Wolbachia symbiosis and insect immune response

Wolbachia and insect immune response 89

© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Science, Insect Science, 15, 89-100

Abstract Bacterial intracellular symbiosis is very common in insects, having significantconsequences in promoting the evolution of life and biodiversity. The bacterial group thathas recently attracted particular attention is Wolbachia pipientis which probably representsthe most ubiquitous endosymbiont on the planet. W. pipientis is a Gram-negative obligatoryintracellular and maternally transmitted α-proteobacterium, that is able to establish symbi-otic associations with arthropods and nematodes. In arthropods, Wolbachia pipientisinfections have been described in Arachnida, in Isopoda and mainly in Insecta. They havebeen reported in almost all major insect orders including Diptera, Coleoptera, Hemiptera,Hymenoptera, Orthoptera and Lepidoptera. To enhance its transmission, W. pipientis canmanipulate host reproduction by inducing parthenogenesis, feminization, male killing andcytoplasmic incompatibility. Several polymerase chain reaction surveys have indicated thatup to 70% of all insect species may be infected with W. pipientis. How does W. pipientismanage to get established in diverse insect host species? How is this intracellular bacterialsymbiont species so successful in escaping the host immune response? The present reviewpresents recent advances and ongoing scientific efforts in the field. The current body ofknowledge in the field is summarized, revelations from the available genomic informationare presented and as yet unanswered questions are discussed in an attempt to present acomprehensive picture of the unique ability of W. pipientis to establish symbiosis and tomanipulate reproduction while evading the host’s immune system.

Key words Drosophila, immune response, insects, symbionts, Wolbachia

89

Insect symbiosis

Symbiosis is ubiquitous on this planet and has been de-scribed for many multicellular organisms. Several lines ofevidence suggest that symbiosis has had significant conse-quences in promoting the evolution of life and biodiversity(Gray & Doolittle, 1982; Margulis, 1970; Margulis, 1993;Margulis & Fester, 1991). Insects seem to be the animalgroup which has successfully established most differentsymbiotic associations both inside and outside their bodies(Bourtzis & Miller, 2003; Bourtzis & Miller, 2006; Buchner,1965). These symbiotic associations have been shown to

Correspondence: Kostas Bourtzis, Department of Environ-mental and Natural Resources Management, University ofIoannina, Agrinio 30100, Greece. Tel: +30 26410 74114; fax:+30 26410 39571; email: [email protected]

Wolbachia symbiosis and insect immune response

Stefanos Siozios, Panagiotis Sapountzis, Panagiotis Ioannidis and Kostas BourtzisDepartment of Environmental and Natural Resources Management, University of Ioannina, Agrinio 30100, Greece

affect different aspects of the insect life cycle andphysiology, including development, nutrition, reproduc-tion and speciation, defense against natural enemies andhost plant preference, thus aiding insects in developing andmaintaining the most diverse lifestyles of all animals(Bourtzis & Miller, 2003; Bourtzis & Miller, 2006; Buchner,1965; Dale & Moran, 2006).

Insect symbionts can be classified into two main groups.The first group is called “primary” symbionts. Thesesymbionts reside within a bacteriome and are maternallytransmitted to the progeny. Representatives of this groupinclude Buchnera (the primary symbiont of aphids),Wigglesworthia (tsetse flies), Tremblaya (mealy bugs),Carsonella (psyllids), Portiera (whiteflies), Blochmannia(ants), Blattabacterium (cockroaches) as well as Baumanniaand Sulcia (the co-resident primary symbionts of xylem-feeding sharpshooters) (Akman et al., 2002; Bandi et al.,1994; Baumann, 2006; Gil et al., 2003; Hunter & Zchori-

© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences

Insect Science (2008) 15, 89-100, DOI 10.1111/j.1744-7917.2008.00189.x

Invited review

90 S. Siozios et al.


Fein, 2006; Moran et al., 2005b; Shigenobu et al., 2000;Wu et al., 2006). These primary symbionts have a long co-evolutionary history (an ancient association) with theirinsect host, which is reflected in the fact that phylogenetictrees based on bacterial genes are similar to the trees basedon host insect genes (Aksoy, 2003; Baumann, 2006).Several studies have conclusively shown that the primarysymbionts have the capacity to provide their hosts withnutrients such as essential amino acids, vitamins and othercofactors, thus establishing an obligatory mutualistic sym-biotic association (Akman et al., 2002; Buchner, 1965;Douglas, 2003; Gil et al., 2003; Nakabachi et al., 2005;Shigenobu et al., 2000; Wu et al., 2006; Zientz et al., 2004).

