Download Anopheles gambiae Drosophila melanogaster Bo Lindberg

Novel Modes of Immune Activation in Anopheles gambiae
and Drosophila melanogaster
Bo Lindberg
Novel Modes of Immune Activation
in Anopheles gambiae and Drosophila
melanogaster
Bo Lindberg
Department of Molecular Biosciences, The Wenner-Gren Institute
Stockholm University, Sweden 2014
© Bo Lindberg, Stockholm 2014
ISBN 978-91-7447-859-4 (includes pages 1-81)
Printed in Sweden by Universitetsservice US-AB, Stockholm 2014
Distributor: Department of Molecular Biosciences, The Wenner-Gren Institute
SAMMANFATTNING
Malaria är en parasitsjukdom som orsakas av släktet Plasmodium och drabbar varje år runt en halv miljard människor varav nära en miljon avlider.
Nästan samtliga dödsfall rör barn under fem år. Sjukdomen är numera i princip utrotad i västvärlden men är mycket vanligt förekommande i subtropikerna, särskilt i Afrika där också den dödligaste parasitarten Plasmodium
falciparum är utbredd. Malaria sprids till människor genom saliven hos
myggor av släktet Anopheles. Myggan smittas i sin tur av människor som har
parasiter i blodet. För att kunna överföras till en människa måste malariaparasiten ta sig igenom en serie utvecklingsstadier i myggan och vandra från
tarmen till salivkörtlarna, en process som tar tre veckor. Under denna period
attackeras parasiten av myggans immunförsvar och lider ofta stora förluster.
Det är dock inte utrett om, och i så fall exakt hur myggans immunförsvar
känner igen parasiten eller om det i själva verket är myggans tarmbakterier
som alarmerar om att något är fel. Till skillnad från däggdjur saknar insekter
ett så kallat adaptivt immunförsvar och kan därför inte tillverka antikroppar.
De förlitar sig istället på ett medfött immunsvar där vissa återkommande
strukturella drag hos sjukdomsalstrande organismer känns igen av specialiserade receptorer hos immuncellerna. En av huvudmålen med det här arbetet
var att utforska nya tillvägagångssätt för insekter att känna igen inkräktare. I
den första artikeln visar vi att immunförsvaret hos myggan Anopheles gambiae reagerar på ett ämne som förkortas HMBPP. Denna lilla molekyl tillverkas av både malariaparasiten och många bakterier, men inte av flercelliga
djur, och används bland annat i tillverkningen av hormoner och vitaminer.
Vi visar bland annat att HMBPP utsöndras av malariaparasiten och att immunförsvaret aktiveras hos myggor när ämnet tillsätts i deras blodmål vilket
också påverkade antalet tarmbakterier. I artikel II användes bakterien Escherichia coli och bananflugan Drosophila melanogaster som modellorganismer för att visa att radioaktivt bestrålade bakterier fortsätter att producera
och utsöndra ämnen som kan aktivera immunsvar hos insekter. I den tredje
artikeln kartlades hela arvsmassan hos bakterien Elizabethkingia anophelis,
en vanligt förekommande bakterie i myggtarmen. Informationen i artikel III
användes sedan i artikel IV för att beskriva bakteriens karaktärsdrag, bland
annat hur den besitter gener för att hjälpa myggan att bryta ned komplexa
kolhydrater i nektar samt den genetiska bakgrunden till bakteriens antibiotikaresistens.
ABSTRACT
Malaria is a disease of poverty and continues to plague a great part of the
world’s population. An increased understanding of the interactions between
the vector mosquito, the malaria parasite, and also the mosquito gut microbiota are pivotal for the development of novel measures against the disease.
The first aim of this thesis was to gain a deeper knowledge of the microbial
compounds that elicit immune responses in the main malaria vector Anopheles gambiae and also using the model organism Drosophila melanogaster.
The second aim was to analyze the genome characteristics in silico of a bacterial symbiont from the mosquito midgut.
In Paper I, we investigated the immunogenic effects of (E)-4-hydroxy-3methyl-but-2-enyl pyrophosphate in Anopheles. This compound is the primary activator of human Vγ9Vδ2 T cells and is only produced by organisms
that use the non-mevalonate pathway for isoprenoid synthesis, such as Plasmodium and most eubacteria but not animals. We show that the parasite releases compounds of this nature and that provision of HMBPP in the bloodmeal induces an immune response in the mosquito.
In Paper II, we investigated whether bacteria inactivated by gammairradiation could still stimulate potent immune responses in Drosophila cells.
We show that E. coli retains the capacity to synthesize and release peptidoglycan de novo for several days after the irradiation event. When cells
were stimulated with supernatants from irradiated bacteria, however, a
unique response was observed.
In Paper III, we presented the draft genome sequence of Elizabethkingia
anophelis, a predominant gut symbiont of An. gambiae recently described in
our lab and subsequently found in another laboratory strain of the mosquito.
The genome data were then annotated in Paper IV to gain insights into the
symbiotic characteristics of the bacterium, as well as the genetic background
for its strong antibiotic resistance. In conclusion, this thesis work has shed
light on novel modes for stimulating immune responses in insects and also
led to the characterization of a predominant bacteria in the mosquito gut that
may be used in future malaria intervention strategies.
LIST OF PUBLICATIONS
I.
Lindberg, B.G.., Merritt, E., Olofsson, B., Faye, I.
Immune eliciting and antioxidant properties of a microbe-associated
isoprenoid precursor in Anopheles gambiae.
PLOS ONE. 2013 Aug 13;8(8):e73868.
II.
Lindberg, B.G.. Oldenvi, S., Steiner, H.
Medium from γ-irradiated Escherichia coli bacteria stimulates a
unique immune response in Drosophila cells.
Manuscript, submitted.
III.
Kukutla, P.*, Lindberg, B.G.*, Pei, D., Rayl, M., Yu, W., Steritz,
M., Faye, I., Xu, J.
Draft genome sequences of Elizabethkingia anophelis R26T and
Ag1 from the midgut of the malaria mosquito Anopheles gambiae.
Genome Announc. 2013 Dec 5;1(6):e01030-13.
IV.
Kukutla, P.*, Lindberg, B.G*, Pei, D., Rayl, M., Yu, W., Steritz,
M., Faye, I., Xu, J.
Insights from the genome annotation of Elizabethkingia anophelis
from the midgut of the malaria mosquito Anopheles gambiae.
Manuscript, revised and submitted.
*Contributed equally
ABBREVIATIONS
AMP
APC
APL1
Cact/CACT
DAMP
DAP
Dif
DUOX
ERK
FOXO
GNBP
GPI
HMBPP
Hz
IL
Imd/IMD
IPP
Jak/JAK
JNK
LPS
LRR
MAMP
MEP
NF-κB
NOS
p70S6K
PAMP
PGN
PGRP
PI3K
PKC
PM
PRR
RNS
ROS
STAT
antimicrobial peptide
antigen presenting cell
Anopheles Plasmodium-responsive LRR 1 protein
cactus
danger-associated molecular pattern
diaminopimelic acid
dorsal-related immune factor
dual oxidase
extracellular signal-regulated kinase
forkhead box O
Gram-negative binding protein
glycosylphosphatidylinositol
(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate
hemozoin
interleukin
immune deficiency
isopentenyl pyrophosphate
janus tyrosine kinase
c-Jun N-terminal kinase
lipopolysaccharide
leucin-rich repeat
microbe-associated molecular pattern
2-C-methyl-D-erythritol 4-phosphate
nuclear factor kappa-light-chain-enhancer of activated B cells
nitric oxide synthase
p70S6 kinase
pathogen-associated molecular pattern
peptidoglycan
peptidoglycan recognition receptor
phosphatidylinositol 3-kinase
protein kinase C
peritrophic matrix
pattern recognition receptor
reactive nitrogen species
reactive oxygen species
signal transducer and activator of transcription
TCR
TCT
Tep/TEP
TGF
TLR
TNF
T cell receptor
tracheal cytotoxin
thioester-containing protein
transforming growth factor
Toll-like receptor
tumor necrosis factor
TABLE OF CONTENTS
INTRODUCTION ................................................................................... 13
Insect immunity .................................................................................. 14
Molecular pattern recognition ............................................................ 15
Pattern molecules ............................................................................ 16
Pattern recognition receptors........................................................... 17
Immune signaling pathways ............................................................... 20
The Toll pathway ............................................................................. 20
The Imd pathway ............................................................................. 23
Other immune-related pathways ...................................................... 25
Malaria ................................................................................................ 26
Parasite development in the mosquito.............................................. 27
Antiplasmodial responses in Anopheles gambiae ............................ 28
Anatomical barriers ......................................................................... 29
Recognition and signal transduction ............................................... 30
Imd and Toll signaling .................................................................. 31
Additional immune signaling pathways ......................................... 32
Effector responses ........................................................................... 33
The TEP1 system........................................................................... 33
Antimicrobial peptides .................................................................. 35
ROS and nitric oxidase.................................................................. 36
Gut bacteria, the third player ........................................................... 37
Elizabethkingia anophelis ............................................................ 39
Plasmodium-derived elicitors........................................................... 40
Isoprenoid precursors ..................................................................... 41
Stimulation of Vγ9Vδ2 T cells ........................................................ 42
Isopentenyl pyrophosphate and HMBPP ....................................... 44
Recognition and signal transduction ............................................. 45
THE PRESENT STUDY ......................................................................... 47
Specific aims of the work .................................................................... 47
Results and discussion ........................................................................ 47
Paper I ............................................................................................. 47
Paper II ........................................................................................... 48
Paper III and IV .............................................................................. 50
CONCLUDING REMARKS .................................................................. 51
ACKNOWLEDGEMENTS .................................................................... 52
REFERENCES ........................................................................................ 53
INTRODUCTION
All organisms possess an immune system to defend themselves against potential pathogens. The immune response must be rapid enough and also able
to discriminate intruders from self to avoid excessive damage to host tissues.
In vertebrates, the immune system is divided into two branches termed innate and adaptive immunity. The former is evolutionarily much more ancient
and comprises the immediate, non-specific response to pathogens through
recognition of certain conserved patterns. The latter is solely found in vertebrates and is signified by the production of specific antibodies against antigenic compounds of the pathogen. Another typical feature of the adaptive
immunity is the creation of a memory that will trigger a faster response upon
subsequent exposures to the same intruder.
The first evidence of an inducible immune response in insects was demonstrated by Boman and coworkers (Boman et al., 1972). Insects lack an adaptive immune system in its classical sense and must rely on innate immune
responses to combat pathogens. The innate immune system is remarkably
well conserved from invertebrates to mammals and the lower complexity of
insects without influence of adaptive responses render them excellent models
for subsequent studies of the human counterpart (Ishii et al., 2008;
Medzhitov, 2007). The first indication of a common evolutionary origin was
based on findings that insects and mammals have similar regulatory elements and factors for immune genes (Engstrom et al., 1993; Sen and
Baltimore, 1986; Sun and Faye, 1992). The similarity was subsequently confirmed through the discovery of the Toll receptor in the fruit fly Drosophila
melanogaster and subsequently the orthologous Toll-like receptor (TLR) in
humans, which warranted the Nobel prize in 2011 (Lemaitre et al., 1996;
Medzhitov et al., 1997; Poltorak et al., 1998).
