Transgenesis and reverse genetics of mosquito innate immunity
Department of Entomology, University of California, Riverside, California 92521, USA
* Author for correspondence (e-mail: alexander.raikhel{at}ucr.edu)
Accepted 18 July 2003
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Summary |
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Key words: transgenesis, reverse genetics, mosquito, immunity, defensin, cecropin, Relish, Dorsal, dsDNA, RNAi, Aedes aegypti, vitellogenin gene, transposable element
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Introduction |
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The availability of genomic and EST sequences, together with tools such as
DNA microarrays and proteomics, will make mosquitoes powerful model systems
for studying innate immunity against parasites and pathogens
(Dimopoulos et al., 2002;
Christophides et al., 2002
).
The lack of precise genetic tools, however, has been a serous limitation to
the in-depth analysis of the mosquito immune system. Reverse-genetic analyses,
based on RNAi and transgenic techniques, will fill these deficiencies in the
research of mosquito innate immunity.
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RNAi-based reverse genetics |
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Heritable reverse genetics in mosquitoes |
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Our interest is to explore hemolymph-borne antipathogen factors. We have
centered our efforts on the detailed analysis of the vitellogenin
(Vg) gene as a female- and fat body-specific gene that could drive an
effector molecule to a high level of expression, but only exclusively after a
blood meal. Understanding the internal structure of a promoter for use in
transgenic research is essential in order to reduce possible negative effects
associated with the engineered gene. Knowledge of the presence and location of
precise enhancer elements in the promoter allows its manipulation in order to
achieve desired and predicted expression levels. We undertook a detailed
analysis of the Vg promoter
(Kokoza et al., 2001b). This
analysis revealed three regulatory regions in the 2.1-kb upstream portion of
the Vg gene. The proximal region (-121/-619) is required for the
correct tissue- and stage-specific expression at a low level. The median
region (-619/-1071) is responsible for a stage-specific hormonal enhancement
of the Vg expression. Finally, the distal region (-1071/-2015) is
necessary for the extremely high expression levels characteristic of the
Vg gene (Fig. 1). The
development of the Vg gene-based expression cassette that can drive
the fat body-specific expression in response to a blood meal permits the
testing of numerous effector molecules for their antibacterial and
anti-pathogen properties.
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Utilization of transgenesis for overexpression of antimicrobial
peptides
The sequencing of the An. gambiae genome has revealed that
Anopheles mosquitoes utilize fewer antimicrobial peptide (AMP)
families than Drosophila
(Christophides et al., 2002).
The major AMP families in Anopheles are Defensins and Cecropins, each
consisting of four genes. For Ae. aegypti, Defensins and Cecropins
also appear to be the major AMPs
(Lowenberger, 2001
).
Our research has focused primarily on achieving an understanding of the mosquito immune system utilizing the reverse genetic approach through stable transformation. We have elected to focus on Ae. aegypti because this species is more amenable to transgenesis. Furthermore, Ae. aegypti eggs can be stored over 6 months, which makes it possible to maintain a large genetic stock. Ae. aegypti has long been used to study malaria because it can transmit the avian parasite Plasmodium gallinaceum. Moreover, Ae. aegypti genomic and EST projects soon will bring abundant information concerning immune genes and effector molecules in this vector mosquito (D. Severson, personal communication). Therefore, Ae. aegypti represents an outstanding model system for research on vector-pathogen interactions in mosquitoes.
