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. Author manuscript; available in PMC: 2019 Apr 27.
Published in final edited form as: Environ Microbiol. 2017 Oct 27:10.1111/1462-2920.13964. doi: 10.1111/1462-2920.13964

Mi Casa es Su Casa: How an Intracellular Symbiont Manipulates Host Biology

Tamanash Bhattacharya 1, Irene LG Newton 1
PMCID: PMC5924462  NIHMSID: NIHMS928624  PMID: 29076641

Summary

Wolbachia pipientis, the most common intracellular infection on the planet, infects 40% of insects as well as nematodes, isopods, and arachnids. Wolbachia are obligately intracellular and challenging to study; there are no genetic tools for manipulating Wolbachia nor can they be cultured outside of host cells. Despite these roadblocks, the research community has defined a set of Wolbachia loci involved in host interaction: Wolbachia effectors. Through the use of Drosophila genetics, surrogate systems, and biochemistry, the field has begun to define the toolkit Wolbachia use for host manipulation. Below we review recent findings identifying these Wolbachia effectors and point to potential, as yet uncharacterized, links between known phenotypes induced by Wolbachia infection and it’s predicted effectors.

Introduction

Associations between symbiotic intracellular bacteria and insect hosts are widely prevalent and diverse. Examples range from the obligate mutualistic symbionts observed in aphids (Buchnera aphidicola), sharpshooters (Baumannia cicadellinicola), tsetse flies (Wigglesworthia glossinidia), and carpenter ants (Blochmannia species), to those mutualistic symbionts perceived to be facultative in nature (such as Hamiltonia defensa or Serratia symbiotica and aphids). Still other symbionts are less well defined with regards to their effects host fitness and include the bacterial members of Rickettsiales and Spiroplasma genera, which infect many different insects, and are viewed as either reproductive parasites (Moran et al., 2008) or protective mutualists (Hedges et al., 2008). As these symbionts, regardless of their fitness effect on the host, must live and replicate within host cells and they have evolved mechanisms to allow for their survival and proliferation within complex host microenvironments. In pathogenic systems, well-known examples of cell invasion and colonization strategies include those employed by Salmonella typhimurium, Yersinia pestis, and other enteric pathogens, which involve mechanisms that allow attachment and internalization into the host cell by inducing phagocytosis (Cossart and Sansonetti, 2004). Another example can be found in Listera, where once the pathogen has successfully invaded a host cell, Listeria encoded Listeriolysin O proteins allow the bacterium to escape the vacuole and spread to other host cells (Gedde et al., 2000). Bacteria such as Shigella flexneri and Listeria monocytogenes secrete effector proteins in the host cytoplasm to directly bind and modify the host actin cytoskeleton, allowing motility within the cell and cell-to-cell spread (Goldberg and Theriot, 1995; Smith et al., 1995). However, much less is known about the mechanisms used by symbionts for host manipulation, although effectors have been identified in a few symbiotic systems (Dale et al., 2002; Costa et al., 2012; Rice et al., 2017).

The α-proteobacterium Wolbachia pipientis is arguably the most prevalent intracellular invertebrate infection on the planet, infecting as many as 40% of all insect species alone (Zug and Hammerstein, 2012). Wolbachia are members of a monophyletic lineage that includes genera such as Rickettsia, Anaplasma, and Ehrlichia, all of which are characteristically defined as obligate intracellular bacteria (Correa and Ballard, 2016). Wolbachia are primarily maternally transmitted, although rare instances of horizontal transmission are evident based on incongruent phylogenies between hosts and symbionts (Heath et al., 1999; Vavre et al., 1999). However, unlike the other genera Wolbachia do not infect mammals, and infection with Wolbachia has long been associated with reproductive manipulations of insect hosts (Werren et al., 2008). Through the manipulation of host reproduction, Wolbachia benefit infected matrilines, facilitating its spread in a population (Correa and Ballard, 2016).

Wolbachia’s ability to alter host phenotype has recently attracted considerable interest for several reasons. From an evolutionary perspective, Wolbachia-associated phenotypes may play roles in rapid speciation, and given its widespread distribution, Wolbachia may have ample opportunity to influence insect evolution (Bordenstein et al., 2001). Wolbachia infections are also medically relevant. For example, Wolbachia are obligate symbionts for filarial nematodes and are currently recognized as potential chemotherapeutic drug targets (Slatko et al., 2010). In insect hosts, the wide repertoire of phenotypic changes induced by Wolbachia has led to several successful biocontrol efforts. For example, mosquitoes transinfected with a virus-blocking Wolbachia strain are being used to prevent transmission of several vector-borne human diseases across the globe (Hoffmann et al., 2011; Walker et al., 2011).