Following the breakthrough in the late 90’s, the work on Drosophila faced a
huge rise in popularity. The work was boosted by the early publication of the
complete Drosophila genome (Adams et al., 2000).Today it remains the
most well-described model organism for studying insect immunity. The field
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has, however, rapidly expanded in the last 10 years or so to cover a wide
range of insect species, mostly thanks to the greatly reduced costs and increased speeds of the next-generation sequencing technology. A lot of emphasis is put on anopheline and aedine mosquitoes that act as vectors for
many severe diseases including malaria, dengue fever, west nile virus, and
yellow fever among others. Current prevention measures risk becoming diminished by an increased drug resistance of the pathogens as well as a growing insecticide resistance of the vectors. Also, despite an enduring potential,
effective and affordable vaccines are still lacking.
In this thesis, the general features of the insect immune system are first presented, mainly based on findings in Drosophila. The second part revolves
around malaria, with focus on the immune recognition and response of the
vector mosquito Anopheles gambiae and the role of the gut microbiota in the
outcome of the disease.
Insect immunity
The insect innate immune system can be divided into three main classes:
anatomical barriers, humoral immunity and cellular immunity (reviewed in
(Lemaitre and Hoffmann, 2007)). Anatomical barriers refer to the epithelial
linings of the skin, gut, reproductive tract and trachea that are constantly
exposed to large numbers of microbes. The epithelial cells constitute a physical barrier and secrete antimicrobial compounds locally as a first line of
defence against intruders. Some epithelial compartments such as the gut and
trachea have chitinous meshworks as an additional barricade. In the midgut,
the peritrophic matrix (PM) lines the intestinal lumen and is comparable to
mucous secretion in the vertebrate digestive tract (reviewed in (Lehane,
1997; Merzendorfer and Zimoch, 2003)). The matrix is composed of secreted chitin, proteins and proteoglycans and serves as a semi-permeable barrier
against enteric pathogens (Kuraishi et al., 2011).
Humoral responses encompass the secretion of antimicrobial compounds
locally as well as into the hemocoel, the insect body cavity. The latter is
referred to as the systemic immune response and is mainly regulated by the
insect fat body, an organ comparable to the vertebrate liver and adipose tissues and the major production site of antimicrobials. Systemic responses are
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classically thought to become triggered when intruders have managed to
breach the anatomical barriers.
The hemocoel is packed with blood cells (hemocytes), which exert the cellular immune response. The terminology for classifying hemocytes differs
between insect orders and voices for a more uniform nomenclature have
been raised (Castillo et al., 2006). In Drosophila, hemocytes are divided into
three cell types: plasmatocytes, crystal cells and lamellocytes (Lanot et al.,
2001). Most abundant are the plasmatocytes that mediate phagocytosis of
bacteria, yeast and several particles. Crystal cells are responsible for depositing melanin through activation of the prophenoloxidase (PPO) cascade,
which causes dark spots at sites of injury or on invading parasites. This event
is referred to as melanization and is thought to promote pathogen killing
during clot formation and wound healing (reviewed in (Eleftherianos and
Revenis, 2011)). Lamellocytes are clearly the largest cell type and are almost
exclusively present in parasitized larvae where they participate in the encapsulation of wasp eggs that are too large to be phagocytosed (reviewed in
(Lemaitre and Hoffmann, 2007)).
Molecular pattern recognition
The concept of non-self pattern recognition was originally introduced by
Charles Janeway Jr. in 1989. At the time, the adaptive immune system was
at the forefront of immunological research. In order to achieve an adaptive
immune response, however, immunologists had to mix antigens with socalled adjuvants that among other components also contained heat-killed
bacteria. The reason why was not known, and Janeway referred to it as the
“dirty little secret” of the community. According to his model, the adaptive
immune system needed an initial signal delivered by the innate immune system. Furthermore, he suggested that the sensing of pathogens was in fact
carried by innate, germline-encoded pattern recognition receptors (PRRs)
that recognized conserved motifs, termed pathogen-associated molecular
patterns (PAMPs) (Janeway, 1989). The model was later confirmed through
the findings of the Toll (Lemaitre et al., 1996) and TLR receptors (Poltorak
et al., 1998). Nowadays, microbe-associated molecular pattern (MAMP) is
also a commonly used term due to the additional presence in non-pathogenic
microbes. A compound is classified as a MAMP if it fulfills a set of criteria.
First, it must act as ligand for recognition by host pattern recognition recep15
tors (PRRs). Second, the compound has to be strongly conserved and essential for microbial survival.
Compared to the somatically rearranged receptors and antibodies of the
adaptive immune system, PRRs are present in both vertebrates and invertebrates and are evolutionary conserved. Depending on the receptor type, an
activated PRR either acts immediately by triggering phagocytosis or endolytic degradation of the target or triggers a downstream signaling event. In
insects, the receptor-mediated signal activation leads to expression of antimicrobial effectors. The antimicrobial peptides (AMPs) (Steiner et al., 1981)
are the classical example, although several other factors are also involved in
the response.
Pattern molecules
Drosophila senses bacteria through recognition of the peptidoglycan (PGN),
a central cell wall component and a designated MAMP (Fig. 1). Several other bacterial compounds act as MAMPs in humans, including lipopolysaccharides (LPS) (Poltorak et al., 1998), flagellin (Hayashi et al., 2001), lipoteichoic acid (LTA) (Schwandner et al., 1999) and CpG-DNA (Krieg et al.,
1995), but have yet to display clear-cut immunogenic activity in Drosophila.
Recognition of fungi and yeast is mediated by PRR recognition of the cellwall polysaccharide β-(1,3)-glucan, a designated MAMP for the silkmoth
Bombyx mori (Ochiai and Ashida, 2000) and Drosophila (Ligoxygakis et al.,
2002). In addition, certain virulence factors from yeast and bacteria are recognized by PRRs (see below). Bacterial PGN associates with insect PRRs
termed peptidoglycan recognition proteins (PGRPs), a receptor family that is
highly conserved in evolution (see below; reviewed in (Kurata, 2014)).
The general PGN-structure is derived from a disaccharide backbone of Nacetylglucosamine and N-acetylmuramic acid attached to short stem peptides
that are in turn cross-linked. The resulting mesh-like layer aids in maintaining the conformational integrity and offsets the osmotic pressure of the cytoplasm. In Gram-negative bacteria, the PGN stem peptides are directly crosslinked and contain meso-diaminopimelic acid (DAP-PGN) at the third amino
acid position. Most Gram-positive bacteria have L-lysine (Lys-PGN) instead
and additional cross-linking peptides between the stem peptides. Exceptions
do exist; for example Bacillus species produce DAP-PGN. Gram-positive
cells also possess a much thicker PGN-layer with greater structural variation
than Gram-negatives (Schleifer and Kandler, 1972). The latter group have in
16
turn an additional, outer cell wall that coats the PGN. Hence, it has been
debated whether hemocytes need to first phagocytose Gram-negative bacteria in order for PGRPs to reach the PGN. Karlsson et al. demonstrated, however, that during logarithmic growth Escherichia coli constantly shed and
release monomomers of PGN to allow cellular restructuring (Karlsson et al.,
2012). The soluble monomeric form named tracheal cytotoxin (TCT) is a
potent MAMP for certain PGRPs (Kaneko et al., 2004). Cell-cell contact is
therefore not necessary for activation of downstream immune responses in
this context.
Pattern recognition receptors
The arsenal of PRRs for PGN recognition in mammals constitute of TLRs
and NOD-like receptors. Insects on the other hand rely on PGRPs, which
were first isolated by Ashida’s group and subsequently cloned by Kang et al.
who also showed that this family of proteins is conserved from insects to
humans (Kang et al., 1998; Yoshida et al., 1996). The relative contribution
of PGRPs to the insect immune system is reflected in the number of family
members expressed; Drosophila has 13 genes encoding 19 proteins (Werner
et al., 2003; Werner et al., 2000), An. gambiae has seven genes and nine
proteins (Christophides et al., 2002), whereas human and mouse have four
genes each.
In Drosophila, PGRPs are divided into long (PGRP-L) and short (PGRP-S)
forms. Another feature that separates PGRPs is the ability to degrade PGN
through amidase activity (Mellroth et al., 2003). Both PGN and TCT can
diffuse into the hemolymph (Gendrin et al., 2009). Amidase active PGRPs
are therefore scavenging PGN from the host microbiota to prevent chronic
immune activation (Bischoff et al., 2006; Mellroth et al., 2003; Paredes et
al., 2011; Zaidman-Remy et al., 2006). The known modulators include
PGRP-LB, SC1a, SC1b and SC2, which all cleave DAP-PGN (reviewed in
(Kurata, 2014)). In addition, PGRP-SC1/2 recognize and degrade Lys-PGN
(Bischoff et al., 2006; Garver et al., 2006; Mellroth et al., 2003). A few catalytic PGRPs mediate immune responses. Degradation of PGN by PGRPSC1a enhances phagocytosis of the Gram-positive and Lys-PGN-containing
Staphylococcus aureus (Garver et al., 2006). The amidase activity of PGRPSB1 is directly bacteriolytic (Mellroth and Steiner, 2006) although this effect
seem redundant for defence against bacterial infections (Zaidman-Remy et
al., 2011).
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Among the other members of the long form, PGRP-LC and PGRP-LE recognize DAP-PGN (see below) (Kaneko et al., 2004; Leulier et al., 2003;
Stenbak et al., 2004; Werner et al., 2003) and are receptors for the Imd
pathway (Choe et al., 2002; Gottar et al., 2002; Ramet et al., 2002; Takehana
et al., 2002) and the melanization cascade (Schmidt et al., 2008; Takehana et
al., 2002). PGRP-LA and PGRP-LD both contain transmembrane regions
(Werner et al., 2000) and the former has been proposed to promote AMP
expression in the trachea (Gendrin et al., 2013). The sixth member of the
long form, PGRP-LF, cannot bind PGN and instead acts as a negative regulator of Imd signaling through its interaction with the PGRP-LCx-isoform to
prevent dimerization (Basbous et al., 2011; Persson et al., 2007).
Besides PGRP-LCx, two other isoforms, PGRP-LCa and PGRP-LCy, exist
that differ in their extracellular PGRP domains and hence in their PGN binding specificities. PGRP-LCx has a deep PGN-binding cleft, is able to bind
both mono- and polymeric PGN and likely forms clusters (Kleino and
Silverman, 2014). The cleft is however occluded in PGRP-LCa, which instead interacts with both TCT and PGRP-LCx and acts as an adaptor for the
latter through heterodimerization (Chang et al., 2006; Chang et al., 2005;
Kaneko et al., 2005; Mellroth et al., 2005). The expression of PGRP-LCy is
much lower than for the other two isoforms and the receptor has been proposed to have a minor antagonistic function (Neyen et al., 2012). PGRP-LE
lacks a transmembrane domain and is instead released both intra- and extracellularly where it exclusively recognizes DAP-PGN and either mediates
Imd pathway activation, PPO-induced melanization (Takehana et al., 2002),
or Imd-independent autophagy (Yano et al., 2008). It has also been shown
that PGRP-LE/LC act synergistically and might form heterodimers
(Takehana et al., 2004).
The remaining short PGRPs, PGRP-SD and PGRP-SA, are secreted into the
hemolymph and mediate the activation of the Toll and PPO pathways (Fig.
1, 2; see next section) (Bischoff et al., 2004; Michel et al., 2001). The Toll
pathway is also activated by fungal β-(1,3)-glucans (Ligoxygakis et al.,
2002; Ochiai and Ashida, 2000) as well as subtilisin-like virulence factors
from yeast (Gottar et al., 2006) and bacteria (El Chamy et al., 2008), which
are recognized by the PRRs GNBP3 and Psh, respectively. Bacterial virulence factors have also been linked to manipulations of Imd-pathway activity. The E. coli-derived cytotoxic necrotizing family 1 (CNF1) modifies the
18
RhoGTPase, Rac2, that in turn initiates immune responses by interacting
with Imd and Rip-kinases (Boyer et al., 2011).