To express Aedes Defensin A, we employed the Hermes
transposable element as a vector and the Drosophila cinnabar gene as
a marker to transform the white-eye Ae. aegypti host strain
(Kokoza et al., 2000). We used
the 2.1 kb 5' upstream region of the vitellogenin (Vg)
gene to drive expression of the Defensin A (DefA) gene. The
Vg-DefA transgene insertion was stable and that the Vg-DefA
transgene was strongly activated in the fat body after a blood meal. The mRNA
levels reached a maximum at 24 h post-blood meal, corresponding to the
expression peak of the endogenous Vg gene. High levels of transgenic
Defensin A were accumulated in the hemolymph of blood-fed female mosquitoes
and persisted for 20-22 days after a single blood feeding
(Fig. 2C). Purified transgenic
Defensin A showed antibacterial activity similar to that of Defensin isolated
from bacterially challenged control mosquitoes. This work made it possible to
use a tissue-specific inducible promoter for the overexpression of immune
genes in the center of innate humoral immunity, the fat body.
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Further progress has depended on developing an efficient, routine gene
transformation for vector insects by using the piggyBac transposable
vector pBac[3xP3-EGFP afm] and the selectable marker EGFP under the 3xP3
promoter for transformation of Ae. aegypti
(Kokoza et al., 2001a).
Selection was performed from immediately hatched, first-instar larvae of
G1 progeny, and this larval selection, in combination with the use
of a vigorous wild-type mosquito strain, significantly improved the efficiency
of the labor-intensive transgenic technique
(Fig. 2A). Based on developed
transgenic techniques, we have successfully generated transgenic mosquitoes
containing the AMPs Defensin A and recently Cecropin A
(Fig. 2B)
(Kokoza et al., 2001a
; A.
Ahmed, S. W. Shin, I. Lobkov, V. Kokoza and A. S. Raikhel, manuscript in
preparation). In survival tests, transgenic mosquitoes carrying either
Vg-DefA or Vg-CecA transgenes exhibited resistance to the
Gram-negative bacterium Enterobacter cloacae that was nearly twice as
high as that of the wild-type mosquitoes
(Fig. 2D) (A. Ahmed, S. W.
Shin, I. Lobkov, V. Kokoza, and A. S. Raikhel, manuscript in preparation).
Anti-malarial activities have been described for the natural cationic AMPs,
Cecropins and Defensins. Studies using exogenous Defensins and Cecropins have
demonstrated that these antibacterial peptides possess potent
anti-Plasmodium activity
(Shahabuddin et al., 1998;
Gwadz et al., 1989
).
Furthermore, Defensin has been implicated in the local innate immune response
of An. gambiae midgut to Plasmodium infection
(Richman et al., 1997
;
Tahar et al., 2002
). These
observations suggest that some AMPs could be involved in anti-malarial defense
and therefore could be explored as potential effector molecules in transgenic
mosquitoes to block transmission of vector-borne diseases. In contrast, dsRNA
knock-out of the Defensin gene in An. gambiae had no effect
on the development of Plasmodium
(Blandin et al., 2002
). In our
preliminary tests, two independent transgenic Ae. aegypti strains
overexpressing Defensin A exhibited 65-70% inhibition of P.
gallinaceum oocyst growth (V. A. Kokoza, M. Shahabuddin, A. Ahmed and A.
S. Raikhel, unpublished results). Further studies are required to implicate
AMPs in anti-Plasmodium activity.
Development of dominant negative knock-out for the IMD/Relish pathway
in Ae. Aegypti
In Drosophila, three types of Rel regulatory molecules can affect
the expression of numerous immune genes, including AMP genes
(Hoffmann et al., 1996;
Hultmark, 2003
). They are
involved in two distinct pathways: the Toll pathway, which activates primarily
anti-fungal and anti-Gram-positive responses and is mediated by Dorsal-related
Immunity Factor (Dif) and Dorsal; and the Imd pathway, which is regulated by
Relish and predominantly directed against Gram-negative bacteria.
We have characterized the Ae. aegypti Relish gene
(Shin et al., 2002). The
primary structure of the Aedes Relish gene exhibited three unique
features compared with Drosophila Relish: (1) the mosquito
Relish gene encodes three alternatively spliced transcripts that give
rise to different proteins, (2) a `Death Domain' is present at the extreme C
terminus, and (3) a short His/Gln-rich stretch followed by a long S-rich
region is present at the putative N-terminal transactivation domain.