Although Wolbachia is such a widespread symbiont, we are just beginning to identify the mechanisms of it’s host-symbiont interaction, largely owing to the fact that Wolbachia’s obligately intracellular lifestyle makes it a difficult organism to study. Indeed, for many years the distribution and population biology of Wolbachia infections has been the focus of scientific studies in the field, leaving a substantial gap in our understanding of the molecular determinants underlying Wolbachia-host symbioses (Serbus et al., 2008). However, advances in biochemical and molecular methods have allowed researchers to begin to define the Wolbachia infection toolbox (Newton, 2017). For example, leveraging the genetic tools in Drosophila melanogaster, components of host cell biology have been identified as important for Wolbachia colonization. In Drosophila, Wolbachia heavily infect the reproductive tract, colonizing a region of the developing oocyte, the germarium, which allows Wolbachia to infect the developing germ line (Figure 1) (Serbus et al., 2008). Although different Wolbachia strains invade the germ line at different stages of oogenesis, all Wolbachia have adopted the strategy of host germ line invasion in order to efficiently infect the next generation (Genty et al., 2014). The mechanisms by which Wolbachia target this tissue and persist in the host are not well defined. However, it is likely that Wolbachia use a Type IV Secretion System (T4SS) to modify host cell processes during infection.

Figure 1. Wolbachia colonizes the reproductive tract of Drosophila melanogaster.

Figure 1

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(A) The ovaries can be dissected to reveal individual ovarioles (denoted by bracket), each containing progressive stages of egg development found along the length of the ovariole (B). Wolbachia strain wMel colonize this tissue in D. melanogaster in a characteristic pattern, strongly infecting the germarium and localizing to the developing oocyte (shown in green). (C) Wolbachia is visualized in green within one ovariole using fluorescence in situ hybridization.

The Wolbachia Type IV Secretion System

The bacterial type IV secretion system (T4SS) is a membrane-associated, multiprotein complex used by bacteria to deliver cargo (in the form of DNA and/or protein) to another cell. Translocated protein substrates are referred to as effectors, because they can exert an effect on the host cell. These secretion systems are related to conjugation machines and can work in the transport of cargo between bacterial cells but are perhaps most famous for their involvement in inter kingdom transfer of DNA and protein (Cascales and Christie, 2003; Ding et al., 2003; Backert and Meyer, 2006; Whitaker et al., 2016). For example, Agrobacterium tumefasciens transforms plants through the secretion of T-DNA and virulence (vir) factors via T4SS (Pitzschke and Hirt, 2010).

A T4SS was first identified in Wolbachia pipientis strains via Southern blot using a Rickettsia prowazekii virD4 (the locus encoding the type IV coupling protein) probe and verified with genomic library sequencing (Masui et al., 2000). In wMel, the T4SS loci exist in multiple operons (Figure 2A) Masui et al. showed that the T4SS in various Wolbachia strains is encoded in multiple operons, as the virB8-11-virD4-wsp operon is nonsyntenic with a second T4SS locus, virB4. Subsequently, the structure of these two main operons (from virB8 to wsp and virB3-B6 including four unannotated loci) was identified and expression of VirB6 confirmed through immunofluorescence in vivo (Rances et al., 2008) and genomic sequencing of wMel coupled to PCR amplification of other strains (wRi and wAlbB), confirmed this genetic architecture is conserved across the Wolbachia genus (Wu et al., 2004). Outside of these previously identified operons in wMel are other components of the T4SS, including homologs of VirB8 (WD0817), VirB4 (WD1173), and multiple, syntenic, co-expressed, VirB2 homologs (WD1288, WD1289, WD1290) (Figure 2A,C). These loci are referred to as vir loci based on homology to T4SS components from known models (such as Agrobacterium) and otherwise, are referred to using the wMel locus tag. Interestingly, analysis of high-throughput RNAseq data for Wolbachia infected flies shows that some components of the apparatus (virB2 most strikingly) are upregulated during host pupation as well as in male adult flies (Figure 2C). Wolbachia is therefore cuing in on some developmental signal to regulate the expression of these loci. The pattern of expression of these T4SS genes, and their conservation throughout Wolbachia evolution (Pichon et al., 2009; Li and Carlow, 2012) suggests that this secretion machinery may be important; it is perhaps one mechanism by which Wolbachia manipulates host cell biology.

