Wolbachia filarial interactions

Microreview

Wolbachia filarial interactions

Mark J. Taylor,* Denis Voronin, Kelly L. Johnstonand Louise FordFilariasis Research Group, Parasitology Department,Liverpool School of Tropical Medicine, Pembroke Place,Liverpool L3 5QA, UK.

Summary

Wolbachia pipientis is a widespread intracellularbacterial symbiont of arthropods and is common ininsects. One of their more exotic and unexpectedhosts is the filarial nematodes, notable for theparasites responsible for onchocerciasis (riverblindness), lymphatic filariasis (elephantiasis) anddirofilariasis (heartworm). Wolbachia are onlypresent in a subgroup of the filarial nematodes anddo not extend to other groups of nematodes eitherparasitic or free-living. In the medically and veteri-nary important species that host Wolbachia, thesymbiont has become an essential partner to keybiological processes in the life of the nematode tothe point where antibiotic elimination of the bacte-ria leads to a potent and effective anti-filarial drugtreatment. We review the cellular and molecularbasis of Wolbachia filarial interactions and high-light the key processes provided by the endosym-biont upon which the nematodes have becomeentirely dependent. This dependency is primarilyrestricted to periods of the lifecycle with heavymetabolic demands including growth and develop-ment of larval stages and embryogenesis in theadult female. Also, the longevity of filarial parasitesis compromised following depletion of the symbi-ont, which for the first time has delivered a safeand effective treatment to kill adult parasites withantibiotics.

Wolbachia and filarial nematodes

All filarial nematodes of medical and veterinary impor-tance rely on Wolbachia symbiosis, with the exception ofLoa loa (Taylor et al., 2005a). The species responsible for

lymphatic filariasis (Wuchereria bancrofti and Brugiamalayi), onchocerciasis (Onchocerca volvulus) and heart-worm (Dirofilaria immitis) have been shown to host Wol-bachia in all lifecycle stages and geographical isolatesand show close co-evolutionary histories with their nema-tode host consistent with their mutualistic dependency. Arecent study of a broader range of filarial nematodegroups has shown that the symbiotic relationship is appar-ently absent from some individuals and species bothwithin the Onchocercidae and in more ancestral groupsinfecting lizards, amphibians and birds (Ferri et al., 2011).Taken with the recent genomic analysis which highlightsdifferent roles for Wolbachia within its symbiosis withfilarial nematodes (Darby et al., 2012), this suggests abroader range of symbiotic relationships exists betweendifferent Wolbachia clades.

Intracellular niche and bacterial structure

Wolbachia are obligate endosymbiotic a-proteobacteriaclosely related to other rickettsial organisms such as Ehr-lichia, Anaplasma and Rickettsia. They are pleiomorphic,ranging from 0.2 to 4 mm in size, and reside in an obligateintracellular niche, within host-derived vacuoles, through-out the syncytial hypodermal cord cells (Kozek, 1977;Taylor et al., 1999) and in the ovarian tissues, oogonia,oocytes and developing embryos within the uterus(Kozek, 1977; Taylor et al., 1999) (Figs 1 and 2). Indi-vidual bacteria reside in a single vacuole and during divi-sion the vacuole expands along the bacteria to form twoseparate vacuoles. During rapid periods of growth, clus-ters of Wolbachia are observed in a single vacuole. Thesebacteria are usually undergoing division and can assem-ble three to six bacteria per vacuole. This phenomenonappears unique to Wolbachia from filarial nematodesand is mostly observed in young adult worms (Fig. 1).Wolbachia generally have a moderately electron-densematrix with distinct ribosomes and DNA. Some bacteriahave a spore-like structure with very high electron densityof the matrix, smaller size (0.5 mm), and are surroundedby a multi-membrane envelope (Fig. 1).

