Introduction
The group’s
ongoing and future research program broadly focuses on the
innate immune systems of mosquito disease vectors, and comprises
several independent but synergistically interacting projects. We
are interested in understanding the role and mechanisms of the
mosquito’s innate immune system in the defense against human
pathogens such as Plasmodium and the Dengue virus. A
major focus is concentrated on Anopheles gambiae anti-Plasmodium
defense systems that. Towards this, we have contributed with
several pioneering discoveries. Our mission is to characterize
the defense mechanisms employed by mosquito vectors against
human pathogens. Our competitive advantage derives from a unique
blend of core competencies in molecular entomology, innate
immunity and functional genomics, as well as the access to
state-of-the-art research infrastructure at the Johns Hopkins
Malaria Research Institute. The long-term goal of our research
program is to broaden the basic knowledge of this field and
provide new tools for the development of vector borne disease
control strategies.
Development of mosquito gene expression analysis tools
The majority of our research projects involve high throughput
transcription analyses for the study of infection responsive
processes and immune response regulation. My group has acquired
extensive expertise on microarray-based gene expression
methodology, and developed the first full genome microarrays for
Anopheles gambiae, Aedes aegypti, Culex pipiens
and Plasmodium berghei. These genomics tools have been
instrumental in a variety of project including the dissection of
A. gambiae responses to Plasmodium infection, the
annotation and basic characterization of the Ae. aegypti
and P. pipiens transcriptomes, the study of C. pipiens
responses to West Niles virus infection, and Ae. aegypti
responses to dengue virus infection. We have also performed
studies on Plasmodium gene regulation (Dong et al.,
2006b; Mair et al., 2006; Nene et al., 2007). We have also
contributed towards the development of novel bioinformatics
tools for the analysis of microarray data (Heard et al., 2005).
FUTURE DIRECTIONS: My group is continuously monitoring
the development and evaluating the use of new functional
genomics / post genomics technologies for our studies. Our
Agilent Technologies-based mosquito microarrays provide cost
effective and competitive performance for the next 2-3 years.
High throughput sequencing-based gene expression assays are then
expected to become a more powerful approach and will obviously
be adopted for our studies. The current mosquito microarrays
will continually be updated when the mosquito genome sequences
are re-annotated in order to ensure representation of the most
current transcript annotations.
The
malaria parasite Plasmodium has a complex life cycle in
the vector mosquito that involve several developmental
transitions, interactions and invasion of epithelial tissues and
evasion of the mosquitos' immune system. Gametocytes (GC) are
ingested with an infectious blood meal and fertilize in the
mosquito midgut to develop into a zygote which later transforms
into a motile ookinetes (OK). The ookinetes will invade the
midgut epithelium through a specific mechanism to reach the
basal side where they develop into oocysts (OC). Thousands of
sporozoites (S) will develop within the oocysts and disperse
throughout the mosquito’s' hemolymph. From there the sporozoites
will invade the salivary glands in which they can remain for
weeks, prior to transmission to a new host. Salivary gland
invasion also appears to be mediated by specific interaction.
Relationships
between An. gambiae immune defenses against different
Plasmodium species.
Most studies addressing An. gambiae responses to
Plasmodium infection have utilized the rodent Plasmodium
berghei model system, which is more amenable to experimental
manipulation than is the human parasite Plasmodium falciparum.