Figure 2. Wolbachia encode and express a complete type IV secretion system in several operons.

Figure 2

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(A) The three operons for Wolbachia T4SS components and (B) the predicted location of these proteins within the Wolbachia inner membrane (IM) and outer membrane (OM) as well as the host membrane (HM) (C) Wolbachia expresses these loci during host development from embryogenesis through to adult male and female flies. For each locus, the heat map illustrates no or low expression in red and high expression in blue. Expression data published in (Gutzwiller et al., 2015).

Wolbachia Secreted Substrates and Phenotypes Induced in the Host

Since the genome of strain wMel was first sequenced, researchers have identified potential Wolbachia effectors using bioinformatics analyses (Wu et al., 2004). The Wolbachia genome is noteworthy for the large number of encoded Ankyrin repeat domains, a 33-residue domain that was thought to be restricted to eukaryotes and predicted to be involved in a wide array of protein-protein interactions (Mosavi et al., 2004). Within the Wolbachia genus, the Ankyrin repeat domain-containing proteins are quite variable and may undergo domain shuffling (Iturbe-Ormaetxe et al., 2005; Siozios et al., 2013). However, as genomic sequencing became more commonplace, it came to light that many microbes encode Ankyrin repeats (Al-Khodor et al., 2010), and some of these are likely involved in host-microbe interactions. For example, in Legionella pneumophila, several secreted substrates encode Ankyrin repeat domains (Ensminger and Isberg, 2009).

However, domain architecture alone is not sufficient to identify secreted effectors in known model systems, as many effectors encode no known domains and are hypothetical proteins of unknown function (Collmer et al., 2002; Bruggemann et al., 2006). Four approaches have been recently employed to identify candidate Wolbachia effectors. In the first study, in order to identify proteins that interact with the secretion machinery, a heterologous secretion assay using the E. coli conjugation machinery combined with a chimeric type IV coupling protein (VirD4) identified three Wolbachia loci that encode proteins which are likely secreted: WD0636, WD0811, WD0830 (Whitaker et al., 2016). Another study used a growth assay in Drosophila S2 cells, identifying Wolbachia loci that kill the cells upon expression (Ote et al., 2016). That screen identified and characterized the wMel protein TomO, which is implicated in germ line stem cell proliferation. The third study identified Wolbachia loci associated with the reproductive manipulation known as cytoplasmic incompatibility (CI; identifying proteins found in mosquito sperm (Beckmann and Fallon, 2013) and loci associated with strains that cause CI (LePage et al., 2017)). Two loci (cifA and cifB) were identified and transgenically expressed in flies to recapitulate CI. Finally, bioinformatics was combined with a large-scale yeast growth assay to identify Wolbachia secreted effectors from strain wMel (Rice et al., 2017). As part of that work, 84 candidate effectors were screened and 14 predicted effectors were identified, some encoding Ankyrin repeat domains but many encoding other domains primarily known from eukaryotic systems (Table 1). One of these candidate effectors, WD0830, had been biochemically characterized as an actin bundler (Sheehan et al., 2016). However, this list does not include the reproductive cytoplasmic incompatibility loci (cifA and cifB) (LePage et al., 2014; Beckmann et al., 2017) nor tomO (Ote et al., 2016). Below we provide examples of Wolbachia-induced phenotypes most directly linked to specific Wolbachia effectors. We end by suggesting potential mechanistic links between domains found in candidate effectors (Table 1) and known phenotypes.

Table 1. Predicted and characterized Wolbachia effectors.

Wolbachia wMel locus number is shown as well as encoded Pfam domains.