Due to the obligate intracellular niche and lack of arequirement to infect cells, Wolbachia have lost much ofthe cell wall structures common to more typical Gram-negative bacteria. Peptidoglycan has not been reliably

Received 9 October, 2012; revised 26 November, 2012; accepted26 November, 2012. *For correspondence. E-mail [email protected]; Tel. (+44) 151 705 3112; Fax (+44) 151 705 3371.

Cellular Microbiology (2013) 15(4), 520–526 doi:10.1111/cmi.12084First published online 24 December 2012

© 2012 Blackwell Publishing Ltd

cellular microbiology

detected, and while the Wolbachia (wBm) genome pre-dicts all the genes required for biosynthesis of lipid II, themonomer that will be polymerized into peptidoglycan(Foster et al., 2005), it also reveals that Wolbachia lackthe transglycosylases that catalyse the formation of linearglycan chains of peptidoglycan (Foster et al., 2005; Hen-richfreise et al., 2009). However, as shown in Chlamydia,lipid II biosynthesis does appear to be biologically impor-tant, and in Wolbachia this pathway has been shown to besensitive to fosfomycin, an antibiotic that blocks synthesis

of lipid II (Henrichfreise et al., 2009). Wolbachia may havea very atypical cell wall structure, which may providean anchor point between the inner and outer membranesfor outer membrane proteins; alternatively, it has beenproposed that lipid II may be involved in cell division(Henrichfreise et al., 2009). Lipoproteins are essentialstructural and functional components of bacteria so arelikely to be crucial in Wolbachia structure; analysis of thewBm genome indicates that the Wolbachia lipoproteinsare diacylated since the necessary lgt and lspA genes are

Fig. 1. Ultrastructural localization of Wolbachia within their filarial host.A–C. Population dynamics of increasing bacterial numbers in the lateral cord cells of adult worms. Bacteria are distributed mostly on theinner side of the hypodermal cells oriented towards the pseudocoelom. Localization of bacteria (highlighted in green) in the cytoplasm ofhypodermal cord and cells lining the uterus in a young adult female (A).D and E. Wolbachia (green) in developing eggs (D) and a few Wolbachia (black asterisks) within a microfilaria (E).F–H. Morphological features of Wolbachia in Brugia malayi. A single bacteria (black asterisks) each within a host vacuole (F); Wolbachia(white asterisk) with an electron-dense bacterial matrix and surrounded by a multi-membrane envelope, representing the morphology ofspore-like structures (G); and several Wolbachia (black asterisks) are clustered in a shared single vacuole (H).I and J. Specific localization of Wolbachia in hypodermal cells, which are oriented towards the pseudocoelom and close to a secretory canal.Short arrows show physical contact between the hypodermal cell and basal membrane of the uterus; long arrows represent the borderbetween two hypodermal cells. Scale bar = 5 mm (A–E) and 1 mm (F–J).

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© 2012 Blackwell Publishing Ltd, Cellular Microbiology, 15, 520–526

predicted, while the third gene responsible for triacylation(lnt) appears absent (Turner et al., 2009). Wolbachiapeptidoglycan-associated lipoprotein (wBmPAL), which isabundantly expressed on the cell membranes, and is apotent stimulator of the innate and adaptive inflammatoryresponses associated with filarial pathogenesis, may playan important role in membrane structural integrity (Turneret al., 2009). Both the Wolbachia lipid II and lipoproteinpathways are being pursued as anti-filarial targets sincethey have been shown to be sensitive to specific inhibition(Henrichfreise et al., 2009; Johnston et al., 2010).

