Detection of the Wolbachia protein WPIP0282 in mosquito spermathecae: Implications for cytoplasmic incompatibility

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Highlights

  • Peptides encoded by wPip_0282 occur in sperm from dissected mosquito spermathecae.

  • Homologs of wPip_0282 are only present in CI-inducing Wolbachia strains.

  • wPip_0282 and wPip_0283 comprise a two gene operon that has duplicated and diverged.

  • wPip_0283 encodes a C-terminal, eukaryotic SUMO protease domain.

Abstract

Cytoplasmic incompatibility (CI) is a conditional sterility induced by the bacterium Wolbachia pipientis that infects reproductive tissues in many arthropods. Although CI provides a potential tool to control insect vectors of arthropod-borne diseases, the molecular basis for CI induction is unknown. We hypothesized that a Wolbachia-encoded, CI-inducing factor would be enriched in sperm recovered from spermathecae of female mosquitoes. Using SDS-PAGE and mass spectrometry, we detected peptides from the 56 kDa hypothetical protein, encoded by wPip_0282, associated with sperm transferred to females by Wolbachia infected males. We also detected peptides from the same protein in Wolbachia infected ovaries. Homologs of wPip_0282 and the co-transcribed downstream gene, wPip_0283, occur as multiple divergent copies in genomes of CI-inducing strains of Wolbachia. The operon is located in a genomic context that includes mobile genetic elements. The absence of wPip_0282 and wPip_0283 homologs from genomes of Wolbachia in filarial nematodes, as well as other members of the Rickettsiales, suggests a role as a candidate CI effector.

Introduction

Wolbachia are Gram-negative, alpha proteobacteria (family Anaplasmataceae, order Rickettsiales) that infect a high percentage of arthropod species, up to 76% in one survey (Jeyaprakash and Hoy, 2000). Wolbachia are also mutualistic endosymbionts of the nematodes Brugia malayi and Onchocerca volvulus, which in humans cause lymphatic filariasis and onchocerciasis, respectively (Taylor et al., 2012). Recently, it has been shown that Wolbachia can cause significant immune responses in humans when liberated from microfilariae (Brattig et al., 2004, Shiny et al., 2011, Bazzocchi et al., 2007). In mosquitoes, Wolbachia represent a potential means of controlling vector populations because they manipulate and distort reproduction, causing cytoplasmic incompatibility (CI). CI skews offspring ratios in a way that provides a reproductive advantage to females infected by Wolbachia, facilitating the organism's spread through naïve insect populations (Serbus et al., 2008) and providing a gene drive system for population replacement (Rasgon et al., 2006). In addition, some Wolbachia infections shorten vector life spans (McMeniman et al., 2009), reduce vector competence (Frentiu et al., 2010, Vavre and Charlat, 2012), and interfere with immune mechanisms that facilitate maintenance of pathogens (Teixeira et al., 2008). Elucidation of the molecular mechanisms that cause CI would represent an important advance towards use of Wolbachia for biological control of insect pest populations and vector-borne disease.

CI occurs when sperm from Wolbachia-infected males fertilize eggs from uninfected females. Clark et al. (2003) suggest that during development in Wolbachia-infected testes, spermatocytes acquire a Wolbachia strain-specific modification that can be rescued if the male pronucleus matures in cytoplasm of eggs infected with the same (compatible) strain of Wolbachia. However, if a modified sperm matures in cytoplasm of eggs from uninfected females, the modification cannot be corrected, and developing embryos show disruptions in cell cycle synchrony (Serbus et al., 2008, Callaini et al., 1997); in diploid insects such as mosquitoes, eggs from a CI cross fail to hatch. CI is complex, and in Culex pipiens mosquitoes, 17 different cytotypes have been described, with both unidirectional and bidirectional mating incompatibilities among mosquitoes from different geographical locations (Laven, 1967). Thus far, mechanisms by which Wolbachia strains cause the incompatibility patterns observed in crossing experiments are unknown, and recent comparisons of Wolbachia genomes from geographically isolated strains of C. pipiens from Johannesburg (JHB) and Sri Lanka (Pel) have uncovered few differences that might account for CI (Klasson et al., 2008; Salzberg et al., 2008).