The second group is called “secondary” symbionts.These symbionts are primarily also maternally transmitted;however, they can often colonize naïve host individualsand species through horizontal transmission. These sym-bionts usually establish facultative symbiotic associationsthat can be deleterious or beneficial (Dale & Moran, 2006).Representatives of this group include, among others, Sodalisglossinidius (Toh et al., 2006), Serratia symbiotica (Moranet al., 2005a), Halmitonella defense (Moran et al., 2005a),Regiella insecticola (Moran et al., 2005a), Fritscheabemisiae (Everett et al., 2005), Wolbachia pipientis(Wolbachia for the purpose of this review; Hertig, 1936; Loet al., 2007), Cardinium hertigii (Zchori-Fein et al., 2004),and male-killing bacteria, like Rickettsia, Arsenophonusand Spiroplasma (Hurst et al., 2003b). Both positive andnegative effects on the host have been observed in symbi-otic associations involving secondary symbionts. The posi-tive effects include the capacity of infected hosts to surviveheat stress, to develop resistance to parasitic wasps, and toexhibit altered host plant preference (Montllor et al., 2002;Oliver et al., 2005; Oliver et al., 2003; Scarborough et al.,2005; Tsuchida et al., 2004). In other cases, the facultativesymbionts negatively affect growth, reproduction and lon-gevity of the host (Chen et al., 2000; Min & Benzer, 1997;Stouthamer et al., 1999).

During the last three decades, it has become increas-ingly evident that several intracellular bacteria (Wolbachia,Cardinium, Spiroplasma, Arsenophonus, Rickettsia) caninfluence the reproduction of their insect hosts (Bourtzis &Miller, 2003; Bourtzis & Miller, 2006). The bacterialgroup that has attracted particular attention in almost allareas of life sciences, from sociobiology to molecularbiology, is Wolbachia (Stouthamer et al., 1999).

Insect-Wolbachia symbiosis

Wolbachia is a group of Gram-negative obligatory intrac-ellular and maternally transmitted α-proteobacteria that

infect a number of invertebrate species, including mites,spiders, crustaceans, filarial nematodes, and especiallyinsects (Stouthamer et al., 1999). Wolbachia was firstreported as a rickettsial infection of the gonads of themosquito Culex pipiens (Hertig, 1936). Wolbachia have along and successful history of symbiotic associations witharthropods and nematodes, collectively the “Ecdysozoa”.In arthropods, Wolbachia infections have been describedin Arachnida, and Isopoda, but mainly in Insecta. Severalsurveys have indicated that up to 70% of all insect speciesmay be infected with these bacteria, rendering Wolbachiathe most ubiquitous intracellular symbiotic organism onEarth (Jeyaprakash & Hoy, 2000; Werren & Windsor,2000).

Two evolutionary trajectories are recognized for thesevertically transmitted Wolbachia strains: mutualism orreproductive parasitism (Dedeine et al., 2003; Dobson,2003). Two examples of mutualistic Wolbachia associa-tions have been reported to-date: (i) in filarial nematodes,Wolbachia is necessary for normal development and fertil-ity (Taylor et al., 2005); and (ii) in the wasp Asobaratabida, Wolbachia is needed for normal oogenesis (Dedeineet al., 2001). On the other hand, multiple examples ofreproductive parasitism have been reported in arthropodsand, particularly in insects. These reproductive manipula-tions include cytoplasmic incompatibility, parthenogenesis,feminization and male-killing (Bourtzis & Miller, 2003;Bourtzis & Miller, 2006; Stouthamer et al., 1999; Werren,1997). The reproductive manipulations induced byWolbachia are of great interest in two important scientificareas: (i) for the development of novel and environmen-tally friendly strategies for the control of agricultural pestsand disease vectors, as well as for the manipulation ofbeneficial arthropods (Ruang-Areerate & Kittayapong,2006; Xi et al., 2005; Zabalou et al., 2004b); and (ii) aspotential mechanisms of speciation (Bordenstein, 2003;Jaenike et al., 2006; Koukou et al., 2006; Werren, 1998).