Locus Pfam domains Reference
WalE1 Synuclein Sheehan et al., 2016
WD0033 PAZ Rice et al., 2017
WD0290 DUF2207 IncA PAZ Rice et al., 2017
WD0292 Ankyrin Rice et al., 2017
WD0338 -- Rice et al., 2017
WD0353 IncA Zip Rice et al., 2017
WD0385 Ankyrin Rice et al., 2017
WD0438 Ankyrin Rice et al., 2017
WD0462 IncA HAUS-augmin3 Rice et al., 2017
WD0465 DUF812 Rice et al., 2017
cifA VirJ, DUF3243, STE LePage et al., 2017
cifB PDDEXK, Ulp-1 LePage et al., 2017
WD0811 -- Rice et al., 2017
WD1171 DUF3534 Rice et al., 2017
TomO Ankyrin Ote et al., 2016
WD1223 -- Rice et al., 2017
WD1321 -- Rice et al., 2017
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A Wolbachia deubiquitinase responsible for cytoplasmic incompatibility in Drosophila melanogaster

The molecular mechanisms behind reproductive manipulations induced by Wolbachia have been a mystery for decades. The most common reproductive manipulation induced by Wolbachia is cytoplasmic incompatibility (CI), whereby infected offspring that result from infected males and uninfected females are not viable (Werren et al., 2008). CI is thought to result from the manipulation of sperm by Wolbachia, a modification that is only “rescued” by infected females. Hints as to the cell biological phenomenon behind CI have been explored in various systems. For example, when infected Nasonia males are crossed with uninfected females, unviable embryos result from a mistiming of nuclear envelope breakdown for the male and female pronuclei (Tram and Sullivan, 2002). Although the phenomenon of cytoplasmic incompatibly was originally characterized in the mid 20th century (Yen and Barr, 1971), the genes facilitating it – cifA and cifB – have only recently been identified (Beckmann et al., 2017; LePage et al., 2017). These two loci (WD0631 and WD0632 in wMel) are syntenic and associated with Phage WO; they are part of the “Eukaryotic Association Module” which is packaged into phage particles (Bordenstein and Bordenstein, 2016). When expressed in the fly, the cif loci induce phenotypes that are canonically associated with CI, such as chromatin bridging and regional mitotic failure (LePage et al., 2017). Most importantly, these defects are rescued when transgenic males expressing the CI loci are crossed with Wolbachia infected females (LePage et al., 2017). Indeed, expression of cifA or cifB alone increase CI and neither locus alone could rescue the CI-induced defects. With regards to function, the wMel and wPip homologs of cifB encode functional deubiquitinase domains (Beckmann et al., 2017), although the deubiquitinase (DUB) domain is not universally conserved among cifB homologs across the Wolbachia phylogeny (Lindsey et al., 2017). In Drosophila melanogaster, the DUB domain in the wPip cifB homolog is critical for the induction of cytoplasmic incompatibility; transgenic flies expressing a the construct lacking the catalytic residue do not exhibit a phenotype (Beckmann et al., 2017). The two Cif proteins seem to work synergistically, binding to each other in vitro (Beckmann et al., 2017), and increasing CI when expressed together in D. melanogaster (LePage et al., 2017). Although the cif loci are syntenic and this genomic organization is conserved broadly across the Wolbachia phylogeny, there is little evidence of co-transcription in wMel (Lindsey et al., 2017). Future mechanistic detail about Wolbachia cytoplasmic incompatibility will be revealed by answering the following questions: How do these proteins induce CI? How are they transferred to the host cytosol? What proteins rescue the phenotype? The answers to these questions, as well as the host target(s) for the Cif proteins, are unknown, but will reveal more about how Wolbachia targets and manipulates host reproduction.

A Wolbachia Ankyrin repeat containing protein rescues germ line stem cell proliferation in Drosophila

Despite the fact that Wolbachia possess small genomes of 0.9–1.5 Mbp, they encode a large number of proteins containing Ankyrin repeat domains (ANK) (23 in wMel and 60 in wPip) which, while common in eukaryotic and viral genomes, are rarely observed in bacterial genomes (Al-Khodor et al., 2010). Intracellular bacteria within the Rickettsiales such as Wolbachia, Rickettsia, Orientia, Anaplasma and Ehrlichia encode a large number of ANK-containing proteins, but only a few other bacterial species outside of this clade are known to encode such a large number of proteins containing ANK domains, namely Legionella pneumophila and Coxiella burnetii (Ensminger and Isberg, 2009; Al-Khodor et al., 2010; Chen et al., 2010). Considered to be one of the most flexible protein-protein interaction domains present in eukaryotes, ANK domains are involved in a wide array of cellular functions such as signal transduction, transcriptional regulation, cytoskeletal interactions, cell cycle regulations and tumor development (Al-Khodor et al., 2010). Given the array of functions in eukaryotes, it is hypothesized that Wolbachia ANK proteins are likely mediators of host manipulation.