Several lines of evidence are consistent with the hypothesis that Wolbachia secrete one or more effector protein(s) that associates with sperm DNA and interferes with male pronuclear chromatin architecture. The streamlined Wolbachia genomes have retained genes encoding all of the components for a functional bacterial type IV secretion system (T4SS), which is known to mediate extracellular export of proteins and DNA (Rances et al., 2008). Moreover, in developing sperm and in ovarian tissues, Wolbachia have been shown to localize around the nucleus and directly contact the nuclear envelope, consistent with the possibility that they secrete molecules into the nucleus (Ferree et al., 2005, Clark et al., 2002, Clark et al., 2003). Presgraves (2000) demonstrated that in Drosophila, CI is induced by an unknown modification on paternal chromatin; more recently, Landmann et al. (2009) reported impaired maternal histone deposition on male pronuclear chromatin in Drosophila. Severity of the CI phenotype is associated with high Wolbachia titers in the testes (Clark et al., 2002, Clark et al., 2003, Veneti et al., 2003, Veneti et al., 2004), while increased mating decreases CI penetrance within Drosophila simulans, implying depletion of an effector chemical that must have time to accumulate in developing sperm (Karr et al., 1998).

We recently undertook extensive proteomic studies with the goal of identifying potential CI inducing protein candidates in mosquito reproductive organs, including the Wolbachia DNA binding protein HU beta (Beckmann et al., 2013). To extend these studies, we hypothesized that CI effector proteins might be expressed at levels sufficient for detection by mass spectrometry in mature spermatozoa recovered from the spermathecae of female mosquitoes. Unlike the diverse developmental stages of sperm recovered from dissected testes, mature spermatozoa, transferred to a female during mating and stored in her spermathecae, are free of Wolbachia itself, which are discarded in “waste bags” that eliminate excess cytoplasmic material as the spermatids elongate (Serbus et al., 2008). Thus, we reasoned that dissected spermathecae would be enriched for secreted Wolbachia proteins accumulated during spermatogenesis and retained by virtue of association with organelles or nuclear DNA, but would not include Wolbachia structural proteins, or proteins involved in Wolbachia replication and maintenance. Using SDS polyacrylamide gel electrophoresis (SDS PAGE) and mass spectrometry we identified peptides encoded by wPip_0282 in C. pipiens spermathecae as well as in ovarian tissues. In-silico analysis revealed that wPip_0282 is expressed from a two-gene operon, which has undergone duplication and divergence in Wolbachia genomes for which data are available. RT-PCR data confirmed polycistronic translation of the operon and its duplicate homolog as one mRNA. Intriguingly, wPip_0282/0283 operons occur only in insect-associated, CI-inducing Wolbachia, and map to genomic regions characterized by mobile genomic elements, ankyrin repeats, and WO phage genes.

Section snippets

Mosquito colonies and maintenence

Colonies of C. pipiens pipiens (Buckeye strain) mosquitoes were maintained at 25 °C as described previously (Beckmann and Fallon, 2012). Larvae were fed pulverized rat chow and yeast. Adults were allowed to feed on 10% sucrose in water. C. pipiens mosquitoes are naturally infected with wPip. A cured colony of mosquitoes was established by tetracycline treatment. Infection status was verified by PCR as detailed previously (Beckmann and Fallon, 2012). Aedes albopictus mosquitoes (Houston strain,

Detection of WPIP0282 in spermathecae and ovaries

To search for Wolbachia proteins in sperm enriched extracts we dissected sperm-filled spermathecae (approximately 2400, from 800 female mosquitoes per lane; each individual mosquito contains 3 round spermathecal lobes that individually store sperm), and total protein was extracted for analysis by SDS-PAGE. Analysis showed two bands that migrated between 45 and 66 kDa in protein from spermathecae filled with Wolbachia modified sperm (Fig. 1, lane 1), relative to unmodified sperm from

Wolbachia pipientis 0282 as a CI effector candidate

We initiated these studies to explore whether the Wolbachia-encoded DNA binding protein, HU beta (Beckmann et al., 2013), was enriched in mature sperm from Wolbachia infected male C. pipiens. To do this we dissected spermathecae from females mated with infected males and subjected protein extracts to SDS PAGE and mass spectrometry. We did not detect the presence of the HU beta in the spermathecal extracts, but we did detect peptides from a 56 kDa hypothetical protein encoded by wPip_0282.

Acknowledgments

We thank Todd Markowski and Bruce Witthuhn at the University of Minnesota's Center for Mass Spectrometry and Proteomics for help with mass spectrometry detection and gel staining, Cassie Kurtz and Elissa Carroll for help with mosquito maintenance, and Gerald Baldridge, Rod Felsheim, and Tim Kurtti for helpful discussions. This work was supported by NIH grant AI081322 and by the University of Minnesota Agricultural Experiment Station, St. Paul, MN.

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