Two lines of evidence suggest that Wolbachia can readilyinvade novel host species and establish symbioticassociations. The first is based on inferences from phylog-enies and host distributions. It has been shown that closelyrelated bacterial strains infect evolutionarily distant hostspecies and that on the other hand the same host can carrydiverse Wolbachia strains (Werren et al., 1995). The sec-ond line of evidence comes from experimental transfers ofWolbachia strains, mostly through embryonic injections,into evolutionarily close and/or distantly related naïvehosts. The majority of these newly established host-Wolbachia symbiotic associations is stable and expressesthe expected reproductive phenotype (Boyle et al., 1993;Braig et al., 1994; Poinsot et al., 1998; Xi et al., 2005;Zabalou et al., 2004a).



The above observations indicate that host co-adaptationis not necessary and that Wolbachia carry all the necessarymechanisms to invade host cells and establish symbiosis,including invasion of developing eggs or embryos (Dale &Moran, 2006). How has Wolbachia been so successful inestablishing such a great number of symbiotic associations?How do these bacteria move horizontally so efficiently?How do they cope with the immune system of their hosts?Have they evolved mechanisms to manipulate the immunesystem or are they hidden from it? Are they perhapsmimicking host components and thus are not recognized asinvaders? Or, do they carry the necessary molecular toolswhich enable them to manipulate and/or overcome hostimmune response?

Insect immunity

Insects are able to mount a sophisticated innate immuneresponse against bacterial, fungal and other parasitic in-truders (Brennan & Anderson, 2004; Bulet et al., 1999;Dimopoulos et al., 2001; Hoffmann, 2003; Levashina,2004). This innate immune system is usually divided intocellular and humoral mechanisms involving phagocytosis,encapsulation, activation of proteolytic cascades and syn-thesis of antimicrobial peptides in the fat body, followed bythe secretion of those peptides into the hemolymph (Brennan& Anderson, 2004; Bulet et al., 1999; Hoffmann, 2003;Lanot et al., 2001; Lavine & Strand, 2002).

Studies in various insect species have identified numer-ous inducible antimicrobial peptides including Drosomycin,Metchnikowin, Cecropin, Defensin, Attacin, Diptericin,Drosocin in Drosophila and the three families of Defensins,Cecropins and Gambicins in mosquitoes (Brennan &Anderson, 2004; Bulet et al., 1999; Dimopoulos et al.,2001; Hoffmann, 2003; Levashina, 2004). The antimicro-bial activities of these peptides are specific to certainmicrobes. Some are active against fungi (Drosomycin,Metchnikowin, Cecropin, Gambicin), others against Gram-positive bacteria (Defensin, Metchnikowin, Gambicin) orGram-negative bacteria (Attacin, Cecropin, Diptericin,Drosocin, Gambicin) (Brennan & Anderson, 2004; Buletet al., 1999; Dimopoulos et al., 2001; Hoffmann, 2003;Levashina, 2004). Genetic and molecular studies havedefined components of the signaling pathways which re-sult in the production of these inducible antimicrobialpeptides. These components belong to two signaling path-ways that control the activation of NF-κB-like factors inresponse to infection: the Toll and the Imd pathways. Forreviews on these signaling pathways and the production ofinducible antimicrobial peptides see Brennan & Anderson(2004) and Hoffmann (2003).