However, An. gambiae is not the natural vector of the
P. berghei parasite that differ significantly from the human
pathogen with regard to a variety of biological processes (Dong
et al., 2006b). In order to address potential differences in
mosquito defense responses to the two parasite species, and
thereby validate the usefulness of the rodent model, we
conducted a study were the transcriptional immune responses to
infection of the An. gambiae gut was compared between the
two parasite species. The results showed remarkably diverse
responses to infection with the two parasite species that can be
attributed to differences in infection level and in the biology
of interaction between the two parasite species and the
mosquito. The smaller number of induced putative immune genes
upon P. berghei infection may indicate that the
mosquito’s immune surveillance system is more capable of sensing
P. falciparum, or P. berghei may in some way
suppress the mosquito’s immune response, and that could partly
explain the significantly higher infection levels of the rodent
parasite in An. gambiae. Assays have also been conducted
to address the mosquito responses to malaria infected blood in
absence of parasite stages that infect mosquito tissues, and
showed broad effects on gene regulation, that indicates the
extensive qualitative differences between infected and
non-infected blood (Becker et al., 2004, Lim et al., 2005). The
capacity to mount an immune response to infected blood, in the
absence of Plasmodium invasion of the mosquito gut, is
likely beneficial in controlling Plasmodium infection
since it would allow for enrichment of anti-Plasmodium
factors prior to epithelial invasion. These experiments
generated a wealth of information on novel putative anti-Plasmodium
factors that can be pursued for functional dissection. This
analysis was also extended to the level of gene function for
twelve selected immune genes that were assessed for effect on
infection with both parasite species through RNAi gene silencing
methodology. Seven of these genes were found to influence
mosquito resistance to both parasite species while two genes
were specifically involved in defense against P. falciparum
and another two were more specific for P. berghei. This
study showed that the mosquito is utilizing both parasite
species-specific and universal defense mechanisms to combat
Plasmodium (Dong et al., 2006b).
FUTURE DIRECTIONS: The discovery of significant
differences between infection responses to the two parasite
species have prompted us to exclusively use the human pathogen
P. falciparum in our ongoing and future studies. We are
now addressing the immune system and anti-Plasmodium
responses of natural field mosquitoes and have established
collaboration with a research group in Cameroon to initially
study the differences in immune gene expression between lab and
field mosquitoes, and between different field mosquito
populations.
Relationships between the anti-bacterial and anti-Plasmodium
defense systems and functional versatility of anti-Plasmodium
factors.
The mosquito’s immune system is most likely predominantly
devoted to combating the bacterial and fungal pathogens that are
present in its external environment and intestinal flora, and it
was until recently unclear whether defense mechanisms have
evolved that are specific for Plasmodium (Dong et al.,
2006b). One can argue that the mosquito’s immune surveillance
system and antimicrobial effectors have to cope with a
particularly broad spectrum of pathogens, including the
Plasmodium parasite, as a result of its complex life style
and diverse ecological niches. To address the relationships
between the transcriptional immune responses to Plasmodium
and bacterial immune challenges, we implemented a study that
compared the gene expression in mosquitoes injected with
Escherichia coli and Staphylococcus aureus to
Plasmodium-infected mosquitoes. This study showed a
significant overlap between the mosquito’s response to bacteria
and Plasmodium infection and the bacteria
infection-responsive transcripts included eight anti-Plasmodium
genes. This observation supported the hypothesis that the
mosquito is utilizing some of the same immune pathways and
mechanisms for defense against these two classes of pathogen.
RNAi gene silencing assays were also conducted to address
potential overlaps in anti-Plasmodium and antibacterial
gene function; this study showed that the genes displaying anti-Plasmodium
activity also influenced the mosquito’s resistance to
bacterial infection, while several genes with an effect on
resistance to bacterial infection could not influence
Plasmodium development. These findings suggest the mosquito
is mainly employing its antimicrobial defense system in the
fight against the malaria parasite (Dong et al., 2006b).
Although certain immune gene allele frequencies have been
correlated with Plasmodium exposure in the field, there
is little reason to believe that the mosquito would have
undergone major adaptations to the malaria parasite and evolved
highly specific anti-Plasmodium defense mechanisms (Luckhart
et al., 2003, Morlais et al., 2004).