Figure 1. Schematic overview of microbial recognition and subsequent activation of
downstream responses in Drosophila.
19
While several receptors in insect humoral immunity have been identified,
less is known regarding the mechanisms of recognition that precede cellular
responses. The Drosophila phagocytic receptor Eater is exclusively expressed on the cell surface of plasmatocytes and binds directly to Grampositive, but not Gram-negative bacteria (Chung and Kocks, 2011). Membrane disrupting treatments of the bacteria by the insect AMP Cecropin A,
however, results in binding of Eater to the latter Gram-type as well, indicating a collaborative response of humoral and cellular immunity. The putative
MAMP for Eater is not known, although extracellular PGRP-SC1a has been
proposed to act as an opsonin (Kurata, 2014). Another phagocytic receptor,
Draper, has however been found to interact with LTA of S. aureus, indicating a true PAMP-PRR relationship (Hashimoto et al., 2009). The production
of cell wall teichoic acids (WTAs) by the same bacterium, on the other hand,
masks its PGN layer from recognition by PGRP-SA and SD, which indicates
an evolutionary arms race of hide and seek (Atilano et al., 2011).
Immune signaling pathways
The production and secretion of antimicrobial peptides (AMPs) in response
to microbial challenge is the classic hallmark of humoral immune response
in insects. Expression is typically induced by the Toll pathway, the immunodeficiency (Imd) pathway or a combination of both. The different PRRs of
the two pathways allow discrimination between PGN-types; Lys-PGN is
thought to mainly activate the Toll-pathway, whereas DAP-PGN is preferentially recognized by Imd pathway receptors (Fig. 2).
The Toll pathway
The Toll pathway is required for dorsoventral patterning during embryonic
development as well as in the immune defence against Gram-positive bacteria and fungi in Drosophila. The Drosophila genome encodes nine Toll
genes including the Toll pathway receptor Toll. In contrast to human TLRs,
Toll is in insects a cytokine receptor rather than PRR. Pattern recognition is
instead mediated by the above mentioned extracellular receptors, PGRP-SA,
PGRP-SD, the Gram-negative binding proteins GNBP1 and GNBP3, and
Persephone (Psh). In addition to maturation of the Toll ligand Spätzle (Spz)
to its active form, the Toll pathway is also dependent on endocytosis for
activation (Huang et al., 2010).
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Figure 2. The Toll (left panel) and Imd (right panel) signaling pathways in Drosophila. Abbrevations not mentioned in the main text include ESCRT-0, endosomal
sorting complexes required for transport; TAB2, TAK1-binding protein; dnr1, defense repressor 1; U, ubiquitination; P, phosporylation.
Upon recognition of Lys-PGN, PGRP-SA forms a complex with GNBP1 (a
misnomer) that in turn hydrolyzes the PGN to present new glycan reducing
ends to PGRP-SA (Gobert et al., 2003; Michel et al., 2001; Wang et al.,
2006). Although PGRP-SA can bind monomeric Lys-PGN, at least a dimeric
21
muropeptide is required for Toll pathway activation (Filipe et al., 2005). A
plausible explanation is that PGRP-SA requires clustering through the binding of several receptors to the same Lys-PGN-fragment for activation of
downstream pathway in insects (Park et al., 2007). PGRP-SD also participates in the sensing of Gram-positive bacteria, although it has been proposed
to confer a partially redundant role to the PGRP-SA-GNBP1 complex
(Bischoff et al., 2004). Later, it was found that PGRP-SD participates in
increasing the interaction of GNBP1 to both PGRP-SA and Lys-PGN (Wang
et al., 2008). In addition, the crystal structure of PGRP-SD suggests that it
preferentially binds to DAP-PGN and thus represents a route for activation
of the Toll pathway by Gram-negative bacteria (Leone et al., 2008).
The activation of PRRs recruits a modular serine protease and induces a
proteolytic cascade that leads to cleavage and activation of Spz by the protease Spz processing enzyme (SPE) (Jang et al., 2006). An alternative route is
present where activated Psh becomes proteolytically matured to directly
activate SPE (El Chamy et al., 2008; Gottar et al., 2006). The processed and
mature Spz binds to the Toll receptor that in turn dimerizes. The intracellular
domains of Toll will then recruit the adaptor protein MyD88, Tube, and the
kinase Pelle which will form a trimeric complex (Sun et al., 2002). Upon
formation, the complex activates Dorsal or Dorsal related immune factor
(Dif). Both factors belong to the family of the nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB). The exact mechanism for
Dif/Dorsal activation is not resolved but includes phosphorylation and subsequent degradation of Cactus (Cact), an ortholog to inhibitor of kappa B
(IκB) in humans (Wu and Anderson, 1998), likely mediated by Pelle (Huang
et al., 2010; Towb et al., 2001). The active forms of Dif/Dorsal are then able
enter the nucleus and initiate transcription of AMP genes.
Dorsal was originally linked to dorsoventral patterning but is expressed in
the fat body of larvae and adults where it is induced (Reichhart et al., 1993)
and participates in the response to microbial challenge (Lau et al., 2003). In
contrast, Dif is only involved in mediating transcription of immune effectors
(Ip et al., 1993). In larvae, there seem to be a redundancy between the two
factors (Manfruelli et al., 1999; Rutschmann et al., 2000), whereas only Dif
induces the antifungal AMP Drosomycin (Drs) in adults (Lemaitre et al.,
1996; Rutschmann et al., 2000). In the commonly used macrophage-like S2
cell line, however, Dorsal has been suggested as the most important transcription factor for Drs expression (Valanne et al., 2010).
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The Imd pathway
The first indication of a second immune pathway in Drosophila was based
on the repeated failure to link Dif to the expression of the AMP gene Diptericin (Dpt) (Ip and Levine, 1994; Lemaitre et al., 1995b). Around the same
time, the Immune deficiency (Imd) gene was discovered in which a mutation
strongly reduced the expression of antimicrobial peptides (Corbo and
Levine, 1996; Lemaitre et al., 1995a). The different components of the Imd
pathway were subsequently step-wise discovered, initially through the employment of forward genetic screening of mutant strains with impaired induction of Dpt, and later using RNA interference (RNAi) and yeast twohybrid screens (reviewed in (Imler, 2014)). Although not known at the time,
the NF-κB of the Imd pathway, Relish, had already been discovered (Dushay
et al., 1996) and was later shown to be crucial for AMP induction and bacterial resistance (Hedengren et al., 1999). Analogous to human NF-κBs, p100
and p105, Relish both contain a Rel domain and inhibitory ankyrin repeats
and thus differ from Dif/Dorsal, which are dependent on Cact for inhibiton.
The Imd pathway has been likened to human tumor necrosis factor alpha
(TNFα) signaling (Kaneko and Silverman, 2005; Lemaitre and Hoffmann,
2007). In contrast to the Toll pathway, the Imd pathway is only known to
participate in immunity and seems to be the only immune cascade involved
in local induction of AMP expression (Ferrandon et al., 1998; Tzou et al.,
2000). Another striking difference is the much faster onset of the Imd pathway with nuclear translocation of Relish occurring within minutes (Paquette
et al., 2010) and peak expression of AMPs within a few hours after bacterial
challenge (Lemaitre et al., 1997). The Toll-mediated response in comparison
can take up to hours for activation and the expression of AMPs can instead
be sustained for days (Lemaitre et al., 1997). It has hence been speculated
that the Imd pathway is involved in acute responses and to more quickly
growing pathogens than the Toll pathway (Lemaitre and Hoffmann, 2007).
Pathway activation is achieved through sensing of Gram-negative and Grampositive bacilli (Lemaitre et al., 1997; Stenbak et al., 2004) through recognition of DAP-PGN by the membrane bound PGRP-LCx and PGRP-LCa as
well as intra- or extracellularly through PGRP-LE. Receptor activation probably causes dimerization or multimerization (Mellroth et al., 2005) and leads
to recruitment of Imd (Choe et al., 2005). Imd in turn acts as an adaptor protein and recruits dFADD (Leulier et al., 2002) and the apical caspase Dredd
(Leulier et al., 2000). Upon complex formation, Dredd is proteolytically
23
activated through ubiquitination (Meinander et al., 2012) and cleaves Imd,
which in turn also gets ubiquitinated (Paquette et al., 2010). These events are
thought to mediate recruitment of the mitogen activated protein kinase
(MAPK) TAK1, which in turn phosphorylates and activates the IκB kinase
(IKK) as well as the c-Jun N-terminal kinase (JNK) branch of the pathway
(reviewed in (Kleino and Silverman, 2014)).
The exact mechanism behind Relish activation is not entirely resolved, but
involves phosphorylation by IKK (Silverman et al., 2000) and proteolytic
removal of the ankyrin repeats, probably by Dredd (Meinander et al., 2012;
Stoven et al., 2000; Stoven et al., 2003). The N-terminal fragment of Relish
subsequently translocates into the nucleus to promote transcription of AMP
genes, typically Dpt, Attacin (Att) and Cecropin (Cec) (Lemaitre et al., 1997;
Stoven et al., 2000) through binding to specific upstream κB-sequences
(Busse et al., 2007; Sun et al., 1991a; Sun et al., 1991b). However, the control regions of many cecropin as well as attacin genes often contain various
different promoter sites. It is therefore not surprising that the expression of
these genes has often been reported as somewhat promiscuous (HedengrenOlcott et al., 2004)). Later, it was shown that AttA, among other immune
genes, is regulated equally by both pathways through upstream κB-regions
for both Dif and Relish (Busse et al., 2007). Moreover, Dif and Relish are
able to form heterodimers to induce transcription of genes that were originally thought to be more strictly regulated by either pathway (Tanji et al.,
2010).
The Imd pathway needs constant regulation to avoid chronic immune activation. Extracellular regulation is mediated by the above mentioned scavenging PGRPs and inhibitory PGRP-LF. Interestingly, the amidase-active
PGRPs seem to only regulate the Imd pathway, despite the obvious recognition of Lys-PGN by some of them (Bischoff et al., 2006). A plausible explanation is that the Imd, but not the Toll pathway, is involved in local responses and thus needs additional modes of fine-tuned regulation to maintain tolerance to the normal flora. In light of this, it is surprising that a member of
the Toll receptor family, Toll-8/Tollo, has been found to constitute a receptor
for a signal cascade that antagonizes Imd-pathway activation in the trachea
of Drosophila (Akhouayri et al., 2011). Additional regulation of the pathway
occurs at various intracellular levels. Constitutive activation is inhibited by
Caspar, which prevents processing of Relish by Dredd (Kim et al., 2006).
Upon pathway activation, the regulator Pirk is induced before AMPs and
24
participates in a negative feedback loop, likely by interfering with the ImdPGRP-LC complex (Aggarwal et al., 2008; Kleino et al., 2008). In the gut of
Drosophila, PGRP-LE has been proposed to mediate negative feedback inhibition of the pathway through the constitutive induction of Pirk and PGRPLB (Bosco-Drayon et al., 2012). Additional inhibitors of Dredd as well as
several proteins involved in ubiquitin-mediated proteasome degradation have
been linked to the regulation of the pathway (reviewed in (Kleino and
Silverman, 2014)).