Aedes Relish transcripts were induced by bacterial injection, and
their product bound to
B motifs located within the promoters of insect
AMP genes (Shin et al.,
2002
).
We generated genetically immune-deficient transgenic mosquitoes by
overexpression of a dominantly negative construct of Aedes Relish
(Shin et al., 2003).
Relish-mediated immune deficiency (RMID) phenotype was created by transforming
an Ae. aegypti with the
Rel driven by the Vg promoter
using the pBac[3xP3-EGFP afm] vector (Fig.
3A). A stable, transformed strain had a single copy of the
Vg-
Rel transgene, the expression of which was highly
activated by blood feeding. These transgenic mosquitoes were extremely
susceptible to infection by Gram-negative bacteria
(Fig. 3B)
(Shin et al., 2003
). In order
to establish whether or not RMID was dominant-negative, we crossed the
wild-type (UGAL strain) mosquitoes with the RMID transgenic mosquitoes. The
heterozygous RMID/UGAL mosquitoes exhibited the same susceptibility to the
E. cloacae or E. coli infection as the RMID transgenic
mosquitoes (Fig. 4A). These
experiments clearly showed that the RMID phenotype originated from the
Vg-
Rel transgene and that it was genetically
dominant. A hypothetical model of how dominant-negative
Rel is
expressed is presented in Fig.
5.
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Reverse genetics and transgenesis in future studies of mosquito
innate systemic immunity
The development of a reverse genetic approach as described above has opened
a door for further exploration of the mosquito immune system. Using
transgenesis has many advantages, the principal one being the establishment of
genetically stable, transgenic mosquito lines that can be crossed with other
transgenic and wild-type mosquitoes to produce new genetic variations.
With Drosophila, the genetic approach was extensively explored in
order to identify the functional relevance of individual AMPs for both
IMD/Relish- and Toll-mediated innate immunity
(Tzou et al., 2002). The
development of reverse genetic tools for Ae. aegypti has permitted us
to use a similar method for studying mosquito immunity. We generated
transgenic hybrids with the Vg-DefA and
Vg-
Rel transgenes
(Fig. 3C). In these hybrid
transgenic mosquitoes, Vg-mediated activation of Defensin A, which is
independent of Relish-mediated activation, restored the resistance to E.
cloacae (Shin et al.,
2003
). Thus, the RMID phenotype of the transgenic mosquitoes could
be directly linked to the lack of AMP expression. Another hybrid cross that
has been established carries two transgenes: Vg-DefA and
Vg-CecA. Both these AMPs are highly expressed in these hybrid
transgenic mosquitoes, which exhibited an enhanced resistance to certain
microorganisms (A. Ahmed, S. W. Shin, I. Lobkov, V. Kokoza and A. S. Raikhel,
manuscript in preparation).
Surprisingly, the published genome sequences of An. gambiae do not
carry a Dif ortholog. Dif is important in the activation of many
Drosophila immune genes and its apparent absence in mosquitoes raises
a question about the difference between their immune responses and that of
Drosophila. The translated primary sequences of Relish from Ae.
aegypti and An. gambiae (str. PEST, agCP3820, and agCP3732) show
more complicated structures when compared with Drosophila Relish or
mammalian NF-B/I-
B compound proteins, p100 and p105
(Fig. 6). Due to the absence of
Dif in the mosquito immune repertoire, complex alternative splicing and the
additional N-terminal structure of mosquito Relish may in part function as
substitutes for Dif. In order to initiate studies of Toll-mediated innate
immunity in Ae. aegypti, we cloned the gene homologous to
Drosophila dorsal (S. W. Shin, unpublished data). Aedes
dorsal has two isoforms that show high structural similarity to
Drosophila Dl and Dl-B splice variants
(Gross et al., 1999
).