(Mosquito breeding site in the
field)
In order to further address the role of the mosquito’s
endogenous bacterial flora in a potential priming of the immune
system, we used antibiotic-treatment to generate
“microorganism-free” mosquitoes and tested these for
permissiveness to Plasmodium infection. P. falciparum
infection was 4- to 5-fold higher in bacteria-free compared to
septic An. gambiae, suggesting that the bacteria flora of
mosquitoes can in some way influence its susceptibility to
Plasmodium and hence its vectorial capacity. Microarray gene
expression assays were also used to investigate the effect of
the mosquito microbial flora on immune gene expression by
comparing antibiotic-treated to non-treated adult female
mosquitoes. These assays showed that the endogenous microbial
flora was responsible for a significant level of basal immune
gene expression, some of which was likely to influence the
mosquito’s susceptibility to Plasmodium. Other
experiments have shown that the bacteria in the mosquito gut
also exert a direct anti-Plasmodium activity which,
however, does not explain the large difference in susceptibility
between antibiotic-treated and non-treated mosquitoes (Dimopoulos
group, unpublished data).
We know from studies in Drosophila that the insect innate immune
responses to bacterial infection involve the activation of the
intracellular immune signaling pathways Toll and Imd, that are
highly conserved throughout insect phyla (Waterhouse et al.,
2007). In order to address the implication of these pathways in
the defense against P. falciparum a RNAi gene silencing
strategy was employed that targeted the negative regulators
Cactus and Caspar to activate the Toll and Imd pathways,
respectively, upon parasite infection (Meister et al., 2005,
Frolet et al., 2006, Kim et al., 2006). Both pathways were
capable of controlling susceptibility to infection and
activation of the Imd pathway had the strongest effect with a
~90% reduction in P. falciparum infection level (Dimopoulos
group, unpublished data). This finding further support the
extensive overlaps between the mosquito’s antibacterial and
anti-Plasmodium defense systems. We pursued the analysis
further to address other immune defense reactions such as
melanization, which is a major Plasmodium killing
mechanism in refractory mosquitoes. We used a Sephadex bead
melanization assay to test the implication of various immune
genes in the melanization mechanism. These experiments showed
that the RNAi –mediated silencing of the TEP1 and LRIM1 genes,
which encode proteins known to mediate Plasmodium killing
in malaria-susceptible mosquitoes, significantly compromised the
ability of mosquitoes to melanize the beads. In contrast,
silencing of two Plasmodium protective c-type lectins,
CTL4 and CTLMA2, did not affect bead melanization (Osta et al.,
2004; Blandin et al.,2004). This data suggest that the anti-Plasmodium
factors have multiple functions, as determinants of both
Plasmodium and bacteria killing as well as melanization of the
parasite and other foreign bodies, while the Plasmodium
protective factors are specifically utilized by the parasite for
evasion of mosquito defense mechanisms (Warr et al., 2006).
FUTURE DIRECTIONS: Our working hypothesis is that the
presence of bacteria and fungi in the mosquito primes or
activates immune activity against Plasmodium in addition
to having a direct effect on parasite development in the
mosquito gut. Consequently, the mosquito’s anti-Plasmodium
defense system is largely shaped by its microbial exposure in
nature, which is continuous, in contrast to the relatively low
exposure rate to Plasmodium. Current and future projects
aim at the identification of the mechanism(s) and stage(s) at
which the presence of bacteria negatively impacts upon the
development of malaria parasites. We have initiated the
functional characterization of some of the bacteria induced
immune genes using RNAi gene silencing and other assays to
address their specific role in defense (Dimopoulos group,
unpublished data). We will also address the mosquito’s microbial
exposure in nature since it is likely to vary significantly
between different ecological niches and thereby be responsible
for some of the differences in vectorial capacity between
different mosquito populations.