Other immune-related pathways
A few other signaling pathways have been implicated in insect immune responses. The underlying microbial triggers and effector responses are, however, not always entirely understood. The janus tyrosine kinase/signal transducers and activators of transcription (Jak/STAT) pathway has been proposed as functionally analogous to the interferon system in mammals
(Kingsolver et al., 2013). The signaling pathway is involved in various cellular processes and mediates both cellular and humoral responses to viruses,
parasites and some bacteria. The three major proteins of the pathway are the
receptor Domeless, Jak, and the transcription factor Stat92E. Nuclear translocation of the latter has been linked to viral and bacterial infections in mosquitoes (Barillas-Mury et al., 1999; Souza-Neto et al., 2009) and Drosophila
(Dostert et al., 2005). In Drosophila, the Jak/STAT pathway regulates several immune genes such as the antibacterial peptide gene Listericin (in corporation with PGRP-LE) (Goto et al., 2010) as well as Turandot and the complement like factor Thioester-containing protein 2 (Tep2), of which the exact
immune functions remain to be established (Agaisse et al., 2003; Boutros et
al., 2002; Lagueux et al., 2000). The pathway also plays a central role during
wasp parasitization as it regulates the differentiation of prohemocytes into
lamellocytes (Makki et al., 2010).
The Akt/PI3K pathway is involved in various cellular processes including
metabolism, proliferation, longevity and immunity. The pathway is in insects
triggered by insulin like peptides (ILPs), human insulin and likely other
growth factors, and contains at least two key transcription factors, p70S6
kinase (p70S6K) and forkhead box O (FOXO). In absence of stimuli, the
transcription factor FOXO remains active in the nucleus to transcribe target
genes. FOXO maintains low level expression of AMPs during starvation
(Becker et al., 2010) and also regulates autophagy and muscle aging (Bai et
al., 2013; Demontis and Perrimon, 2010). Upon pathway activation, Akt
25
phosphorylates FOXO, which in turns translocates to the cytoplasm were it
is degraded. Instead, p70S6K becomes active to promote protein translation.
The three major MAPK pathways, extracellular-signal-regulated kinase
(Erk), p38 and JNK are involved in various cellular processes. They have
also been associated with immune responses and are in this sense reviewed
in the malaria section.
Malaria
Malaria is an ancient disease that continues to plague a great part of the
world population. The word malaria stems from the expression for “bad air”
in medieval Italian and refers to the miasma theory coined by Hippocrates
that certain diseases were spread through contaminated air rather than by
human contact. At the end of the 19th century, the mystery behind the disease
was gradually unravelled. The first breakthrough was the sighting of the
disease-causing parasite in 1880 by Alphonse Laveran, who also hypothesized that the disease was spread by mosquitoes. At the same time, the protozoan genus Plasmodium was described by Angelo Celli and Ettore Marchiafava and was later found to be the causative agent for all malaria. Subsequently, Sir Ronald Ross demonstrated the role of mosquitoes as vectors for
the parasite and also deciphered the mosquito life cycle of avian malaria in
1897 for which he received the Nobel Prize in 1902. Around the same time,
the Italian physician and zoologist, Giovanni Battista Grassi, described the
human malaria parasite, P. falciparum, and demonstrated that transmission
to humans is mediated by mosquitoes of the genus Anopheles (reviewed in
(Desowitz, 1999).
According to the World Health Organization (WHO), the number of malaria
episodes world-wide were in 2012 estimated to be 207 million, resulting in
approximately 627 000 deaths (WHO malaria report, 2013). About 80 % of
the cases occurred in sub-Saharan Africa, where also 90 % of death cases
were reported. Malaria is especially dangerous for children under the age of
five as 77 % of the global deaths occurred in this age group. These numbers,
while still huge, represent a decline in the last 10-year period and in part
correlates with increased efforts to control malaria. Insecticide-treated mosquito bed nets and indoor residual spraying with insecticides have together
with new artemisinin-based combination therapies proven successful. It is,
26
however, difficult to interpret to what extent these measures correlate with
the roll-back of malaria as environmental factors have to be taken into account. Growing resistance to current insectides and antimalarial drugs has
also been reported in several countries and is a rapidly growing concern. In
addition, WHO have faced critique for grossly underestimating the mortality
numbers (Murray et al., 2012). Alternative methods aimed at controlling,
eliminating and ultimately eradicating malaria are thus urgently needed.
The malaria situation has generated an increasing interest in transmission
blocking and vector control strategies. During the last 15 years or so, great
efforts have been put into deciphering the genetic and biochemical backgrounds of the complex interactions between the mosquito and the malaria
parasite. Novel genetic tools have also led to the development of transgenic
strategies for either controlling the vector population or inhibiting disease
transmission (reviewed in (David et al., 2013)). The former typically aims at
blocking the reproductive capacity, for example by mass release of sterile
males or females. On the downside with this approach, the possible sideeffects on the ecosystem are hard to foresee and could also likely result in
rapid species reestablishment or replacement. In the latter approach, the ecological impact might be less pronounced since the mosquito population
would be stably maintained. Mosquitoes are instead genetically engineered
to either express antiparasitic factors or boost the endogenous immune response. Paratransgenesis is another interesting, albeit controversial, strategy
that has received growing attention lately. The aim is to block disease transmission by the introduction of a symbiotic microbe that has been transformed to specifically express antiparasitic factors. The aforementioned
strategies aim at ultimately introducing genetically modified organism in
nature, which is not without risks. The introduced trait can potentially confer
an unwanted selective advantage to the modified organism and inserted
genes can be horizontally transferred to other species. It is hence important
to aim at thoroughly addressing such issues in the laboratory or semi-field
facilities before proceeding further. It is also likely that a combination of
several approaches will have to be applied simultaneously to control and
ultimately eradicate vector-borne diseases.
Parasite development in the mosquito
The malarial life cycle within the mosquito is highly complex and involves
several developmental and replicative stages, which last for about 3 weeks
(reviewed in (Aly et al., 2009)). Upon ingestion of blood from a contagious
27
host by the female mosquito, gametocyte stages of Plasmodium will end up
in the midgut, transform into gametes and subsequently mate (Fig. 3). This is
the only transitional stage where the parasite undergoes sexual reproduction.
Mosquitoes are therefore in biological terms regarded as the “primary”
Plasmodium host whereas humans and other mammalian hosts are seen as
“secondary” (reviewed in (Marois, 2011)).
The resulting zygote differentiates into a highly motile ookinete that invades
and traverses the gut epithelium, resulting in substantial structural and osmotic epithelial damage. Upon reaching the basal surface of the epithelium,
the ookinete ceases movement and transforms into a spherical oocyst (“egg
sac” in Greek). Immediately after formation, the oocyst will commence multiple rounds of mitotic divisions. This process is known as sporogony and
results in massive amplification of asexual, highly motile parasites referred
to as sporozoites. Consequently, the enormous increase in ookinete size will
cause membrane rupture and release of sporozoites into the mosquito hemocoel. From here, sporozoites will migrate to and invade the salivary glands
in order to get injected along with the saliva into a new host.
Antiplasmodial immunity in Anopheles gambiae
Malaria infection often causes severe illness in humans. Several of the clinical symptoms are due to hyperactivation of host immune responses by the
parasite. Mosquitoes on the other hand are much more tolerant to the infection. A plausible explanation is that the much shorter generation time of
mosquitoes have adapted them to better cope with the parasite throughout
the evolution. Nevertheless, Plasmodium infection induces a strong immune
response and affects mosquito survival, reproduction and fitness (Ahmed
and Hurd, 2006; Ahmed et al., 2001; Anderson et al., 2000; Hogg and Hurd,
1997). In the Anopheles genus, only around 40 out of more than 400 mosquito species permit the development of Plasmodium parasites (Service, 1993).
Even within laboratory-reared strains selected for susceptibility, the individual vector capacity often varies dramatically. To understand why and how
mosquitoes differ in their susceptibility, a review of the Anopheles immune
responses against the parasite is presented.
28
Figure 3. Schematic review of the developmental stages of Plasmodium within the
mosquito (left panel) and the antiplasmodial responses mounted by the mosquito
(right panel). DTN, dityrosine network; PM, peritrophic membrane; BL, basal lamina.
Anatomical barriers
The mosquito gut harbours natural bacteria that proliferate up to 1000-fold
after a blood meal, presumably due to the increase in nutrients (Pumpuni et
al., 1996). Inducing immune responses to control such a vast amount would
divert a lot of resources from egg development. It is therefore of vital importance to restrict the contact with the microbiota. A common response to
blood ingestion in hematophagous insects is to secrete a PM from the midgut
epithelium (Fig. 3). In Anopheles, the PM reaches maximum thickness approximately 24 h after blood ingestion, roughly overlapping with the gut
29
bacterial growth peak. The time point also coincides with the ookinete midgut invasion, which can be explained by parasite expression and secretion of
chitinases that locally dissolve the PM (Langer and Vinetz, 2001; Moreira et
al., 2004). Vaccines designed to target ookinete-secreted proteins could thus
be used in transmission-blocking strategies and still represent an interesting,
albeit challenging approach (reviewed in (Lavazec and Bourgouin, 2008)).
An additional barrier composed of dityrosine crosslinks is formed by oxidase-peroxidase enzymes including dual oxidase (DUOX) upon blood feeding (Kumar et al., 2010). The resulting network prevents microbial elicitors
from reaching the gut epithelium and thus aids the mosquito from chronic
immune activation. On the other hand, the barrier also shields developing
parasites and could therefore be regarded as a double-edged sword for the
mosquito.
Invaded midgut cells are either lysed directly or undergo programmed cell
death and are extruded from the epithelium. It is doubtful whether cell extrusion is an active antiplasmodial response rather than a general tissue repair
mechanism. For invading ookinetes, however, it is of crucial importance to
escape into the basal lamina before this event (reviewed in (Baton and
Ranford-Cartwright, 2005)).
Recognition and signal transduction
The immune signaling pathways are strongly conserved between An. gambiae and D. melanogaster. At recognition and effector phases, however, genes
are clearly more divergent and species-specific suggesting that each species
has adapted fine-tuned immune responses against their natural pathogens
(Waterhouse et al., 2007). The Toll, IMD and JAK-STAT pathways have all
been identified in An. gambiae through orthology to Drosophila
(Christophides et al., 2002; Waterhouse et al., 2007). Interestingly, there
seems to exist a pathway discrepancy in the response to different Plasmodium-species; the IMD response is crucial for responses to P. falciparum
whereas the Toll pathway plays a key role in responses to P. berghei (Dong
et al., 2006; Garver et al., 2009; Mitri et al., 2009). The latter is a rodent
malaria species and does not infect An. gambiae in nature due to the mosquito’s preference for human blood. Nevertheless, P. berghei has regularly been
employed as a model species as it normally gives rise to much higher oocyst
numbers than P. falciparum. A potential problem with employing nonnatural vector-parasite combinations in basic research is that immune re30
sponses rarely involved in natural infections are over-emphasized. Indeed,
infection with either Plasmodium species induces quite different transcriptome responses in the mosquito (Dong et al., 2006).
IMD and Toll signaling
Similar to Drosophila, the An. gambiae IMD and Toll pathways are thought
to be activated through PGRP recognition of PGN. Interestingly, AgPGRPLC (PGRPLC according to the HUGO nomenclature, which is slightly modified from that of Drosophila (Christophides et al., 2002)) can potentially
bind both DAP- and Lys-PGN (Meister et al., 2009). Challenge with bacteria
of either Gram types also activates the IMD pathway, which is required for
mosquito survival (Meister et al., 2009; Meister et al., 2005). These findings
suggest that IMD signaling is a general response to bacteria in An. gambiae.