The term “cellular immune response” refers to the ac-

tivity of insect blood cells (hemocytes) against infection.Insects produce different classes of hemocytes which havedistinct functions. The most dominant class is plasmatocytes(phagocytic macrophage-like cells) which comprises about90% of the blood cell population (Lanot et al., 2001;Lavine & Strand, 2002). Microbial pathogens are generallyphagocytosed by plasmatocytes, whereas parasitoids andother bigger parasites are encapsulated (Schmidt et al.,2001). Phagocytic uptake of invaders by blood cells ininsects seems to be a vital defense strategy as in mammals.Genetic and molecular studies have also revealed that anautophagic machinery plays a protective role against intra-cellular bacteria, thus acting as an intracellular innateimmune mechanism. Autophagy is a cellular membrane-traffic housekeeping mechanism involved in size regulation,recycling of cellular proteins, as well as removing mal-formed or superfluous organelles (Reggiori & Klionsky,2002). Autophagy is also essential for cells to surviveduring nutrient limitation (Scott et al., 2004). During thisprocess, regions of the cytoplasm as well as organelles arefirst enclosed by double- or multiple-membrane structurescalled autophagosomes. The trapped cytoplasmic materialis completely separated from the rest of the cytoplasm anddelivered to autolysosomes, which are formed by thefusion of autophagosomes with lysosomes for hydrolyticdegradation. Several reviews on autophagy have recentlybeen reported (Amano et al., 2006; Kirkegaard et al., 2004;Levine, 2005; Mizushima, 2005).

Host immunity and Wolbachia

It has been shown that Wolbachia is present in bothgonadal and somatic tissues including the haemolymph(Dobson et al., 1999). Wolbachia is able to move horizon-tally between different insect species (Heath et al., 1999;O’Neill et al., 1992; Werren et al., 1995). Experimentaltransfers of Wolbachia strains, mostly through embryoniccytoplasmic injections, between diverse hosts have re-sulted in stable symbiotic associations that express theexpected reproductive phenotype (Boyle et al., 1993; Braiget al., 1994; Poinsot et al., 1998; Xi et al., 2005; Zabalouet al., 2004a; Zabalou et al., 2004b). In addition, in a recentand very elegant study, Frydman and colleagues (2006)showed that injected (and/or transplanted) Wolbachia intothe Drosophila haemocel are able to cross several tissuesand finally, through the somatic stem cell niche, reachgerm line, where they establish themselves (Frydman etal., 2006). Thus, Wolbachia is expected to get in contactwith major components of the insect immune system andtrigger a defense. If so, how do Wolbachia infections theninvade and persist in insect hosts?

There are two alternative explanations of the lack of



detection by the host: (i) Wolbachia is being recognized aspart of the host; however, insect immune response istriggered by bacterial surface antigens rather than directrecognition of non-self; (ii) lack of immune response maybe due to a mechanism of evading immunity and/or ab-sence of elicitors of immunity (Hurst et al., 2003a).

It was demonstrated that Wolbachia does not activate theproduction of diptericin and cecropin, known antimicro-bial peptides against Gram-negative bacteria, in the Droso-phila simulans Riverside (DSR) strain, despite the fact thatthis host is heavily infected with Wolbachia in both go-nadal and somatic tissues (Bourtzis et al., 2000). On theother hand, E. coli-challenged Wolbachia-infected D.simulans lines retained their ability to mount an immuneresponse, as was indicated by the significantly increasedlevels of expression of both diptericin and cecropin genes.Similar results were obtained from immune challengeexperiments of single and double Wolbachia infected linesof the mosquito Ae. albopictus. These results suggest thatWolbachia neither induces nor suppresses insect humoralimmune response system (Bourtzis et al., 2000). However,it was recently reported that the virulent strain Wolbachiapopcorn can up-regulate the expression of the host antimi-crobial peptide Cecropin C and lysozyme based on Droso-phila microarray analysis (McGraw et al. personalcommunication, cited in McGraw & O’Neill, 2004). Thisactivation is probably due to the fact that the uncontrolledreplication of the Wolbachia popcorn strain results in thelyses of host cells, thus allowing the bacterium to get incontact with receptors regulating the host immune re-sponse (McGraw et al. personal communication, cited inMcGraw & O’Neill, 2004).

Members of the genus Spiroplasma also form symbioticassociations with a wide range of insect hosts establishingthemselves in different tissues, including haemolymph,and have been associated with male-killing phenotypes(Hurst et al., 2003b). Likewise Wolbachia, expression ofantimicrobial genes in Spiroplasma-infected flies did notup-regulate either in naturally Spiroplasma-infected hostsor after their challenge with the Gram-negative bacteriumE. coli or spores of the fungus Beauveria bassiana com-pared to uninfected and/or unchallenged controls (Hurst etal., 2003a; Hurst et al., 2003b). These data also suggestedthat Spiroplasma neither induces nor suppresses the hostimmune response. However, ectopic induction of antimi-crobial gene expression by either septic shock or via theTI10b mutation that up-regulates the expression of aroundhalf of the genes in the antimicrobial cascade, leads to thereduction of the parasite titer (Hurst et al., 2003a; Hurst etal., 2003b).