Population biology and dynamics

In B. malayi, Wolbachia have been detected in all lifecy-cle stages, individual adult worms and isolates from dif-ferent endemic regions (Kozek, 1977; Taylor et al., 1999;Fenn and Blaxter, 2004; McGarry et al., 2004). Unlike thesituation in most arthropods, the presence of Wolbachiawithin a filarial species generally appears fixed with allindividual nematodes infected (Bandi et al., 1998; Tayloret al., 2005a), suggestive of a mutualistic relationship.However, the level of infection varies considerably duringfilarial development, illustrating dynamic growth of thebacterial population at certain stages of the nematodeslifecycle (Fenn and Blaxter, 2004; McGarry et al., 2004;Fischer et al., 2011). Wolbachia numbers are at theirlowest (~ 70–100) and constant in microfilariae (Mf) and

the insect-borne larval stages (L2 and L3). Shortly aftertransmission to the mammalian host a dramatic increasein the bacterial population occurs (McGarry et al., 2004)and this appears to play an essential role in larval devel-opment and establishment, as demonstrated by thearrested larval growth and development in response toantibiotic treatment (reviewed in Taylor and Hoerauf,2001; Taylor et al., 2012). This rapid multiplication contin-ues throughout L4 development, so that the major Wol-bachia population growth occurs within the first monthfollowing infection of the mammalian host (Fenn andBlaxter, 2004; McGarry et al., 2004; Fischer et al., 2011).This proliferation serves to populate the hypodermalcords of adult worms with ~ 2–5 ¥ 105 bacteria in adultmale and 4–14 ¥ 106 in female worms (McGarry et al.,2004). In female worms, Wolbachia numbers continue toincrease as the worms mature and the ovary and embry-onic stages become infected (McGarry et al., 2004), sup-portive of their role in oogenesis and/or embryogenesisand consistent with the observed effects of antibiotictreatment on these processes. While the studiesdescribed above demonstrate dynamic changes in Wol-bachia load in filarial lifecycle stages, there also appearsto be considerable variation in bacterial numbers of indi-vidual worms (McGarry et al., 2004). Higher levels ofWolbachia infection within a worm may potentially conferselective advantages in terms of worm development orfecundity.

The periods of rapid growth of Wolbachia coincidingwith larval and embryonic growth and developmentsuggests that the fundamental role of Wolbachia is theprovision of nutrients or metabolites essential for thesemetabolically demanding processes. This accounts for therapid arrestment of larval growth and embryogenesisseen soon after antibiotic depletion. Nevertheless, otherbiological processes become compromised much laterfollowing loss of the symbiont including the partial block intransmission of the parasite in the insect vector (Alberset al., 2012) and the macrofilaricidal effects which occurbetween 12 and 24 months post treatment (Taylor et al.,2010).

Autophagy regulates Wolbachia populations

In order to understand the process by which the hostnematode regulates the population growth of Wolbachiaat a sufficient level to maintain the symbiosis, yet to avoidfitness costs or the pathological consequences of bacte-rial overgrowth, the role of autophagy, a conservedintracellular defence mechanism and regulator of cellhomeostasis, was investigated (Voronin et al., 2012). Acti-vation of autophagy coincided with the onset of rapidbacterial growth and expansion, which shows that in spiteof their mutualistic association, the nematode’s immune

Fig. 2. Association of autophagy membrane protein (ATG8a,green) and Wolbachia (small red dots) in the hypodermal cord cellsof the filarial nematode Brugia malayi (large red structures arenematode nuclei). Activation and recognition of Wolbachia byautophagy occurs during the periods of rapid bacterial expansionand growth. Image taken using 63¥ objective.

522 M. J. Taylor, D. Voronin, K. L. Johnston and L. Ford


system recognizes Wolbachia as a ‘pathogen’. Geneticand chemical modulation of autophagy activation orsuppression resulted in a corresponding decrease orincrease in bacterial populations. Indeed, activation ofautophagy produced a bactericidal activity of similar mag-nitude to antibiotic elimination, which could be exploitedas a novel mode of action for bactericidal drugs. In orderto maintain its population, Wolbachia must evade or cir-cumvent autophagic destruction, which may be achievedby a balance between the rate of bacterial growth and therate of autophagic elimination, possibly through modifica-tion or mimicry of components of the autophagy pathway(Voronin et al., 2012).