Molecular characterization of the hyper-variable pattern
recognition receptor AgDscam function in anti-Plasmodium
defense
The innate immune system of the mosquito, unlike that of
vertebrates, appears to lack the capacity to transiently adapt
its pattern recognition repertoire toward the recognition of
specific pathogens or to produce immunological memory. Instead,
it relies on the limited diversity of its repertoire of ~130
germ line-encoded pattern recognition receptors (Waterhouse et
al., 2007). Malaria transmission in nature involves a variety of
mosquito populations and parasite strains that differ in their
genetic makeup. This variability complicates the development of
malaria control strategies based on transgenic expression of
anti-Plasmodium factors in vector mosquitoes, since their
parasite-killing activity may vary depending on parasite strain-
and species-specific differences (Aguilar et al., 2005). A
broad-spectrum anti-Plasmodium factor would therefore be
preferred for use in such control strategies. One of these
recently identified anti-Plasmodium factors is the Down syndrome
cell adhesion molecule (Dscam). Like its D. melanogaster
ortholog, Dscam in Anopheles gambiae (AgDscam) possesses
82 alternatively spliced Ig (immunoglobulin) domain exons, which
have the potential for generating 31,920 alternative splice
forms with different interaction specificities. We have shown
that AgDscam is an essential hypervariable receptor of the
Anopheles gambiae immune surveillance system, demonstrating
phenotypic plasticity in response to different spectra of
pathogen exposure. We have shown that AgDscam produces splice
form repertoires that are pathogen challenge-specific in the
sense that they are enriched with receptor molecules having
increased affinity and defense specificity for the eliciting
pathogen (Dong et al., 2006a; Watson et al., 2005). We have also
shown that AgDscam splicing is regulated by the Toll and Imd
immune pathways (Dimopoulos group, unpublished data). AgDscam’s
broad range of anti-pathogen activity renders it a particularly
interesting candidate for the development of anti-Plasmodium
control strategies based on transgenic expression in mosquitoes;
it has a high probability to display activity against a variety
of Plasmodium strains and species, and thereby overcome the
constraints of the more “specific” anti-Plasmodium
factors in the field where diversity is large.
FUTURE DIRECTIONS: A better understanding of AgDscam’s
anti-Plasmodium activity with regard to its
infection-stage specificity, splice form specificity, and
immune-related regulation are the first essential steps towards
the complete dissection of its biology, which can permit the
development of control strategies based on this gene. We are
currently pursuing several studies that aim at a detailed
understanding of AgDscam function in anti-Plasmodium
defense; we have shown that AgDscam is capable of associating
with Plasmodium in the mosquito gut epithelium (Fig. 1)
and are now focusing on the splice form specificity of this
association (Dimopoulos group, unpublished data). We are also
addressing the roles of the Toll and Imd immune signaling
pathways in regulating AgDscam splicing further, and its
potential implication in killing the later sporozoite stage
plasmodia. [Figure 1: Confocal microscopy images of ookinetes in
the gut epithelium. Samples have been DAPI-stained and subjected
to immunohistochemical analysis using the anti-Pfs25 and AgDscam
antibodies. The AgDscam pre-immune serum produced no detectable
staining of the ookinetes (not shown).]
Molecular characterization of the Imd pathway mediated
resistance to P. falciparum
The microarray based expression analyses on mosquito responses
to Plasmodium infection, in conjunction with the
RNAi-based screens for anti-Plasmodium genes, provided
hints that the Imd pathway is a major player in the defense
against P. falciparum. We have shown that transient
activation of the Imd pathway regulated transcription factor
Rel2 will result in resistance to P. falciparum in three
diverse malaria vector species. We have also identified some of
the effectors that are mediating the Imd pathway’s anti-Plasmodium
activity, and are currently focusing on the dissection of this
defense system through transgenic methodologies and functional
assays.
FUTURE DIRECTIONS: We have initiated projects that will
address the suitability of the Imd pathway anti-Plasmodium
defenses for the development of malaria control strategies based
on genetically modified mosquitoes.
The Aedes aegypti anti viral defense system
The molecular biology of Dengue virus – mosquito interaction is
largely unknown. We have made use of our A. aegypti
genome microarrays in conjunction with other functional genomics
tools to study the mosquito’s responses to infection with the
Dengue virus (Nene et al., 2007). A comparison of gene
expression between non-infected and Dengue (DENV-2) virus
infected mosquitoes, at the stage when the virus spreads from
the mosquito gut to other tissues, identified 240 up-regulated
and 192 down-regulated genes in the non-gut tissues and 28
up-regulated and 35 down-regulated genes in the gut tissue. A
significant proportion of these genes have predicted functions
in the mosquito’s innate immune system with a particular bias
towards components of the Toll immune signaling pathway. The
negative regulator Cactus was down-regulated while several
pattern recognition receptors, signaling modulation and
transduction factors and effector molecules were up-regulated.