Fytrou and colleagues (2006) recently reported negativeeffects of Wolbachia infection on immunity-related traits

of D. simulans and of the parasitoid wasp Leptopilinaheterotoma (Fytrou et al., 2006). The authors showed thatthe presence of Wolbachia in D. simulans significantlyreduced the efficiency of the host’s cellular immune re-sponse against the eggs deposited by the parasitoidLeptopilina heterotoma. In addition, the presence ofWolbachia in L. heterotoma resulted in significantly re-duced numbers of surviving parasitoid offspring withinthe D. simulans host, as a result of increased encapsulation.This finding was rather unexpected, since other maternallyinherited bacteria such as Hamiltonela defense, Serratiasymbiotica and Regiella insecticola of the pea aphidAcyrthosiphon pisum have a major positive effect on hostresistance to parasitoid wasps (Ferrari et al., 2004; Oliveret al., 2005; Oliver et al., 2003; Scarborough et al., 2005).This difference may be due to alternative strategies em-ployed by different symbionts in order to invade hostpopulations. The pea aphid symbionts may spread byincreasing their host’s fitness while Wolbachia relies onthe manipulation of host reproduction.

Wolbachia infections have also been described in filarialnematodes, where they establish an obligatory mutualisticassociation (Foster et al., 2005; Taylor et al., 2005). Treat-ments of nematodes with antibiotics of the tetracyclinefamily produce the following results: First, the antibioticsclear Wolbachia. Second, removal of bacteria blocksembryogenesis, resulting in sterility. Third, Wolbachia arereleased in the systemic circulation of the filaria carrier,presumably after the death of the nematodes. This releaseis associated with the induction of a potent inflammatoryresponse by unknown components of Wolbachia, otherthan lipopolysaccharide (LPS)-like molecules, since theseare not present in Wolbachia (Foster et al., 2005; Taylor etal., 2005; Turner et al., 2006; Wu et al., 2004). Wolbachiahave also been associated with severe inflammatory reac-tions including the pathogenesis of both human and caninefilariasis. In addition, individuals with lymph edema orhydrocele have significantly higher levels of anti-WSP(Wolbachia Surface Protein) antibodies than those withoutthese diseases, suggesting that anti-WSP antibody re-sponses are associated with chronic filarial morbidity butnot filarial infection status (Brattig et al., 2004; Rao &Pandalai, 2004; Taylor et al., 2005; Turner et al., 2006).

It is worth noting that WSP was shown to activate thehuman innate immune system through interaction withToll-like receptors (TLRs). This interaction triggered theactivation of NF-κB, resulting in the activation of genesencoding inflammatory cytokines including TNF-α andseveral interleukins (Brattig et al., 2004). Interestingly,WSP was recently found to be capable of delaying apoptosisin human polymorphonuclear cells (PMNs) (Bazzocchi etal., 2007). Polymorphonuclear cells are essential for the



innate immune response against invading bacteria throughtheir ability to phagocytose microbial pathogens and totrigger a cytokine network thus resulting in inflammatoryand specific immune responses. The exposure of PMNs toagents such as cytokines and bacterial products throughTLR2 stimulation can influence cell survival and delayapoptosis, which contributes to the persistence of inflam-mation (Power et al., 2004). It has also been suggested thatWSP-mediated inhibition of apoptosis in human neutro-phils is linked to reduced caspase-3 activity in WSP-treated cells compared to control samples (Bazzocchi etal., 2007). Finally, Morchón and collegues (2007) reportedthat WSP but not GroEL (a Wolbachia heat shock protein)is able to stimulate the iNOs mRNA expression and nitricoxide (NO) production in BALB/c mice following subcu-taneous inoculation (Morchon et al., 2007). The produc-tion of NO, along with the presence of reactive oxygenspecies, constitutes an important component of the innateimmune response of both insects and mammals. They exerttoxic effects on bacteria and also have the ability tostimulate other components of the immune response system.For example, NO is implicated in the blood cell-dependentinduction of Diptericin in the fat body of Drosophila inresponse to Gram-negative infections (Bogdan, 2001; Foley& O’Farrell, 2003; Nappi et al., 2000).