Wolbachia is known to influence germ line stem cell proliferation (Fast et al., 2011) and rescues the fertility defect in sterile Sex lethal (Sxl) mutant Drosophila melanogaster (Starr and Cline, 2002; Sun and Cline, 2009). A protein underpinning these phenomena was recently identified as the ANK domain containing Wolbachia effector TomO (Ote et al., 2016). This candidate effector was discovered in a functional genomic screen for Wolbachia genes that induce deleterious effects to the host following ectopic expression in Drosophila cells. Subsequent analysis of native expression showed TomO localization in the host cytoplasm (Ote et al., 2016). Germ-line specific overexpression of TomO (locus WD1278) restored fertility in sterile Sex-lethal (Sxl) mutant females by enhancing germline stem cell (GSC) maintenance, perhaps explaining the above-mentioned observations in the literature. Interestingly, overexpressed tomO in germ cells was found to associate with cytoplasmic P-bodies, thought to be involved in mRNA degradation, alongside components like Me31B and Cup. A subsequent search for potential host RNAs enriched in the presence of TomO resulted in the identification of the nos transcript, which is a member of the translation repressor complex shown to support GSC maintenance by preventing differentiation (Wang and Lin, 2004). Binding between TomO and nos was also accompanied by elevated Nos protein expression, which the authors postulated was a result de-repression of nos mRNA translation. Therefore, through the secretion of this specific protein, TomO, Wolbachia modulates host gene expression and germ line stem cell proliferation.

Wolbachia’s vacuolar integrity

Wolbachia are obligately intracellular bacteria and live within host-derived vacuoles when in the cytoplasm. As such, they likely interact with host vesicle trafficking pathways and the host cytoskeleton to maintain vacuole integrity, promote division of the vacuole once the bacteria replicate, and allow for cell-to-cell transfer (Kumar and Valdivia, 2009; Creasey and Isberg, 2014). Wolbachia encode six proteins (WD0073, WD0224, WD0290, WD0353, WD0630 and WD0754) that share homology to Chlamydia tractomatis IncA domains (Rice et al., 2017). In Chlamydia, the IncA proteins play a role in the recruitment of Rab and Rab effectors (Rzomp et al., 2006) and may facilitate homotypic fusion of the Chlamydia vacuole (termed the inclusion) (Delevoye et al., 2008). It is not yet clear if Wolbachia homologs of C. tractomatis IncA perform similar functions, existing evidence regarding IncA’s membrane association might provide insight into the role of Wolbachia IncA proteins in host cell trafficking and cell-to-cell transmission. As Wolbachia is known to use the host phagocytic and endocytic machinery for cell to cell transfer (White et al., 2017), it is possible that some of these effectors are involved in a similar process.

Other wMel predicted effectors that may facilitate vacuolar integrity include those which encode domains involved in cytoskeleton-interaction (synuclein, HAUS-augmin3) (Rice et al., 2017). Interactions between Wolbachia and host cytoskeletal elements have been well characterized. For example, the microtubule cytoskeleton may be critical for Wolbachia transmission. Studies in Drosophila have shown that Wolbachia require host microtubules and the minus- and plus-end motors dynein and kinesin for posterior localization in the mature oocyte, which ensures transmission to the germline of the next generation (Ferree et al., 2005; Serbus and Sullivan, 2007). However, the actin cytoskeleton may also play a role in transmission as host mutations in actin binding proteins alter the efficiency with which Wolbachia are maternally transmitted (Newton et al., 2015). The effector WalE1, was recently characterized as a Wolbachia protein that interacts directly with and bundles F-actin (Sheehan et al., 2016). Secretion of WalE1 was demonstrated via a heterologous assay in E. coli (Whitaker et al., 2016) and upon transgenic expression, WalE1 was shown to increase Wolbachia load in Drosophila melanogaster females, allowing more efficient transmission of Wolbachia to the next generation (Sheehan et al., 2016). Native expression of walE1 mRNA suggests it is upregulated during host pupation, along with components of the type IV secretion system (Rice et al., 2017). Characterization of native WalE1 function, and the host targets of this Wolbachia effector will elucidate whether this actin bundler facilitates soma to germ line transmission or helps to maintain the Wolbachia vacuole.

