurgitate metacyclic promastigotes during bloodfeeding.
[Back to top] The Application of Gene Expression Microarray
Technology to Kinetoplastid Research
Robert
C. Duncan, Poonam Salotra, Neena Goyal, Natalia S. Akopyants, Stephen M.
Beverley and Hira L. Nakhasi
Protozoan
parasites in the order Kinetoplastida cause severe disease primarily in
tropical and subtropical areas. Vaccines to control these diseases have shown
some promise, but none are in active clinical use. Drug treatments are
available for all of the acute infections, but the emergence of resistance and
an unresponsive chronic phase are current problems. Rapid advances in genomic
technology open the possibility of discovering new genes that can contribute to
vaccine initiatives or serve as targets for development of new drugs. The DNA microarray
is a genomic technology, which is being applied to new gene discovery in
kinetoplastid parasites. Both cDNA and genomic microarrays for Leishmania
major have identified a number of new genes that are expressed in a
stage-specific fashion and preliminary results from a L. donovani
genomic microarray also demonstrated new gene discovery. A microarray of Trypanosoma
brucei genomic fragments identified new genes whose expression differs
between the insect borne stage and the human infectious stage of the parasite.
The next few years, building on this foundational work, should witness the most
exciting stage as microarrays are applied to questions such as the basis of
drug resistance, post kala azar dermal leishmaniasis, the regulation of
differentiation to infectious stages, linking coordinately regulated pathways
of genes and development of genetically defined parasites that may have
potential as live attenuated vaccines.
[Back to top] The Structure and Replication of Kinetoplast
DNA
Joseph
Shlomai
Kinetoplast DNA
(kDNA), the mitochondrial DNA of flagellated protozoa of the order
Kinetoplastida, is unique in its structure, function and mode of replication.
It consists of few dozen maxicircles, encoding typical mitochondrial proteins
and ribosomal RNA, and several thousands minicircles, encoding guide RNA
molecules that function in the editing of maxicircles mRNA transcripts. kDNA
minicircles and maxicircles in the parasitic species of the family
Trypanosomatidae are topologically linked, forming a two dimensional
fishnet-type DNA catenane. Studies of early branching free-living and parasitic
species of the Bodonidae family revealed various other forms of this remarkable
DNA structure and suggested the evolution of kDNA from unlinked DNA circles and
covalently-linked concatamers into a giant topological catenane. The
replication of kDNA occurs during nuclear S phase and includes the duplication
of free detached minicircles and catenated maxicircle and the generation of two
progeny kDNA networks that segregate upon cell division. Recent reports of
sequence elements and specific proteins that regulate the periodic expression
of replication proteins advanced our understanding of the mechanisms that
regulate the temporal link between mitochondrial and nuclear DNA synthesis in
trypanosomatids. Studies on kDNA replication enzymes and binding proteins
revealed their remarkable organization in clusters at defined sites flanking
the kDNA disk, in correlation with the progress in the cell cycle and the
process of kDNA replication. In this review I describe the recent advances in
the study of kDNA and discuss some of the major challenges in deciphering the
structure, replication and segregation of this remarkable DNA structure.
[Back to top] Surface Determinants of Leishmania Parasites and their Role in
Infectivity in the Mammalian Host
Thomas
Naderer, James E. Vince and Malcolm J. McConville
Leishmania are intracellular protozoan parasites that reside
primarily in host mononuclear phagocytes. Infection of host macrophages is
initiated by infective promastigote stages and perpetuated by an obligate
intracellular amastigote stage. Studies undertaken over the last decade have
shown that the composition of the complex surface glycocalyx of these stages
(comprising lipophosphoglycan, GPI-anchored glycoproteins, proteophosphoglycans
and free GPI glycolipids) changes dramatically as promastigotes differentiate
into amastigotes. Marked stage-specific changes also occur in the expression of
other plasma membrane components, including type-1, polytopic and peripheral
membrane proteins, reflecting the distinct microbicidal responses and
nutritional environments encountered by these stages. More recently, a number
of Leishmania mutants lacking single or multiple surface components have
been generated. While some of these mutants are less virulent than wild type
parasites, many of these mutants exhibit only mild or no loss of virulence.
These studies suggest that, 1) the major surface glycocalyx components of the
promastigote stage (i.e. LPG, GPI-anchored proteins) only have a transient or
minor role in macrophage invasion, 2) that there is considerable functional
redundancy in the surface glycocalyx and/or loss of some components can be
compensated for by the acquisition of equivalent host glycolipids, 3) the
expression of specific nutrient transporters is essential for life in the
macrophage and 4) the role(s) of some surface components differ markedly in
different Leishmania species. These mutants will be useful for
identifying other surface or intracellular components that are required for
virulence in macrophages.
[Back to top] Leishmaniasis: Current Status of Vaccine Development
Bindu
Sukumaran and Rentala Madhubala
Leishmaniasis, a
spectrum of diseases caused by various forms of Leishmania has become a
major health problem all over the world. Vaccination against leishmaniasis has
passed through many developmental stages beginning with the ancient practice of
‘leishmanization’. Due to various problems and difficulties associated with
traditional vaccines, the interest has been shifted to novel approaches of
vaccination like DNA vaccination, vaccination with live vectors encoding
leishmanial antigens and finally to designer vaccines. In an effort towards
developing an anti-leishmanial vaccine, our laboratory has been working on
various genes present in an amplified locus of Leishmania known as the
‘LD1 locus’. Two genes, ORFF and BT1 (previously ORFG), are part of the
multigenic LD1 locus on chromosome 35. BT1 encodes a biopterin transporter,
while the function of ORFF gene product is unknown. Immunization of mice with
recombinant ORFF (rORFF) and BT1 proteins, individually, or in combination,
conferred partial protection against challenge with Leishmania donovani.