The above studies suggest that Wolbachia has the abilityto escape the host immune system and to form stablesymbiotic associations in both insects and nematodes. Thecurrently available data indicate that the bacterium doesnot actively suppress host immune response. In addition, itseems that certain Wolbachia surface components like thenon-LPS-like Limulus amebocyte lysate (LAL)-reactiveproteins and the major surface protein, WSP, are able totrigger host immune responses. Evolutionary studiesshowed that the wsp gene is under strong positive selection,suggesting the WSP protein may play an important role inhost-Wolbachia interactions (Jiggins et al., 2002).

Host-derived vacuole: the ideal cellularenvironment for Wolbachia?

Being an obligate intracellular bacterial symbiont,Wolbachia must coordinate a range of functions with itshost that include movement and partitioning during celldivision, nutrient acquisition, removal of waste products,regulation of its replication rate and, most importantly,avoidance of immune detection (McGraw & O’Neill,2004). Electron microscopy studies have shown thatWolbachia is a coccid to bacilliform organism which issurrounded by an inner plasma membrane and an outer cellwall. It is located in the cytoplasm of host cells within a

vacuole of host origin (Louis & Nigro, 1989; Wright et al.,1978; Yen & Barr, 1973). The extent to which the host-derived vacuole is modified by Wolbachia and the natureof these modifications are currently unknown (McGraw &O’Neill, 2004). The host-derived vacuole probably playsan important role for the interactions of Wolbachia with thehost cell. The nature of these interactions is also unknown.However, it has been shown that Wolbachia have theunique ability to interact with the host cytoskeleton duringoogenesis and early embryogenesis (Callaini et al., 1994;Ferree et al., 2005; O’Neill & Karr, 1990; Veneti et al.,2004).

It is known that bacteria are initially sequestered intophagosomes (endosome-like vacuoles). These phagosomesnormally mature through a dynamic interaction withendosomal compartments. This interaction includes a se-ries of maturation steps as well as many budding and fu-sion events, which ultimately end with lysosome fusionand the formation of the phagolysosome. This promotesdeath and degradation of most microbes and provides ahost defence against intracellular pathogens (Tjelle et al.,2000; Zerial & McBride, 2001). However, intracellularbacteria have evolved a variety of sophisticated mecha-nisms to invade eukaryotic cells, to establish themselvesand to evade degradation (Hackstadt, 2000; Meresse et al.,1999): (i) human pathogens such as Actinobacillusactinomycetemcomitans, Listeria monocytogenes, Rick-ettsia and Shigella escape from the phagocytotic vacuoleand enter into the cytoplasm (Gouin et al., 1999; Meyer etal., 1999); (ii) other intracellular pathogens includingMycobacterium tuberculosis and Salmonella entericaserovar Typhimurium survive and thrive within modifiedendosome-like vacuoles by preventing the fusion eventsrequired for their maturation (Dorn et al., 2002; Garcia-delPortillo & Finlay, 1995; Meresse et al., 1999); (iii) a thirdgroup of bacteria that includes Brucella abortus, Legionellapneumophila and Porphyromonas gingivalis “adopt” theautophagic pathway during their intracellular life cycle(Dorn et al., 2002); and (iv) there are also intracellularbacteria such as Coxiella burnetii, which transit throughthe normal endo/phagocytic pathway but actively interactwith autophagosomes at early times after infection. Thisintersection with the autophagic pathway delays fusionwith the lysosomal compartment, possibly favouring intra-cellular differentiation and survival of the bacteria (Romanoet al., 2006).

It can therefore be expected that Wolbachia has evolvedthe necessary mechanisms to modify the vacuole it residesin, in order to avoid the degradation process in thephagolysosome; however, neither the mechanisms nor theactual vacuole modifications, which allow Wolbachia toescape lysis and degradation, are currently known. Future



investigations are required to address these importantquestions. The availability of several complete and partialgenome sequences of Wolbachia strains has already sug-gested potential mechanisms through which Wolbachiamight interact with their hosts (Foster et al., 2005; Salzberget al., 2005a; Salzberg et al., 2005b; Wu et al., 2004).