The pathway also has a profound role in mounting the antiplasmodial response and mainly affects the ookinete stage (Garver et al., 2012). The expression of several antiplasmodial effectors including leucin-rich repeat domain protein 7 (LRRD7), fibronectin 9 (FBN9), members of the TEP1 system and AMPs is regulated by the pathway (see next section) (Blandin et al.,
2004; Dong et al., 2006; Garver et al., 2012; Garver et al., 2009; Meister et
al., 2005; Mitri et al., 2009; Povelones et al., 2009).
Similar to Drosophila, the signal cascade results in the activation of the Relish ortholog, REL2F, through cleavage of its inhibitory ankyrin repeats. In
addition, an alternative splice form denoted REL2S exists, which is not
found in Drosophila, lacks the inhibitory ankyrin repeats and is constitutively active, probably to maintain basal immune expression (Meister et al.,
2005). In the same study, knock down experiments indicated that REL2F
mediates the response to P. berghei. The same splice form was later also
linked to protection against P. falciparum (but not to P. berghei in this
study) as knock down of Caspar, which only inhibits the ankyrin-containing
REL2F, completely aborted parasite development (Garver et al., 2009). In
contrast to these findings, however, another study linked the protection
against P. falciparum to REL2S and not RELF (Mitri et al., 2009). REL2
also has a vital role in the antiplasmodial response in An. stephensi as mosquitoes genetically engineered to overexpress the transcription factor in either midgut or fat body tissues in response to blood ingestion were more
resistant to P. falciparum (Dong et al., 2011).
31
Much less is known about the Toll signaling events in An. gambiae. REL1 is
analogous to Dorsal, but the mosquito surprisingly has no Dif counterpart.
Also, the underlying mechanism for Toll pathway activation in An. gambiae
is not known. AgPGRPS is orthologous to DmPGRP-SA but the mosquito
lacks orthologs for enzymes involved in the proteolytic degradation of AgSPZ (Waterhouse et al., 2007). Toll signaling has nevertheless been associated with responses to Gram-positive bacteria, fungi and Plasmodium. Depletion of the REL1 inhibitor, CACT, blocks P. berghei infection and reduces P. falciparum oocyst numbers (Garver et al., 2009). The Toll-mediated
defence against P. berghei seems dependent on the TEP1 system. Knock
down of the central component Anopheles Plasmodium-responsive LRR 1C
protein (APL1C; see below) abolishes the protection (Riehle et al., 2008).
Also, knockdown of CACT increases basal TEP1-levels and blocks parasite
infection (Frolet et al., 2006).
Additional immune signaling pathways
The An. gambiae JAK/STAT-pathway has a unique antiplasmodial function
as it affects developing oocysts (Gupta et al., 2009). The ancestral STAT
gene (STATA) regulates expression of nitric oxide synthase (NOS) which is
necessary for the antiplasmodial response (see below). A protective role of
the pathway has also been shown against P. vivax in An. aquasalis (Bahia et
al., 2011). Interestingly, Gupta et al. showed that STATA levels are regulated by a second, intronless STAT (STATB) that probably originated from
gene duplication.
The phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK pathways are
beside their involvement in several physiological processes also regulating
responses to Plasmodium infection. Increased PI3K/Akt signaling in the
midgut can almost completely block parasite transmission in An. stephensi
(Corby-Harris et al., 2010). The three classical MAPKs, ERK, p38 and JNK
are often associated with responses to various stress stimuli. All three
MAPKs together with NF-κB are activated via Toll-like receptors and mediate inflammatory responses in human macrophages stimulated with Plasmodium-derived signals (see below) (Lu et al., 2006; Zhu et al., 2005). In the
mosquito, ERK has been implicated in NOS regulation and influences parasite development in the midgut in response to the human growth hormone
transforming growth factor β1 (TGF- β1) (Luckhart et al., 2003;
Surachetpong et al., 2009). The JNK pathway represents a second branch of
the Imd pathway in Drosophila and could hence be regarded as a part of the
32
immune response. The first indication of an antiplasmodial role for this
pathway in An. gambiae was based on knock-down experiments on JNK that
generated more P. berghei oocysts (Jaramillo-Gutierrez et al., 2010). The
pathway was additionally found to indirectly regulate antioxidants and was
crucial for preventing chronic oxidative stress. Later, the same group found
that the refractory laboratory L3-5 strain of An. gambiae was dependent on
JNK signaling for the protection and linked the pathway to epithelial nitration, TEP1 and FBN9 expression and subsequent melanization of the parasite (Garver et al., 2013). Epithelial wounding in Drosophila immediately
activates JNK signaling, which induces the expression of leptin-like cytokines of the Unpaired (Upd) family. Secreted Upds interact with the Domeless receptor to activate the Jak/STAT pathway. As a result, hemocytes are
recruited to the wound site and melanization is commenced (reviewed in
(Wang et al., 2014)). It is likely that the wound response in An. gambiae is
carried in a similar manner and that the separately studied JNK and
JAK/STAT pathways in the mosquito in fact cooperatively mediate the response to epithelial damage caused by the parasite. It also remains to be determined whether JNK signaling has a profound role in the defence against
P. falciparum in nature.
Effector responses
The TEP1 system
The melanization response was early on thought to play a key role in parasite
killing as the L3-5 strain, originally selected for resistance against the primate malaria parasite P. cynomolgi, was found to melanize several Plasmodium species (Collins et al., 1986). Several quantitative trait loci (QTL) were
subsequently linked to the melanotic refractoriness against P. cynomolgi
(Zheng et al., 1997; Zheng et al., 2003). Field caught mosquitoes and susceptible laboratory strain do however seldom melanize parasites. Yet, susceptible mosquitoes kill and subsequently lyse 80 % of parasites (Blandin et al.,
2004). Melanization is also not required for bacterial clearance (Schnitger et
al., 2007). It was therefore postulated that parasite killing in L3-5 was mediated through other mechanisms and that melanization was rather a way for
the mosquito to scavenge already killed parasites.
The first QTLs for resistance against P. falciparum in wild-caught An. gambiae (Niare et al., 2002) was later mapped to a single Plasmodium-resistance
island (PRI) (Menge et al., 2006; Riehle et al., 2007; Riehle et al., 2006). In
33
parallel, the development of RNAi-based gene silencing (Blandin et al.,
2002) was pivotal for the subsequent identification of novel antiplasmodial
genes. The technique led to the finding of a complement-like cascade that
initially was found to control P. berghei loads (Blandin et al., 2004). The
system contains three key proteins: TEP1 that is structurally similar to the
vertebrate complement C3 protein (Blandin et al., 2004; Levashina et al.,
2001) and two LRR proteins, LRIM1 and APL1C (Dong et al., 2006; Osta et
al., 2004; Riehle et al., 2006; Riehle et al., 2008). The TEP-system might
signify a unique adaptation to environmental conditions in the mosquito. In
contrast to the six TEP-genes found in Drosophila, the An. gambiae genome
encodes 15 genes and only one ortholog exist between the two species
(Christophides et al., 2002). Also, the TEPs in Drosophila have yet to be
assigned a role in the immune response (Bou Aoun et al., 2011).
The cascade in An. gambiae is initiated when TEP1 is released into the hemocoel. Here it will be chaperoned by a heterodimer of LRIM1 and APL1C
to promote its microbial specificity (Fraiture et al., 2009; Povelones et al.,
2009). The complex is then recruited by a non-catalytic serine protease,
SPCLIP1, to the surface of microbes and activates a convertase cascade that
results in lysis, phagocytosis or melanization (Baxter et al., 2010; Blandin et
al., 2004; Povelones et al., 2013). Blandin et al. showed that knock down of
TEP1 results in a five-fold P. berghei oocyst increase in L3-5 mosquitoes,
which indicates that TEP1 is a prominent marker for resistance in this strain.
In line with this finding, the L3-5 strain has allelic TEP1 differences compared to the unselected G3 strain and the degree of susceptibility largely
depends on the TEP1 allele (Blandin et al., 2009). TEP1 was originally
found to promote phagocytosis of bacteria in cell lines (Levashina et al.,
2001) but are together with LRIM1 also required for melanization of Sephadex beads (Warr et al., 2006). It is hence possible that the melanization
phenotype often observed in non-natural vector-parasite combinations are
due to host recognition of parasite components as xenobiotics. The TEP1
system also regulates P. falciparum infection (Dong et al., 2006; Garver et
al., 2009; Warr et al., 2008) although no, or at best subtle, effects have been
observed in field-caught mosquitoes (Cohuet et al., 2006; Marois, 2011).
The trimeric complex seems more flexible than initially thought, however,
and might include alternative components not targeted in these studies. It has
for example been shown that that three additional TEPs can interact with
LRIM/APL1C (Povelones et al., 2011), and TEP1 is a highly polymorphic
chimera of at least two other TEP loci (Obbard et al., 2008). Moreover, the
34
APL1 loci also display remarkable diversity with preserved polymorphisms
in the wild (Rottschaefer et al., 2011) and APL1A rather than APL1C is
mediating the response to P. falciparum (Mitri et al., 2009).
It is plausible that the TEP-system has evolved to target various natural
pathogens through the flexible formation of specific complexes. Whether or
not the relatively mild fitness cost of P. falciparum infection can cause a
selective pressure on a specific TEP-complex in nature remains to be clarified. Also, the TEP-specific microbial structures need to be resolved in order
to classify them as true PRRs.
Antimicrobial peptides
Similar to Drosophila, AMPs are important effectors of the humoral immune
response of Anopheles. There is, however, no clear-cut evidence that AMPs
comprise a main weapon in the An. gambiae immune arsenal against the
malaria parasite. Four AMP families have been found in the An. gambiae
genome: four defensins (DEFs), four cecropins (CECs), one gambicin
(GAM1) and one attacin (ATT) (Christophides et al., 2002). Of note, Anopheles AMPs seem to be under regulation of both REL1 and REL2 indicating
that IMD and Toll signaling are more redundant than in Drosophila (Garver
et al., 2009; Luna et al., 2006; Meister et al., 2005). Initial experiments with
purified AMPs from the mosquito revealed that CEC1 (Vizioli et al., 2000)
and GAM1 (Vizioli et al., 2001a) are active against bacteria of both Gram
types whereas DEF1 is mainly active against Gram-positive bacteria and
some fungal species (Vizioli et al., 2001b). A modest activity of GAM1
against P. berghei has been shown (Vizioli et al., 2001a) and knock down of
the gene increases ookinete numbers of this species but has no effect on that
of P. falciparum (Dong et al., 2006). Interestingly, injection of AMPs from
other species reduces parasite loads but only if given prior to, or directly
after the infectious blood meal (Gwadz et al., 1989; Lowenberger et al.,
1999; Shahabuddin et al., 1998). This indicates that AMPs are only effective
in a narrow time-window and that timing is a critical factor. The mosquito
might not produce AMPs in time to inhibit oocyst formation, possibly as a
consequence of the dityrosine mesh formed after blood ingestion.
The antiplasmodial activity of AMPs has also been demonstrated using other
mosquito-Plasmodium combinations. In Aedes aegypti, co-overexpression of
CecA and DefA dramatically reduces P. gallinaceum oocyst formation and
blocks sporozoites from reaching the salivary glands (Kokoza et al., 2010).