Wolbachia–induced changes in host gene expression affect diverse processes

Several predicted wMel effectors include proteins with domains involved in transcriptional regulation (the PAZ si-RNA binding, the Fez1, and the CNOT1 HEAT domains). Does Wolbachia alter host transcription? What host phenotypes may be altered by these changes in gene expression? Persuasive evidence of Wolbachia-mediated regulation of host gene expression comes from both microarray and RNAseq studies comparing infected and uninfected individuals (Hussain et al., 2011; Darby et al., 2012). For example, microarray studies of gene expression conducted in Drosophila cell culture indicated significant upregulation of genes involved in the antimicrobial immune response, ion homeostasis, negative regulation of cell proliferation and larval development, as well as significant downregulation of host unfolded protein response (UPR) genes, and genes involved in positive regulation of transcription, and RNA splicing (Xi et al., 2008). Therefore, it is clear that Wolbachia affect global gene expression in their hosts. Indeed, regulating expression of particular host pathways is likely important for the expression of Wolbachia-induced host phenotypes.

Expression of certain host transcripts may be important for Wolbachia-mediated blocking of RNA virus replication (termed “pathogen blocking”). Initial RNAseq analyses of Wolbachia-infected mosquitos suggested that Wolbachia upregulates the host anti-microbial immune response pathways such as Toll and Imd to block virus replication (Ye et al., 2013). However, it has been shown that these loci are not necessary for Wolbachia to inhibit Dengue virus in D. melanogaster (Rances et al., 2012; Rances et al., 2013). Recently, Wolbachia mediated transcriptional changes at another locus was implicated in pathogen blocking; Wolbachia infection significantly upregulates the Drosophila melanogaster cytosine-methyltransferase gene Mt2, and reduction or ablation of Mt2 function (either by knockdown or knockout) diminishes Wolbachia’s ability to inhibit virus replication (Bhattacharya et al., 2017). The mechanism by which Wolbachia achieves this upregulation, however, is not yet known.

Studies outside of D. melanogaster further illustrate the direct influence of Wolbachia-modulated host gene expression on host phenotype, most notably, in the context of the transcriptional regulator wtrM (Pinto et al., 2013). The gene was identified in the Wolbachia strain native to Culex molestus, wPipMol, through comparative genomic analyses aimed at determining differences in genetic elements between closely related wPip strain variants native to the Culex pipiens complex of mosquitoes (Pinto et al., 2013). Although protein WtrM has not been detected in the host cytosol nor has translocation been confirmed, ectopic expression of this Wolbachia gene in C. molestus female mosquitoes led to significant upregulation of the host gene CPIJ005623, a Culex homolog of the D. melanogaster meiotic cell cycle regulator gene grauzone (Page and Orr-Weaver, 1996). Knockdown of CPIJ005623 in C. molestus females led to an increased incidence of CI (Pinto et al., 2013). Therefore, despite the lack of evidence regarding the subcellular localization of the native Wolbachia WtrM protein inside the host cell, it remains a possibility that Wolbachia-induced transcriptional upregulation of CPIJ005623 leads to CI rescue in C. molestus (Pinto et al., 2013).

Conclusion

Identification of cellular and genetic mechanisms underlying Wolbachia-host symbiosis requires better understanding of the proteins used by Wolbachia to manipulate host biology. Unlike free-living bacteria, the absence of conventional molecular tools has made it relatively difficult to functionally characterize proteins that Wolbachia may use to manipulate its host, of which there are many. However, through the use of heterologous systems (Whitaker et al., 2016), eukaryotic model systems like S. cerevisiae (Sheehan et al., 2016; Beckmann et al., 2017; Rice et al., 2017), and leveraging the power of genetics in Drosophila (Ote et al., 2016; Sheehan et al., 2016; LePage et al., 2017), we can begin to identify symbiont effectors and host cellular processes affected.

Originality and Significance Statement.

This review is the first of its kind to cover the mechanistic insights recently discovered with regards to Wolbachia-host biology.

Acknowledgments

We thank Drs Amelia Lindsey and Eric Smith for their thoughtful feedback on initial drafts of this manuscript. This work was supported by the NSF (grant IOS 1456545) and NIH (grant 5R21AI121849-02) to ILGN.

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