What does the Wolbachia genome tell us abouthost-Wolbachia interactions?

Since Wolbachia is exclusively localized in host cytoplasm,enclosed within a host vacuole, it is believed that thebacterium secretes effector molecules to manipulate thehost environment to facilitate its own persistence andreplication. Such a strategy has been described in othersystems. For example, L. pneumophila transduces a cis-acting signal to establish the replicative vacuole via a typeIV secretion system (T4SS) that is encoded by the icm/dot(intracellular multiplication/defect in organelle trafficking)complex (Joshi et al., 2001; Segal et al., 1999; Vogel et al.,1998). Virulent B. abortus also has a T4SS that is essentialfor the biogenesis of the autophagosomal-like vacuole andintracellular replication (Comerci et al., 2001). Other Gram-negative bacteria, such as Agrobacterium tumefaciens andBordetella pertussi, use T4SS to deliver virulence factorsinto the cytoplasm of host cells (Cascales & Christie, 2004;Weiss et al., 1993). Among α-proteobacteria, T4SS havebeen described in species of the genera Ehrlichia, Ana-plasma and Rickettsia, which are closely related toWolbachia (Andersson et al., 1998; Brayton et al., 2005;Collins et al., 2005). The available Wolbachia genomicinformation showed the presence of a functional T4SSarranged into two separate operons (Foster et al., 2005; Wuet al., 2004). Whether Wolbachia and its close relatives useT4SS for the biogenesis of the autophagosomal-like vacu-ole and intracellular replication like Brucella abortus does,awaits experimental proof.

The recent sequencing of the first Wolbachia genome(wMel strain) provided novel insights into the potentialmolecular mechanisms used by this symbiont to manipu-late its hosts (Wu et al., 2004). One interesting feature ofthis Wolbachia genome is the presence of an unusuallyhigh number of genes containing ankyrin repeat domains(at least 23 genes; Iturbe-Ormaetxe et al., 2005; Wu et al.,2004). This number is in itself astonishing, as most bacteriacode for none or at most a very few proteins with ankyrinrepeats, indicating a putative specific role for the Wolbachialife style. Ankyrin repeats, a tandem motif of around 33amino acids, are found mainly in eukaryotic proteins,where they are known to mediate protein-protein interac-tions (Sedgwick & Smerdon, 1999). It has been shown in

eukaryotic systems that ankyrin repeat-containing proteinsare involved in interactions with cytoskeleton proteins.Therefore, they may determine intracellular traffickingand localization (Beck et al., 1997; Hoock et al., 1997;Mosavi et al., 2004). They have also been shown to act astranscriptional and developmental regulators, as well asinhibitors and toxins. For example, the toxin of the blackwidow spider is an ankyrin repeat-containing protein(Kiyatkin et al., 1993). Interestingly, it was recently shownthat such ankyrin repeat-containing proteins encoded in thegenome of a polydnavirus are responsible for the suppres-sion of the host immune response (Kroemer & Webb,2005; Thoetkiattikul et al., 2005). The potential role of theWolbachia ankyrin repeat-containing proteins in host-Wolbachia interactions is currently under investigation inthe authors’ and other laboratories.

Wolbachia: A master manipulator of apoptosis?