35
Several AMPs of various origins were lately used in a screen for new candidate peptides against P. berghei and P. falciparum in An. stephensi and An.
gambiae, respectively, and were despite promising antiplasmodial effects
against cultured parasites not active to the same extent in vivo (Carter et al.,
2013).
ROS and nitric oxide
The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) have an important role in local, epithelial immunity, but must at
the same time be tightly regulated to avoid excessive host tissue damage.
The involvement of ROS/RNS in Anopheles immune responses has received
attention recently, albeit to a slightly less extent in the last few years. This
might be explained by the difficulty in studying such highly reactive compounds and the large number of cellular processes that they affect. The oxidative status of Anopheles mosquitoes has been associated with Plasmodium
susceptibility (Kumar et al., 2003).The ingestion of heme in the blood meal
is generally thought to generate ROS and the parasite traversal of the midgut
epithelium aggravates the oxidative state. The resistant L3-5 strain have
higher systemic H2O2 levels prior to and after blood ingestion compared to
4A r/r, which has been selected for high susceptibility. Moreover, the refractory phenotype is reversed by supplementation of antioxidants in the blood
meal suggesting that ROS are involved in the antiplasmodial response
(Molina-Cruz et al., 2008). The trade-off of the chronic oxidative state in L35 compared to 4A r/r is reflected in a much quicker drop in fecundity with
age (DeJong et al., 2007).
In Ae. aegypti, heme ingestion has been suggested to instead decrease ROS
through the activation of protein kinase C (PKC) signaling (Oliveira et al.,
2011). It is possible that similar antioxidant responses to heme are shared
between hematophagous insects to quickly neutralize the oxidative burden.
Inhibition of PKC was lately shown to significantly decrease P. falciparum
oocyst numbers in An. stephensi (Pakpour et al., 2013). The contribution of
ROS in this sense has not been studied in anophelines. It is, however, plausible that the oxidative status previously seen in Ae. aegypti plays a major role.
Another critical determinant of Anopheles vector capacity is the NOS enzyme (Luckhart et al., 1998). Invaded midgut epithelial cells upregulate the
expression of NOS (Gupta et al., 2009). NOS translocates in turn to the cell
membrane where it is involved in NO synthesis, but also in nitration of the
36
epithelium together with peroxidases. NO is thought to be directly toxic for
the parasite and epithelial nitration is necessary for TEP1-mediated lysis,
providing an important link between RNS and the TEP1 system (Oliveira
Gde et al., 2012). A proposed explanation is that the nitration event tags
parasites that cross the midgut epithelium, which allows subsequent recognition by TEP1. This could in turn potentially be linked to the aforementioned
late-stage effect on parasites by the JAK/STAT pathway as it has been found
to regulate NOS expression (Gupta et al., 2009).
Involvement of NOS in the antibacterial response has also been demonstrated. It was recently demonstrated that knock-down of EATER (homologous
to the phagocytic receptor Eater in Drosophila) abrogated the expression of
NOS and decreased the ability to combat systemic E. coli infection in An.
gambiae (Estevez-Lao and Hillyer, 2013). This finding opens up for future
investigations concerning a putative involvement of NOS in the oxidative
burst upon bacterial phagocytosis.
Gut bacteria, the third player
The mosquito gut harbours much fewer bacterial species compared to mammals. The gut microbiota also appear to be remarkably similar between different species of mosquitoes and are often dominated by a single bacterial
taxon (Osei-Poku et al., 2012). At the individual level, however, the population composition often differs dramatically and could thus potentially contribute to the varying individual susceptibility to Plasmodium. The relative
simplicity provides a good model for dissecting the tripartite interactions
between host, gut flora and pathogenic microbes. A fine-tuned manipulation
of this system could also have a severe impact on Plasmodium transmission.
Following blood ingestion, the gut bacterial bloom sets additional challenges
for developing parasites in the mosquito midgut. The bacteria may compete
for nutrients and can possibly form physiological barriers, a phenomenon
known from vertebrates. Moreover, immune responses raised by the mosquito against bacteria and Plasmodium overlap to a large degree and there is a
growing body of evidence that host immune responses raised against gut
bacteria also affect the parasite (Dimopoulos et al., 2002). It has for example
been shown that co-feedings of either live or heat-killed bacteria cause a
decrease in P. falciparum oocysts whereas depletion of gut bacteria with
antibiotics yields higher oocyst loads (Dong et al., 2009). Knock down of
PGRPLC inhibits the antiplasmodial effect of bacterial co-feeding, suggest37
ing a key role of the mosquito IMD pathway (Meister et al., 2009). Indeed,
several IMD-regulated effectors such as the TEP1 system are active against
bacteria as well, suggesting that the mosquito mounts similar effectors to
combat both bacterial and plasmodial infections (Dong et al., 2006). It has
been proposed that former bacterial encounters during larval stages might
prime basal immunity, possibly via REL2-S to more quickly combat infections (Frolet et al., 2006).
Symbiotic gut bacteria can also directly exert antiplasmodial activity. A resident Enterobacter species from wild A. arabiensis can directly suppress P.
falciparum survival through ROS production, further underlining the importance of ROS in parasite resistance (Cirimotich et al., 2011). Only a fraction of field caught mosquitoes carry Enterobacter in their intestine, however (Cirimotich et al., 2011). Much attention has lately been focused on arthropod pathogens of the genus Wolbachia. Symbionts have been found, or
introduced in Ae. aegypti that decrease transmission of dengue virus and
Plasmodium (Hoffmann et al., 2011; Moreira et al., 2009). These studies
have proven an overall more successful inhibition of viruses. A proposed
explanation is the shared intracellular nature of the virus and Wolbachia. The
antiviral effect of the bacterium has, however, been linked to the host Tolldependent expression of AMPs by bacterially generated ROS (Pan et al.,
2012). The results obtained in this study points to an extraceullar effect of
indirect nature. The genus Wolbachia seems absent in anophelines (reviewed
in (Bourtzis et al., 2013)). Stable infection and vertical transmission were
nevertheless recently achieved in An. stephensi by microinjecting bacteria
into mosquito embryos (Bian et al., 2013). Females harbouring Wolbachia
were significantly less susceptible to P. falciparum. The inhibitory effect has
been proposed to be more moderate in the field, however, and invasion and
stable transmission of An. gambiae is likely much more challenging
(Barillas-Mury, 2013).
An alternative strategy is to initially isolate and characterize symbionts that
are predominant in both field and laboratory mosquitoes. Promising candidates could subsequently be transformed to express antiplasmodial factors
and finally reintroduced in the vector. In a paratransgenesis study on the
species combination An. gambiae and P. falciparum, the anopheline symbiont Pantoea agglomerans was successfully transformed to inhibit the parasite (Wang et al., 2012). The separate introduction of five different effectors,
38
including a scorpine (a defensin family member), was found to decrease
parasite prevalence with up to 84 %.
Elizabethkingia anophelis
The genus Elizabethkingia was quite recently proposed as a separate lineage
from Chryseobacterium and Flavobacterium, to which it had previously
been classified (Kim et al., 2005). So far, three species have been described:
E. anopheles, E. meningoseptica and E. miricola. All three are Gramnegative, non-motile, non-spore forming rods and opportunistic pathogens
that possess broad antibiotic resistance. Members of Elizabethkingia have
been found in wild anophelines (Boissiere et al., 2012; Osei-Poku et al.,
2012; Wang et al., 2011), in laboratory-strains (Akhouayri et al., 2013;
Kampfer et al., 2011; Li and Tang, 2010; Lindh et al., 2008; Wang et al.,
2011) and in aedines (Osei-Poku et al., 2012; Terenius et al., 2012). Interestingly, Elizabethkingia spp. is predominant in the gut of laboratory-reared An.
gambiae, suggesting that they are able to exploit an introduced niche
(Boissiere et al., 2012).
The most well described species, E. meningoseptica (earlier C. meningosepticum), is ubiquitous in nature as well as in hospital environments and is
mainly known for causing meningitis in infants (King, 1959). Although infections with the bacterium are rare, treatment regimens are often complicated by its multidrug resistance (Balm et al., 2013). The second characterized
species of the genus, E. miricola (earlier C. miricola), was first isolated from
condensation water at the MIR space station, suggesting that these bacteria
can sustain harsh environments and sterilizing treatments (Li et al., 2003).
One clinical case of E. miricola has been reported to date and required the
use of last-resort antibiotics to resolve the infection (Green et al., 2008).
Attempts to find a suitable symbiont for paratransgenics led to the characterization of the third species in the genus, E. anopheles, isolated from the gut
of An. gambiae (Ifakara strain) and was designated R26T (Kampfer et al.,
2011). A second strain, Ag1, of the same species was later also isolated by a
different lab group from the G3 strain (Wang et al., 2011). Like the other
members of the genus, E. anophelis appears to have a broad antimicrobial
resistance (Paper IV) (Kampfer et al., 2011). The capacity of E. anopheles to
infect immunocompromised humans was recently confirmed in two case
reports from the Central African Republic and in a Singapore intensive-care
outbreak, respectively (Frank et al., 2013; Teo et al., 2013).
39
Despite the fact that 16S rDNA-sequence comparisons might not be enough
to separate the members of Elizabethkingia, isolates have often more or less
routinely been referred to as E. meningoseptica. It is thus not yet resolved
which species are most prevalent in the wild and in the laboratory respectively. The draft genomes of both species have recently been sequenced and
will allow better tools for separating between the two (Paper III) (Matyi et
al., 2013). Interestingly, in laboratory-reared An. gambiae, symbiotic E. meningoseptica can turn into a pathogen (Akhouayri et al., 2013). It is possible
that the conditional skew towards favouring Elizabethkingia during laboratory conditions also causes this pathogenic effect. Organic extracts from E.
meningoseptica have been reported to display both antimicrobial and antiplasmodial activity (Ngwa et al., 2013). Such an effect could potentially
contribute to the bacterial predominance in the mosquito gut. In contrast, live
E. anophelis bacteria do not seem to generally inhibit the growth of other
bacterial gut isolates in vitro (unpublished data). In addition, 90 % of the
taxa in wild mosquitoes belong to the phylum Proteobacteria (Boissiere et
al., 2012). The antimicrobial activity of E. meningoseptica is therefore likely
not a strong determinant of the gut community in the wild mosquitoes. The
employment of Elizabethkingia spp. in vector control strategies might be of
consideration if the bacteria would turn out to be natural pathogens of An.
gambiae. Due to their broad antimicrobial resistance and putative pathogenicity, the employment of Elizabethkingia is, however, always questionable.
Plasmodium-derived elicitors
Most work on mosquito-parasite interactions done so far has focused on
effector responses and ultimately transmission in terms of parasite numbers
at different stages. Considerably less is known, however, regarding the underlying mechanisms for recognition and no parasite specific PRRs have yet
been revealed. In fact, the key question whether the mosquito mounts a parasite-specific immune response at all remains elusive (Cirimotich et al.,
2010).
Based on findings in humans and other primates, two putative P. falciparum
MAMPs, glycosylphosphatidyl-inositols (GPIs) and hemozoin (Hz), have
previously been investigated in the mosquito. GPIs function as universal cell
membrane anchors for proteins and are the most common carbohydrate modification in Plasmodium (Gowda et al., 1997). The GPI anchors are indispen40
sable for parasite survival and need to be synthesized de novo during intraerythrocytic stages (Macrae et al., 2013). PfGPIs are regarded as toxins
for humans and are thought to mediate many of the symptoms associated
with severe malaria pathophysiology (reviewed in (Arrighi and Faye, 2010)).