Cell lineage tropism and route of transmission

Work investigating the localization of Wolbachia duringembryogenesis has given an insight into the cell lineageof B. malayi and the journey Wolbachia take to result inthe restricted tissue tropism observed in adult worms.Using the completely defined lineage of Caenorhabditiselegans as a framework and whole mount immunofluo-rescent techniques, Landmann et al. (2010) followed Wol-bachia through fertilization, the initial zygotic divisions andembryogenesis, as well as examining adult worms. It wasobserved that Wolbachia become enriched at the poste-rior pole of the egg following fertilization as early as thepronuclei migration step. In the early zygotic divisions,Wolbachia maintain this asymmetric localization at theposterior which allows them to first preferentially segre-gate into the P1 blastomere and, following its division intoEMS and P2 blastomeres, into the posterior P2. Themajority of the Wolbachia then segregate into the C blas-tomere, which eventually gives rise to their presence inhypodermal cord cells. Furthermore it was observed thatnot only did the Wolbachia titres throughout the hypoder-mal cords vary between worms, confirming previous workconducted using qPCR (McGarry et al., 2004), but theyalso varied within an individual worm. In some instances,only one of the cords was infected, while in others onlyhalf of each cord contained Wolbachia. It was suggestedthat Wolbachia spreads throughout the hypodermal cordduring fusion of the individual hypodermal cord cells toproduce the syncytium (Landmann et al., 2010).

The smaller number of Wolbachia that do not follow theroute to the hypodermal cords was also followed. Thosethat initially reside in the AB blastomere instead of P1become diluted out without replicating, but the final des-tination of the minority of Wolbachia that end up in the P3blastomere instead of C remains unclear. Wolbachia arevertically transmitted from mother to offspring and there-fore a cell lineage-specific pattern of segregation from theP3 blastomere, which leads to the Z2/Z3 germline cells,would ensure that Wolbachia are thereby transmitted to

the next generation. However, it was observed that not allembryos contained Wolbachia in their P4 blastomeres,indicating that there is a loss at some point along theP3-P4-Z2/Z3 route in some cases. This drew the authorsto propose that those embryos with Wolbachia in their P4blastomeres would become female and male wormsrespectively (Landmann et al., 2010). However, a recentstudy demonstrated that Wolbachia are absent from bothmale and female reproductive tissues from at least third-stage larvae to young adult worms (Fischer et al., 2011)indicating that the germline is not infected from the outset.In young female adult worms, the germinative zones inthe ovaries, which contain the oogonia, were found to befree of Wolbachia. Over a period of 3 weeks following theL4 to L5 molt, these germinative zones become populatedwith Wolbachia and it was suggested that this is a result ofinvading bacteria from the adjacent hypodermal cords.Further investigations by Landmann et al. (2012), exam-ining late embryogenesis through to adult, have demon-strated Wolbachia crossing the basal membrane of thecords in both male and female L4 (8–10 days post infec-tion) and subsequently invading the distal gonad of thefemale worms, thereby confirming that invasion doesoccur. Overall it appears that Wolbachia reach the femalereproductive tissue to coincide with oocyte developmentto ensure transmission to the next generation, althoughthe precise mechanisms used by Wolbachia to crosstissue zones and invade the germline remain to bedefined.

Molecular and cellular interactions betweenWolbachia and their filarial hosts

The sequencing of the genomes of both Wolbachia (wBm)(Foster et al., 2005) and its B. malayi host (Ghedin et al.,2007) has provided genomic clues and allowed for an insilico comparison of the biosynthetic capacities of thenematode and its endosymbiont, to highlight potentialcomplementing metabolic capabilities that might contrib-ute to their mutualistic coexistence. This revealed that thenematodes have become dependent on their endosymbi-onts for a diverse range of biological processes (reviewedin Slatko et al., 2010), such as synthesis of metabolitesincluding haem, riboflavin, flavin adenine dinucleotide andnucleotides, which are provided by Wolbachia to thenematode, which cannot synthesize these molecules denovo (Foster et al., 2005; Ghedin et al., 2007).