Some Wolbachia strains, have evolved mutualistic symbi-otic associations, as is the case with the parasitoid waspAsobara tabida (Dedeine et al., 2003). The presence ofWolbachia is here obligatory for host oogenesis (Dedeineet al., 2001). In a recent and very elegant study, Pannebakerand colleagues (2007) showed that Wolbachia controlsprogrammed cell death processes in A. tabida. Apoptosis isan essential component of insect oogenesis, making thepresence of Wolbachia essential for the maturation ofoocytes (Pannebakker et al., 2007). It was shown thatcontrol of apoptosis of the nurse cells is the key mechanismby which Wolbachia regulates oogenesis in A. tabida.Apoptosis is an essential component of insect oogenesis.There are checkpoints during early and mid-oogenesis atwhich various environmental stress factors can trigger celldeath in egg chambers (McCall, 2004). The authors clearlyshowed that in A. tabida, the presence of Wolbachia isessential to progress egg chambers past the mid-oogenesischeck-point by preventing apoptosis of nurse cells(Pannebakker et al., 2007). Programmed cell death is a hostregulatory feature typically targeted by pathogens. Thisfinding is of paramount importance, since it provides anovel and alternative explanation for the evolution of hostdependence and the evolutionary transition from faculta-tive parasitism towards obligate mutualism. It suggeststhat mutualism and parasitism can be controlled by identi-cal cellular pathways, which implies that shifting betweenthese two different lifestyles can be relatively easy(Pannebakker et al., 2007). In a different system, Bazzocchiand colleagues (2006) showed that the major surfaceprotein of Wolbachia, WSP, is involved in the inhibition ofapoptosis in human neutrophils (Bazzocchi et al., 2007). In



addition, they showed that this effect is associated with areduced activity of caspase-3 activity in WSP-treatedsamples. We are tempted to speculate that WSP may be thefactor which inhibits apoptosis in the A. tabida-Wolbachiasymbiotic association. Taken together, these data suggestthat Wolbachia has evolved sophisticated molecular path-ways to manipulate host apoptosis in order to achieveestablishment in host cells.

Concluding remarks and future advances

The past 10 years have seen a considerable increase in ourknowledge of Wolbachia biology and Wolbachia-hostinteraction. We now know a great deal about the phenom-enology of Wolbachia-induced reproductive alterations inarthropods as well as the phylogenetic and evolutionaryrelationships of many different strains (Bourtzis & Miller,2003; Bourtzis & Miller, 2006). There is significant knowl-edge about the role of Wolbachia in fertility and survival offilarial nematodes and in the induction of immune re-sponses associated with inflammatory-mediated patho-genesis and adverse reactions of nematode carriers to anti-filarial drugs (Taylor et al., 2005; Turner et al., 2006).However, there is only scant knowledge about host-Wolbachia interactions (McGraw & O’Neill, 2004).

The recent sequencing and annotation of the genome oftwo Wolbachia strains, wMel and wBm, which infect theinsect Drosophila melanogaster and the filarial nematodeBrugia malayi respectively, has provided us with a wealthof information for the elucidation of host-Wolbachia sym-biosis on the molecular level. The genome of Wolbachiacontains a functional T4SS, a high number of ankyrinrepeat-containing genes and a great variety of surface and/or secreted proteins; all of which are considered importantfactors in the molecular interaction pathways betweensymbiont and host. Unfortunately, there is so far essen-tially no hint about their potential role. For example, theeffector molecules of T4SS are still to be identified. Theankyrin repeat-containing proteins are known to mediateprotein-protein interactions; however, no candidate inter-acting partner for any of them has been identified as yet.There is a wealth of potentially surface and/or secretedproteins in the genome of Wolbachia; however, theirbiological role is still unknown, although we now knowthat WSP, the major surface protein of Wolbachia, prob-ably plays an important role in the establishment of sym-biosis (Bazzocchi et al., 2007; Pannebakker et al., 2007).

Progress during the last few years in genomics, proteomicsand post-genomics has transformed the way we approachbiological questions. Until very recently, unculturablemicro-organisms, which include several important

pathogens, parasites and symbionts, were not amenable tomolecular and genetic study. With the advent of wholegenome analysis, it is now possible to begin decipheringthe biology of fastidious obligatory intracellular bacteria.The genomes of Wolbachia and model host species, suchas Drosophila, are available, as are proteomic and post-genomics tools. The major challenge over the next fewyears will thus be the identification of the partner mol-ecules involved in Wolbachia-host interactions, includingthe elucidation of how it manages to be “invisible” to thehost immune system.

Acknowledgments

Kostas Bourtzis is grateful to the European Union, theInternational Atomic Energy Agency, the Greek Secre-tariat for Research and Technology, the Greek Ministry ofEducation, the Empirikeion Foundation, and the Univer-sity of Ioannina, which have supported the research fromhis laboratory. The authors also thank Stefan Oehler for hiscomments on a previous version of this article.

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Accepted March 1, 2007

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Wolbachia symbiosis and insect immune response