This suggested implication of the Toll pathway in the
anti-Dengue defense. To address this hypothesis at the
functional level, we tested the impact of Toll pathway
activation, through RNAi silencing of the Cactus gene, on virus
infection in the gut according to Frolet et al., 2006. This lead
to an approximately four-fold decrease in infection level
compare to the GFP dsRNA treated mosquitoes (Xi et al., 2008).
Hence, the A. aegypti Toll pathway appears to play a
major role in defending the mosquito against Dengue.
FUTURE DIRECTIONS: Current and future experiments focus
on the dissection of this anti-Dengue immune response through
gene silencing of other genes that show regulation upon
infection and a determination of the temporal and spatial
specificity of virus clearance in the mosquito. Insect immunity
to viruses is poorly described and these studies will therefore
contribute significantly to the field and also provide potential
tools for the development of dengue control strategies.
Molecular analysis of photic inhibition of blood-feeding in
Anopheles gambiae
Anopheles gambiae mosquitoes exhibit an endophilic,
nocturnal blood feeding behavior. Despite the importance of
light as a regulator of malaria transmission, our knowledge on
the molecular interactions between environmental cues, the
circadian oscillators and the host seeking and feeding systems
of the Anopheles mosquitoes is limited. We have shown that the
blood feeding behavior of mosquitoes is under circadian control
and can be modulated by light pulses, both in a clock dependent
and in an independent manner. Short light pulses (~2-5 min) in
the dark phase can inhibit the blood-feeding propensity of
mosquitoes momentarily in a clock independent manner, while
longer durations of light stimulation (~1-2 h) can induce a
phase advance in blood-feeding propensity in a clock dependent
manner. The temporary feeding inhibition after short light
pulses may reflect a masking effect of light, an unknown
mechanism which is known to superimpose on the true circadian
regulation. Nonetheless, the shorter light pulses resulted in
the differential regulation of a variety of genes including
those implicated in the circadian control, suggesting that light
induced masking effects also involve clock components. Light
pulses (both short and long) also regulated genes implicated in
feeding as well as different physiological processes like
metabolism, transport, immunity and protease digestions.
RNAi-mediated gene silencing assays of the light pulse regulated
circadian factors timeless, cryptochrome and three takeout
homologues significantly up-regulated the mosquito's
blood-feeding propensity. In contrast, gene silencing of light
pulse regulated olfactory factors down-regulated the mosquito's
propensity to feed on blood. Our study show that the mosquito’s
feeding behavior is under circadian control. Long and short
light pulses can induce inhibition of blood-feeding through
circadian and unknown mechanisms, respectively, that involve the
chemosensory system (Das and Dimopoulos, 2008).
FUTURE DIRECTIONS: We are interested in gaining a better
understanding of how environmental cues can influence mosquito
behavioral attributes that are important for disease
transmission.
COLLABORATIONS
We are pursuing collaborations with:
- Dr. Mike Strand on An. gambiae hemocyte function in
immunity.
- Dr. Bruce Christensen on Ae. aegypti responses to
filarial infection.
- Dr. Alexander Raikhel on Ae. aegypti immune response
regulation.
- Dr. Mariano Garcia Blanco on dengue host and restriction
factors.
- Dr. Martin Hibbard on immune responses to dengue infection.
- Dr. Andy Waters and Dr. Gunnar Mair on Plasmodium gene
regulation.
- Dr. Nirbhay Kumar on An. gambiae responses to blood
stage Plasmodium.
- The Culex pipiens genome sequencing consortium.
FUNDING
NIH/NIAID
Ellison Medical Foundation
WHO-TDR
NSF
ASM
A STAR
Johns Hopkins Malaria Research Institute
Johns Hopkins School of Public Health
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