Recently molecular analysis of Wolbachia from thecattle filarial nematode, Onchocerca ochengi (wOo), byDarby et al. (2012), involving sequencing of the genomeand analysis of the transcriptome, has revealed certaindissimilarities between wOo and wBm in terms of meta-bolic capability, the density of insertion sequences and therange of repeat motif-containing proteins (Darby et al.,



2012). This work highlights potential roles for wOo in bothenergy production in the somatic tissue, where Wolbachiamay have a mitochondrion-like function generating ATPfor the host, and modulation of the mammalian immuneresponse, but interestingly does not provide strongsupport for the provisioning of vitamins or cofactors by thisstrain (Darby et al., 2012), as shown in wBm (Foster et al.,2005).

Intriguingly, evidence of lateral gene transfer (LGT) ofWolbachia DNA into the nematode genome has beenshown in Wolbachia-infected filariae (Dunning Hotoppet al., 2007; Slatko et al., 2010), and fragments of Wol-bachia genes have also been identified in the nematodenuclear genomes of filarial species free of Wolbachiaindicating LGT occurred prior to the secondary loss ofWolbachia (McNulty et al., 2010). It remains to be dem-onstrated whether these LGT events have complementedthe nematode genome with essential Wolbachia genes oralternatively represent genetic ‘ghosts’ of a parasitic rela-tionship in the past.

In addition to highlighting metabolites provided by Wol-bachia to its host, comparisons of the genomes can alsoidentify metabolites provided to the endosymbiont bythe host nematode as incomplete biochemical pathwaysin Wolbachia suggest a metabolic dependency on thenematode host. Wolbachia appear unable to perform denovo synthesis of several vitamins and cofactors such ascoenzyme A, nicotinamide adenine dinucleotide, biotin,ubiquinone, folate, lipoic acid and pyridoxal phosphate(Foster et al., 2005).

Many filarial genes show expression changes inresponse to Wolbachia clearance following tetracyclinetreatment (Ghedin et al., 2009; Strubing et al., 2010;Rao et al., 2012). Several nuclearly encoded pathwaytranscripts were increased in response to elimination ofWolbachia in adult B. malayi, including those involved inprotein synthesis and the stress response (Ghedin et al.,2009), and this has recently been shown to involve spe-cific regulatory elements present in the promoters of thesegenes (Liu et al., 2011). Changes in the expression levelsof specific filarial genes such as phosphate permease(Heider et al., 2006) and heat shock protein 60 (Pfarret al., 2008) following Wolbachia clearance have alsobeen shown, suggesting that the encoded proteins areinvolved at some level. However, whether these changesare specific to a disruption in the balance of theWolbachia–host interaction or due to indirect effects suchas antibiotic toxicity or a nematode stress response isunclear and needs to be further investigated.

Although the molecular details of the Wolbachia proc-esses essential for the symbiosis remain to be defined,studies on the cellular consequences of symbiont elimi-nation have provided an important insight into the cellularmechanism at the basis of the symbiotic relationship.

Soon after antibiotic elimination of the bacteria extensiveapoptosis in the adult germline, and in the somatic cells ofthe embryos, microfilariae and L4 occurs (Landmannet al., 2011). Apoptosis extends to uninfected cells, sug-gesting an indirect provision of products from the hypo-dermal population is required to prevent cells fromundergoing cell death. This cellular mechanism does notextend to all somatic cells, including those of the hypo-dermal cord cells, where the bacteria reside, although thecytoskeletal arrangement is disrupted. The pattern ofapoptosis activation correlates closely with the stagesmost vulnerable to antibiotic depletion and provides amechanism to account for the rapid anti-filarial effects ofantibiotic treatment.