We also tested the protective efficacy of ORFF DNA vaccine in BALB/c mice model
and found that the level of protection was significantly higher than that of
ORFF protein. Protection conferred by ORFF DNA vaccine correlated with
significant levels of in vitro splenocyte proliferation and low levels
of antigen-specific antibodies. There was a preferential production of IFN-g compared to IL-4, which indicated the
induction of a protective Th1 response, by the DNA vaccine. Thus, DNA
immunization may offer an attractive alternative strategy against
leishmaniasis. We present here the current status of vaccine development
against leishmaniasis.
[Back to top] Macrophage Specific Drug Delivery in Experimental Leishmaniasis
Mukul
Kumar Basu and Sanchaita Lala
Macrophage-specific
delivery systems are the subject of much interest nowadays, because of the fact
that macrophages act as host cells for many parasites and bacteria, which give
rise to outbreak of so many deadly diseases(eg. leishmaniasis, tuberculosis
etc.) in humans. To combat these deadly diseases initially macrophage specific
liposomal delivery system were thought of and tested in vivo against
experimental leishmaniasis in hamsters using a series of indigenous or
synthetic antileishmanial compounds and the results were critically discussed. In
vitro testing was also done against macrophages infected with Leishmania
donovani, the causative agent for visceral leishmaniasis. The common
problem of liposome therapy being their larger size, stability and storage,
non-ionic surfactant vesicles, niosomes were prepared, for their different drug
distribution and release characteristics compared to liposomes. When tested in
vivo, the retention capacity of niosomes was found to be higher than that
of liposomes due to the absence of lipid molecules and their smaller size. Thus
the therapeutic efficacy of certain antileishmanial compounds was found to be
better than that in the liposomal form. The niosomes, being cheaper, less
toxic, biodegradable and non-immunogenic, were considered for sometime as
suitable alternatives to liposomes as drug carriers. Besides the advent of
other classical drugs carriers(e.g. neoglycoproteins), the biggest challenge
came from polymeric delivery vehicles, specially the polymeric nanoparticles
which were made of cost effective biodegradable polymers and different natural
polymers. Because of very small size and highly stable nature, use of
nanoparticles as effective drug carriers has been explored in experimental
leishmaniasis using a series of antileishmanial compounds, both of indigenous
and synthetic origin. The feasibility of application in vivo, when
tested for biological as well as for other physicochemical parameters, the
polymeric nanoparticles have turned out to be the best and thus may be
projected for effective use in the clinics.
[Back to top] Role of Chemokines in Leishmania Infection
Kaushik
Roychoudhury and Syamal Roy
Chemokines are a
growing group of chemoattractant cytokines that play important roles in
physiological as well as pathological processes. Their roles in various aspects
of pathogenesis and inflammation have come to light in the past decade or so.
It is becoming increasingly clear that chemokines play a major, perhaps
decisive role in Leishmania infections. In this review, we recapitulate
important works linking the chemokine system with relation to visceral and
cutaneous leishmaniasis over the past decade and attempt to put it all together
to propose a single yet unfinished model to account for all the findings.
[Back to top] Progress in Vaccine Research and Possible Effector Mechanisms in Visceral
Leishmaniasis
Rajesh
Ravindran and Nahid Ali
Visceral
leishmaniasis represents a serious public health concern in endemic regions and
is rapidly emerging as an opportunistic infection in HIV patients. The disease
is difficult to diagnose and prevent, and available treatment is associated
with toxicity and drug resistance. Even though significant headway has been
made in the development of vaccines against cutaneous leishmaniasis, visceral
leishmaniasis has received limited attention. The fact that a large proportion
of the people living in endemic areas have self-resolving subclinical infection
and individuals once recovered are immune to reinfection provides a rationale
for designing immunoprophylactic strategies against visceral leishmaniasis. The
primary aim of this paper is to review advances in vaccination strategies
against visceral leishmaniasis, suggesting possible effector mechanism leading
to resistance. It also covers the role of immunostimulators and gives an
account of the adjuvants used against visceral leishmaniasis. Vaccine
strategies in different established experimental models have also been dealt
with which can provide potential leads for their application in humans. In
light of the available observations made during the course of studies performed
on experimental models of visceral leishmaniasis there is increasing evidence
that a successful approach towards a vaccine involves the requirement of Th1
subset of CD4+ cells along with Th2, CD8+, and B cells.
In this review we present the possible mechanism of interaction of these cells
and their effector molecules in providing resistance against visceral
leishmaniasis for the future design of effective vaccine against this disease.
[Back to top] DNA Topoisomerases in Life and Death: Implications in Kinetoplastid
Protozoa
Arnab
Roy Chowdhury and Hemanta K. Majumder
Current biomedical
research has its focus on the search for newer intervention strategies to control
public health impact of parasitic diseases. The dramatic advances of molecular
and cellular biology in recent times have provided opportunities for
discovering and evaluating molecular targets for drug designing, which now form
a rational basis for the development of improved anti parasitic therapy. DNA
topoisomerases, the “cellular magicians” involved in nearly all biological
processes governing DNA, have emerged as one such biological target. Over the
last two decades, interest in topoisomerases has expanded beyond the realm of
the basic science laboratory into the clinical arena. This review aims at
providing a comprehensive insight into the biology of DNA topoisomerases and
also focus on its evolution as a drug target in the unicellular kinetoplastids.