Stimulation of macrophages with purified PfGPIs causes proinflammatory
responses through the activation of MAPK/NF-κB pathways and secretion of
TNFα, interleukin-6 (IL-6), IL-12 and NO (Lu et al., 2006; Zhu et al., 2005).
Depending on the GPI structure, these events are mediated through interactions with TLR2, TLR1 and to a lesser extent TLR4 (Zhu et al., 2011). The
PfGPIs are hence fullfilling Janeway’s criteria in humans since they are indispensable for parasite survival and invoke innate responses via interaction
with conserved PRRs. Conversely, provision of PfGPIs in the blood meal
induces NOS through ERK/Akt signaling in the midgut of An. stephensi
(Lim et al., 2005). PfGPIs also induce several AMP genes and affect fecundity in An. gambiae mosquitoes (Arrighi et al., 2009). Interestingly, protozoan GPIs are produced in excess (Ferguson et al., 1994) and the cell surface
density of these anchors are generally much higher than in multicellular eukaryotes (Macrae and Ferguson, 2005). Moreover, PfGPIs have been shown
to mimic insulin in humans and to some degree An. stephensi, indicating that
Plasmodium, and possibly other protozoans might use these compounds to
manipulate host metabolism and immune system (reviewed in (Luckhart and
Riehle, 2007)).
Plasmodium Hz is a dark brown pigment formed during digestion of hemoglobin within host erythrocytes (Francis et al., 1997). Extracted PfHz, as
well as synthesised Hz, induce ERK and NF-κB dependent generation of NO
in human macrophages (Jaramillo et al., 2005; Jaramillo et al., 2003). In a
comparable manner, NOS is induced in PfHz stimulated An. gambiae and
An. stephensi cell lines as well as in An. stephensi midgut tissues (AkmanAnderson et al., 2007). The signaling events induced by Hz thus seem to
overlap, at least in part, the PfGPI response as both involve Akt and ERK
pathway activation.
Isoprenoid precursors
Isoprenoids are widespread in nature and are indispensable to all living cells
(Belmant et al., 2000). This class of molecules are involved in a wide range
of metabolic processes and serve as building blocks in the synthesis of various compounds including cholesterol, steroid hormones and vitamins (Fig. 4;
reviewed in (Morita et al., 2007)). Organisms synthesize isoprenoids via two
41
different pathways. The mevalonate pathway is found in higher eukaryotes,
Archaebacteria and in the cytoplasm of plants. The more recently discovered
2-C-methyl-D-erythritol 4-phosphate (MEP) or 1-deoxy-D-xylulose 5phosphate (DOXP) pathway is found in most Eubacteria, Cyanobacteria and
plant chloroplasts (reviewed in (Rohmer, 1999)). Some bacteria such as Listeria monocytogenes (Begley et al., 2004) and Streptomyces (Hamano et al.,
2002; Kuzuyama et al., 2000) use both pathways whereas Gram-positive
cocci are the most notable example of mevalonate pathway-restricted bacteria (Boucher and Doolittle, 2000). In addition, the MEP pathway is used by
parasitic apicomplexan protozoa, including Plasmodium. As the name implies, these carry apicoplasts, a special organelle sharing common ancestry
with the chloroplast.
There seem to be a general preference for the MEP pathway in intracellular
pathogens and the pathway might have evolved due to its lower energy consumption (reviewed in (Heuston et al., 2012)). Due to its non-host specificity, the MEP pathway is an interesting target for novel antiparasitic drugs.
Fosmidomycin is an antibiotic that targets DOXP reductoisomerase and inhibits growth of blood-stage malaria parasites (Jomaa et al., 1999). Growth
is however restored upon simultaneous supplementation with the central
isoprenoid precursor, isopentenyl pyrophosphate (IPP) (Yeh and DeRisi,
2011). Upon continued co-treatment for a few generations, parasites lose
their apicoplast and are dependent on IPP supplementation for continued
growth. Isoprenoid synthesis thus appears to be the only vital function of this
organ during the parasite blood stages.
Stimulation of Vγ9Vδ2 T cells
T-cells play a central role in most vertebrate immune responses. They are
distinguished from other lymphocytes by the presence of cell membrane
anchored T cell receptors (TCRs) that recognize antigens. The TCR is composed of two glycoprotein chains that form a heterodimer. Depending on the
glycoprotein composition, T cells are divided into αβ or γδ classes. It is still
not entirely resolved how γδ T cells are activated (see below). In contrast to
αβ T lymphocytes, the typical major histocompatibility complex presentation
of antigenic peptides or glycolipids is not required for γδ T cell activation. In
humans and higher primates, the vast majority of γδ T cells in the blood express the Vγ9Vδ2 heterodimer (also termed Vγ2Vδ2). Vγ9Vδ2 T cells are
stimulated by isoprenoid precursors and other small, pyrophosphorylated,
nonpeptide compounds, commonly referred to as phosphoantigens.
42
Figure 4. Schematic figure of the mevalonate and MEP pathways of isoprenoid
synthesis. Isoprenoids (orange boxes) are synthesized from the isoprenoid precursors
(green boxes) IPP and DMAPP and are in turn used in protein prenylations and as
building blocks for various compounds including vitamins, steroids and cholesterol.
Plasmodium cannot, however, synthesise sterols and must import these from its host
(Labaied et al., 2011). HMGCS, 3-hydroxy-3-methyl-glutaryl-CoA synthase;
HMGCR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; MVK, mevalonate kinase;
PMVK, phosphomevalonate kinase; MVD, mevalonate-5-pyrophosphate decarboxylase; Pyr, pyruvate; G3P, glyceraldehyde 3-phosphate; DOXP, 1-deoxy-D-xylulose
5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4diphosphocytidyl-2-C-methylerythritol;
CDP-MEP,
4-diphosphocytidyl-2-Cmethylerythritol 2-P; MEcPP, 2-C-methyl-D-erythritol 2,4-cycloPP; IDI1, isopentenyl pyrophosphate isomerase; GPP(S), geranyl pyrophosphate (synthase); FPP(S),
farnesyl pyrophosphate (synthase); GGPP, geranylgeranyl pyrophosphate.
43
In absence of stimuli, Vγ9Vδ2 T cells normally constitute 2-5 % of the peripheral blood T cells. In response to several bacterial, viral and protozoan
infections including malaria, however, the Vγ9Vδ2 T cell pool expands rapidly and may even exceed the number other lymphocytes within a few days
(reviewed in (Morita et al., 2007)). In addition to the proliferative response,
stimulated Vγ9Vδ2 T cells acquire cytotoxic functions and secrete proinflammatory cytokines. Activated Vγ9Vδ2 T cells are able to kill Mycobacterium tuberculosis-infected macrophages (Chen et al., 2013; Dieli et al.,
2000) and several tumor cell lines (Corvaisier et al., 2005; Guo et al., 2005;
Mattarollo et al., 2007) and act as antigen presenting cells to induce antigenspecific αβ T cell responses (Brandes et al., 2009; Brandes et al., 2005;
Moser and Eberl, 2007). Vγ9Vδ2 T cells thus act at the border of innate and
adaptive immune responses as an important link between the two systems in
humans.
IPP and HMBPP
Isoprenoid precursors represent the most potent natural group of Vγ9Vδ2 T
cell activators. Despite major differences in starting material, metabolites
and enzymes, both isoprenoid synthesis pathways end up in the synthesis of
the central intermediate IPP (Fig. 4). Through analysis of mycobacterial
extracts, IPP was identified as the first natural antigen able to stimulate
Vγ9Vδ2 T cells (Tanaka et al., 1995). Since IPP is also present endogenously, it was early on suggested that Vγ9Vδ2 T cells are able to somehow discriminate between self and foreign isoprenoid precursors to avoid autoimmune responses. Physiological levels of IPP derived from bacteria that use
the mevalonate pathway are, however, not stimulatory (Jomaa et al., 1999).
IPP could instead possibly act as an endogenous damage-associated molecular pattern (DAMP) when present in locally high concentrations following
release from necrotic host cells or wounded tissues. Metabolic intermediates
such as IPP could also serve as specific antigens for a wide range of malignant tumors (Gober et al., 2003). Tumor cells have increased metabolic rates
and are therefore likely to contain higher levels of metabolic intermediates.
Moreover, some tumor cell lines targeted by γδ T cells appear to have a
dysregulated mevalonate pathway. For example, the concentration and efficiency of HMG-CoA reductase, the rate limiting enzyme in the mevalonate
pathway, is elevated during leukemia and lymphoma (Harwood et al., 1991).
Expanded γδ T cells display highly similar Vγ2 chain repertoires following
exposure to either IPP or Daudi cells (a human Burkitt’s lymphoma cell line)
44
Figure 5. The chemical structures of IPP and HMBPP are highly similar.
(Hebbeler et al., 2007). Also, inhibition of farnesyl pyrophosphate synthase,
the enzyme responsible for converting IPP to farnesyl pyrophosphate (FPP)
by bisphosphonates, stimulates γδ T proliferation in most cancer patients,
presumably as a consequence of increased IPP concentrations (Fig. 4)
(Bonneville and Fournie, 2005; Kunzmann et al., 2000; Kunzmann et al.,
1999). The response of γδ T cells to tumors and IPP seems therefore to overlap, at least partly.
The mycobacterial isolates used for IPP identification by Tanaka et al. also
indicated the presence of additional, yet undefined phosphoantigens.
Through successive deletions of bacterial enzymes in the MEP pathway, a
precursor to IPP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate
(HMBPP), was found to be a much more potent Vγ9Vδ2 T cell ligand (Hintz
et al., 2001). Although structurally similar to IPP, the non-self HMBPP has
an approximately 30 000-fold higher bioactivity and stimulates Vγ9Vδ2 T
cell expansion at picomolar concentrations (Fig. 5).
Recognition and signal transduction
The underlying mechanism for the interaction between phosphoantigens and
Vγ9Vδ2 T cells is still not entirely resolved. The Vγ9Vδ2 TCR is necessary
for phosphoantigen recognition. A tetrameric construct of the receptor has
been shown to bind HMBPP (Wei et al., 2008). Moreover, injection of
HMBPP causes TCRs to form nanoclusters on the membrane surface of
clonally expanding Vγ9Vδ2 T cells in cynomolgus macaques (Chen et al.,
2008). Several studies have indicated that IPP and HMBPP do not need to be
processed but must associate with an antigen presenting cell (APC) or a
membrane in order to be recognized by the TCR (Morita et al., 2001; Wei et
45
al., 2008). Although APCs increase Vγ9Vδ2 T cell responses to soluble
phosphoantigens, the presentation can be mediated by non-specialized APCs
(Mookerjee-Basu et al., 2010). Interestingly, infection of APCs with MEP
pathway negative Staphyolococcus aureus yields comparable Vγ9Vδ2 T cell
responses to MEP positive bacteria (Kistowska et al., 2008). Treatment of
infected APCs with statins that block the host, but not bacterial mevalonate
pathway abrogated the Vγ9Vδ2 T cell response. In addition, accumulation of
IPP through bisphosphonate treatment of APCs yielded similar effects to the
bacterial infections. Taken together, these results suggest that Vγ9Vδ2 T cell
activation in response to infected APCs is not always dependent on the MEP
pathway and that the host mevalonate pathway plays an important role. In
support of this it was also found that infected APCs upregulate HMG-CoA
reductase, similar to previously mentioned tumor cells. Primates thus seem
to have evolved the ability to sense bacteria through changes in their own
mevalonate pathway.