Exploitation of the Wolbachia filarial relationship asa therapeutic target

The symbiotic relationship between Wolbachia and theirhost nematode has been exploited with great successusing doxycycline to treat filariae-infected individuals inseveral field studies (Taylor et al., 2005b; Debrah et al.,2006; 2007; Hoerauf et al., 2008; 2009; Supali et al.,2008; Mand et al., 2009; Turner et al., 2010). A 4–6 weekcourse of doxycycline leads to permanent sterilization offemale worms and sustained loss of microfilariae with theeventual death of the adult worms, providing superiorefficacy to existing anti-filarial drugs and the added benefitof improvements in filarial pathology (Taylor et al., 2010).However, the known contraindications of doxycycline andthe logistical constraints which prevent its widespread usein mass drug administration (MDA) control programmeshave prompted the formation of the A·WOL consortiumwhich aims to overcome these obstacles by discoveringnew anti-Wolbachia drugs (http://www.a-wol.com/).

The A·WOL screening strategy uses a pipeline ofapproaches optimized to identify and validate anti-Wolbachia compounds by screening of both focused anti-infective and diversity-based libraries of existing andnovel drugs and natural products. Early in the consorti-um’s inception, a cell-based Wolbachia screening assaywas developed utilizing a Wolbachia-containing Aedesalbopictus cell line (C6/36 Wp) (Turner et al., 2006), in a96-well format, with a quantitative PCR (qPCR) readout toquantify the Wolbachia 16S rRNA gene copy numberfollowing treatment (Johnston et al., 2010). This validatedassay was first used to screen 2664 drugs of the humanpharmacopoeia for potential repurposing and identified121 hits, 70 of which were orally available thus satisfyingthe target product profile defined by A·WOL. The hitscrossed over several drug classes and several have pro-gressed further down the screening pipeline into in vitronematode assays and in vivo screening models. As thecompounds tested in this screen were already registered



for use in humans, this screening strategy represents anopportunity to quickly develop an alternative chemo-therapy for filariasis.

As well as registered drugs, the A·WOL cell-basedscreen has also been used to screen focused drug librar-ies selected based on known and bio-informaticallypredicted essential gene targets (Holman et al., 2009).Focused anti-infective library screening has, thus far,involved A·WOL in vitro screening of 7144 novel com-pounds from eight chemical libraries. To date this hasgenerated 460 diverse hit compounds, a number of whichhave progressed further into the screening funnel.Notably, the ability to identify hit compounds from thesefocused libraries, which are effective at reducing Wol-bachia load and have improved efficacy over doxycycline,is highly supportive of the long-term goal to identifyA·WOL new chemical entities (NCEs).

In order to expand the capacity to discover NCEs,diversity-based library screening has also been a part ofA·WOL’s activities. Three diversity-based chemical librar-ies consisting in total of 60 000+ compounds have enteredthe primary cell-based assay screen. Hits from theselibraries will be tested for narrow-spectrum activity againstWolbachia in order to produce targeted treatments that donot overlap with other anti-bacterial treatment domains.

Overall, the A·WOL consortium has made significantprogress both in optimizing current regimes of existinganti-Wolbachia treatments and in discovering potentialnew therapies through screening. It is hoped that thesediscoveries will ultimately lead to the delivery of an alter-native complementary strategy, which can achieve thegoal of eliminating lymphatic filariasis and onchocerciasis.

Concluding remarks

As discussed in this review, it is clear that Wolbachia playan obligatory role in the biology of medically importantfilarial nematodes involving a number of cellular andmolecular interactions. The essentiality of the symbiosis isprimarily associated with periods of growth and develop-ment with high metabolic demands, but extends to otherbiological processes including transmission through thevector and longevity of the adult worms. Further investi-gation into the nature of these interactions should serve toenhance our ability to exploit this dependency and poten-tially lead to novel treatments for lymphatic filariasis andonchocerciasis.

Acknowledgements

We thank the Bill and Melinda Gates Foundation for their supportof the A·WOL consortium through a grant awarded to the Liver-pool School of Tropical Medicine.

The authors have no conflict of interest.

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Wolbachia filarial interactions

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