|
Establishing inheritable and inducible expression of dsRNA using
transgenesis and specific promoters is a challenging but important goal in the
development of reverse genetics of mosquitoes. Model organisms, such as C.
elegans and Drosophila, provide a roadmap for how this can be
achieved (Kennerdell and Carthew,
2000; Lam and Thummel,
2000
; Tavernarakis et al.,
2000
; Lee and Carthew,
2003
). Double-stranded RNA can be expressed as an extended
hairpin-loop RNA transcribed from a DNA construct encoding inverted dyad
symmetry (IR) of the target gene. In order to stably transform this IR
construct, it is inserted into a transformation vector under the control of a
tissue- and stage-specific promoter (Fig.
7A). Transgenic mosquitoes can then be generated by a
well-established methodology (Kokoza et al.,
2001a
,2001b
).
Advantages of this approach are: (1) an established stably transformed strain
can be maintained indefinitely in a homozygous state; (2) large numbers of
uniform knockout mosquitoes can be used in genetic and genomic analyses; (3)
RNAi-mediated gene suppression is driven by a promoter in a tissue and
stage-specific manner. The availability of promoters that strictly control IR
expression in a tissue- and stage-specific manner is crucial for extending
this technique to the study of mosquito immunity. For example, it is important
to identify promoters acting in the fat body after the cessation of
vitellogenesis in order to drive immune factor-specific dsRNA expression
beyond temporal activity of the Vg gene. Universal inducible
promoters such as heat shock (hsp) can be used only for
systemic temporal activation of the IR expression. In addition, hsp
promoters are `leaky', and in the case of their direct utilization (as
described above) they could create undesirable effects during organism
development.
|
To circumvent this problem, another transgenic RNAi technique has been
developed in Drosophila in which dsRNA- producing transgenes
are expressed through a binary GAL4/UAS system
(Kennerdell and Carthew, 2000;
Lam and Thummel, 2000
;
Lee and Carthew, 2003
). In
this binary system, the target gene linked to UAS remains silent. The
activator, hsp or a selected tissue/stage-specific promoter, is
coupled to GAL4 without a target gene to activate. Therefore, two independent
parental transgenic strains can be maintained without possible adverse effect
of a target gene expression. After crossing the two parental stains, the
target gene is activated via GAL4-UAS system in the progeny and the
phenotypic expression of its expression can be studied
(Brand and Perrimon, 1993
).
Efficiency of RNAi action has additionally been improved by insertion of a
short intron sequence between inverted repeats of RNAi construct
(Kennerdell and Carthew, 2000
;
Lee and Carthew, 2003
).
Recently, we have demonstrated that addition of a short intron from an
unrelated A. aegypti gene to the anti-GATA factor RNAi construct
improves its performance in Ae. aedypti (G. Attardo, S. Higgs, K. A.
Klingler, D. L. Vanlandingham and A. S. Raikhel, submitted). The development
of an heritable and inducible dsRNA-mediated system based on the binary system
in mosquitoes will offer additional advantages in dissecting immune pathways
and mosquito-pathogen interactions. The proposed strategy for generating of
transgenic RNAi based on the binary GAL4/UAS system is presented in
Fig. 7B.
Mosquito innate immunity clearly has an important role in vector-parasite
interactions, particularly in the immune surveillance of the malaria parasite.
The limitation of parasite replication may depend upon the anti-malarial
activity of immune effector molecules. Two immunological activation pathways
in Drosophila, the IMD/Relish pathway and the Toll/Dif pathway, have
been shown to activate numerous immune genes, and the activation and
regulation of the melanization process seems to be closely linked to these two
pathways (Ligoxygakis et al.,
2002). The development of transgenic immune-deficient mosquitoes
to generate knock-out mutants in the mosquito immunological activation
pathways, and consequent studies of immune-deficient vector-parasite
interactions, will provide insight into the development of alternative
disease-control strategies to block parasite transmission by mosquitoes.
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Acknowledgments |
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