Both HMBPP and IPP trigger activation of several intracellular events and
effector responses. Soluble HMBPP induces rapid crosstalk between
Vγ9Vδ2 T cells and autologous monocytes, resulting in production and secretion of cytokines and chemokines (Eberl et al., 2009). The functional
Vγ9Vδ2 T cell response following both HMBPP (Correia et al., 2009) and
IPP (Li and Pauza, 2009) stimulation is dependent on TCR, ERK and
PI3K/Akt pathway signaling. HMBPP additionally activates the other main
MAPKs (JNK and p38) that are central mediators of TCR signaling transduction (Correia et al., 2009). Furthermore, calcium-dependent nuclear
translocation of the NF-κB-related nuclear factor of activated T cells
(NFAT) enhances the IPP-induced response (Li and Pauza, 2009).
46
THE PRESENT STUDY
Specific aims of the work
•
•
•
To explore the immunogenic properties of the isoprenoid precursor
and designated phosphoantigen HMBPP in An. gambiae
To investigate the immunogenicity of conditioned medium from γirradiated bacteria as a potential tool for studying humoral immune
responses in insects
To sequence and annotate the An. gambiae gut symbiont E. anophelis
Results and discussion
The effects of HMBPP in An. gambiae (Paper I)
Primates have evolved a unique T cell subset that specifically recognizes
phosphoantigens. We wanted to explore whether An. gambiae could also
recognize and respond to these compounds. We selected HMBPP due to its
microbial origin and immunogenic potency in primates. Initially, HMBPP
was tested in parallel with PfGPI in Western blot analysis of MAPK and Akt
pathway activation in An. gambiae 4a3B cells and was found to more strongly induce several of these cascades. The cellular response to HMBPP hence
overlapped, to quite a degree, the signal pathway activation in human
Vγ9Vδ2 T cells and led us to continue studying the mosquito responses to
this compound. Importantly, we demonstrated that P. falciparum releases
phosphoantigens, likely HMBPP, that induce a strong clonal expansion of
Vγ9Vδ2 T cells. To our knowledge, this was the first time such a release was
demonstrated and suggests that phagocytosis of the parasite by APCs is not
necessarily required for Vγ9Vδ2 T cell activation. Instead, the released
phosphoantigens could potentially bind to yet unidentified structures or receptors that in turn present them to the TCR. Such a procedure would thus
not necessarily require any initial internalization by APCs.
Upon addition of the compound to the blood meal, mosquitoes responded by
upregulating several of the selected immune genes. Such a broad activation
was surprising, but at the same time pointed towards an Imd/REL2-mediated
47
response. The differential expression patterns did however not completely
overlap that observed following provision of DAP-PGN in the blood meal.
This gives a hint of a potentially unique response to HMBPP. Several gut
bacterial species use the MEP pathway and could hence potentially benefit
from the addition of HMBPP. We quantified the relative 16S rDNA abundance and found that the amount of bacteria was temporally altered. This
could mean that HMBPP act indirectly through effects on the gut microbiota,
which in turn trigger host responses. On the other hand, HMBPP could also
act on the bacteria through direct induction of mosquito responses. IPP has
previously been shown to confer protection against H2O2 in human fibroblast
cells. Such an effect would likely be of benefit for both gut bacteria and
Plasmodium. We found that both HMBPP and IPP conferred such a protection in 4a3B cells, although the protective effect of HMBPP had a delayed
onset compared to IPP. Further studies are needed to resolve the cause and
consequence of this system.
The effects of medium from γ-irradiated E. coli on Drosophila humoral
immunity (Paper II)
Drosophila senses bacteria through pattern-recognition of PGN, a vital component in the bacterial cell wall. The PGN-layer in Gram negative bacteria is
however shielded by an outer layer of LPS. It has therefore been debated
how the host manages to come in contact with the elicitor. In a recent study,
Håkan Steiner’s group demonstrated that supernatants from log-phase bacteria of either Gram-type could induce the AMP gene AttA in Drosophila S2cells (Karlsson et al., 2012). The stimulatory effect was however lost if the
bacteria were allowed to grow to stationary phase. The experiment was repeated using different fractions of the supernatants from the Gram negative
bacterium E. coli. A response was then observed against the fraction containing TCT, a product of DAP-PGN degradation and a potent inducer of
Imd signaling. This suggests that bacteria shed and release PGN during
growth. When nutrients are scarce, however, TCT is absorbed and recycled
by the bacteria.
In the present study, we wanted to explore how γ-irradiation, at a dose to
completely halt cell division, affected the bacterial PGN-metabolism. In
studies of Drosophila immunity, heat or chemical treatments are regularly
employed to inactivate bacterial growth. Inactivation through γ-irradiation is
however mainly targeting DNA and are hence likely to better preserve metabolic features and antigenic structures. In the study by Karlsson et al., 2012,
48
heat killing completely abrogated the release of PGN. One of the original
aims of this study was therefore to evaluate whether γ-inactivation of E. coli
in log-phase would leave the PGN-metabolism intact, and whether it thus
could be used as a novel method to study insect immune responses against
metabolically active bacteria. Initial experiments were performed by Sandra
Oldenvi, a previous member of the Steiner group.
Radioactively labelled DAP was added to the suspensions directly after irradiating the bacterial. This allowed an eventual synthesis de novo of PGN to
be monitored since the DAP is exclusively used in the stem peptide of PGN.
Bacterial supernatants were collected at several time points for up to one
week following the radiation-exposure. The results indicated a retained metabolic activity following the irradiation event. We initially also tested UVCirradiation, which yielded promising results. The main issue was, however,
that 100 % inactivation of the bacteria could not be achieved, which hindered any long-term time-course studies. Parallel cultures were also maintained without the addition of DAP for later use in the S2-cell assay. The
bacteria were initially maintained in a PBS suspension after irradiation without additional nutrients. When collected supernatants were used to stimulate
S2-cells, a strong induction of both AttA/B and CecA1 was observed. We
failed, however, to detect any release of radioactive TCT, indicating that the
observed effect on S2-cells was rather due to bacterial lysis and cumulative
release of elicitors.
The experiment was subsequently repeated using minimal-medium instead
of PBS. Using HPLC in combination with a scintillation counter, we observed a post irradiation-increase in radioactivity over time in the HPLCfraction corresponding to TCT. The fraction was subsequently found to contain TCT using MALDI-TOF. Next, the supernatants were used to stimulate
S2-cells. In contrast to our previous experiments, the expression of AttA/B
decreased whereas CecA1 still increased relative to the post-irradiation incubation time of the supernatants. The expression of both attacins and cecropins have been reported to be dependent on both Toll and Imd signaling.
Toll signaling in Drosophila cell lines is however not activated by external
stimuli, likely due to a lack of the extracellular components of the pathway.
We hence selected supernatants from late time point (72 h) and repeated the
experiment following RNAi-mediated knock-down of Relish, the NF-κB of
the Imd pathway. This resulted in a decreased expression in the S2-cells,
suggesting that the bacterial medium from prolonged incubation still triggers
49
the Imd signaling pathway. As this pattern has not been observed before, we
performed a whole transcriptome analysis. The unique nature of the response
was confirmed and in addition it showed a number of stress genes, e.g. heat
shock genes, to be induced that are not usually part of the innate immune
response. Taken together, these findings render it likely that some of the
elicitors are derived from peptidoglycan. The nature of the elicitor(s) causing
the more stress like part of the response, however, remains elusive.
Genome annotation of E. anophelis (Paper III and IV)
The gut microbiota of An. gambiae have a profound impact on Plasmodium
infection. The isolation and characterization of abundant taxa will aid in
identifying the causative species and also find suitable candidates for vector
control and paratransgenesis strategies. In these studies, we sequenced and
annotated the genome of E. anophelis, a predominant gut symbiont of An.
gambiae. Two different strains were sequenced. The type strain, R26T was
isolated from the Ifakara strain of An. gambiae in our laboratory at Stockholm University and Ag1 was derived from the G3 strain in Xu’s laboratory,
New Mexico. Upon comparison, both strains were with a few exceptions
identical.
The annotation revealed several features of the bacterium that could potentially contribute to its symbiotic relation with the mosquito host. Also, a
large number of genes involved in antibiotic resistance were found, including β-lactamases, metallo-β-lactamases and efflux pumps that appear overall
conserved within the family Flavobacteriaceae. However, we also found
signs of lateral gene transfer of metallo-β-lactamases genes, which has been
shown to occur between other species in nature. The R26T strain has previously been found to display resistance against several antibiotics. Here we
demonstrate additional strong resistance against CecA as well as the previously applied antibiotic ciprofloxacin in the treatment of E. meningoseptica
infection.
Although not included in the present paper, we additionally performed crossstreaks and found that intact and growing E. anophelis inhibited one out of
eight Anopheles midgut isolates. This contrasts a previous report where extracts from lysed E. meningoseptica displayed broad antimicrobial activity
(Ngwa et al., 2013). It is therefore possible that E. anophelis is adapted to
co-exist with other gut symbionts. Recent reports of opportunistic pathogenicity of E. anophelis warrants further studies to elucidate whether these
50
bacteria can spread to humans from mosquitoes or not. In light of this, we
found that the bacterium displays α-hemolytic activity, which could potentially contribute to the mosquito blood digestion but also add to its virulence
in humans.
Taken together, these findings question the potential use of E. anophelis in
future malaria intervention strategies. It might nevertheless be of interest for
the community to investigate the cause and consequence of its predominance
in the gut of laboratory reared anophelines as this could potentially affect the
interpretations of various studies. It would also be of interest to evaluate its
potential antiplasmodial activity in the mosquito gut.
CONCLUDING REMARKS
The global malaria problem is far from resolved, and novel measures for
controlling the disease are desperately needed. Findings from basic research
are constantly increasing knowledge of the disease that could lead to development of new treatment strategies. In this sense, An. gambiae has become
an important model organism, not only for studying malaria, but also for
deciphering insect-parasite interactions and innate immune responses in general. Working with this species has its downsides, however, due to the difficulties in performing forward genetic screens, transformations and standardization of rearing measures for obtaining repeatable infection experiments.
In this sense, Drosophila is an excellent model organism. It is therefore
slightly surprising that so few specialists are performing cross-species collaborations. I have had the chance to join this exciting scientific community
and participate in studies revolving various topics, which forced me to
broaden my knowledge. Although not all commenced projects were successful, they have all allowed me to grow as a scientist. The findings presented
by me and co-workers will hopefully aid in broadening the picture of immune recognition and the contribution of gut symbionts in An. gambiae.
51
ACKNOWLEDGEMENTS
First of all to my supervisor Ingrid Faye for giving me the opportunity to
join the scientific community and for always being supportive and encouraging throughout the project.
To Håkan Steiner, Jiannong Xu and Ingela Parmryd for constructive and
fruitful collaborations.
To my co-supervisor Ylva Engström and the always helpful fly-folks at
Molbio.
To Marita Troye Blomberg and the members of her group for the good support, for the lending of happy and frisky parasites and for backing me up
financially for the EMBL meeting in Heidelberg.
Many thanks also to my group members Melanie and Noushin for all the
support and for keeping up the good spirit.
To my roommates Harald and Ali for sharing the good times and the grumpy
times, and several nice moments in the gym. Good luck with your projects
and do not give up on CRISPR/Cas.
The MBW floorball-players throughout the years. If it were not for you, I
would not have picked up this great sport again after more than 15 years
without touching a stick.
Of course, to all people at the old GMT and the administrative staff, especially Eva and Eva, I-B and Görel.
And finally, but not least to Sanna and my family for your endless support
and patience.
52
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