Wolbachia pipientis is one of the most prevalent obligate, intracellular bacteria in multicellular animals. By causing a diverse array of reproductive alterations in arthropods that favor the fitness of infected females, this bacterium has spread worldwide through hundreds of thousands of species. One of the unique genomic features to Wolbachia is an overabundance of mobile DNA including a temperate double-stranded DNA bacteriophage, called WO-B that has no known function. Our long term goal is to elucidate the mechanisms, relevance, and applications of this bacteriophage to the biology of Wolbachia. The specific hypothesis is that the bacteriophage is a mobile genetic parasite of Wolbachia, capable of integrating into and killing Wolbachia to ensure its own replication as a selfish element. We base that hypothesis on four observations: First, WO-B is widespread and infects 89% of all strains in the two major Wolbachia lineages that infect arthropods. Second, sequence analyses specify WO-B recombination and horizontal transmission between Wolbachia that co-infect the same host. Third, the presence of WO-B does not correlate with the capacity to induce reproductive alterations. Fourth, observations of WO-B particles from purified insect homogenates and from within lysed Wolbachia cells demonstrate that WO-B is active and may kill Wolbachia. Based on these observations, the experimental focus of this proposal is on the molecular evolution and predation of Wolbachia phage WO-B.
The specific aims are to: 1. Determine the molecular evolutionary forces shaping Wolbachia bacteriophage. By completing this aim, we will answer three related questions:
(Aim 1 A) How does an obligate intracellular niche affect phage genome evolution? We will determine how the distinctive ecological conditions of an obligate intracellular bacterium affect genetic diversity in WO-B and we will compare this to the rules of phage genome evolution in free-living bacteria.
(Aim 1 B) What classes of phage genes are under selection and do they correlate with gene functions such as integration, lysis, lysogeny, virulence, or structure? (Aim 1C) What are the number, diversity, and functions of phage genes that laterally transfer to and from Wolbachia co-infections? Our preliminary results suggest rampant horizontal phage transfer coupled with strong purifying selection. 2. Determine if bacteriophage kill Wolbachia and thereby regulate intrahost densities and Wolbachia's effects on hosts. Here, we will answer two questions related to phage function:
(Aim 2 A) In a controlled laboratory environment, do lytic phages prey on Wolbachia endosymbionts? (Aim 2B) Does environmental stress trigger phage lysis and does this variation in lysis correspond to variation in Wolbachia densities and interactions with the host? We propose these subaims as an alternative to the prevailing view that phage may be adaptive to Wolbachia's ability to modify arthropod reproduction. Our preliminary studies suggest their principal role is predation of Wolbachia. ? ? PROJECT NARRATIVE: The study of Wolbachia pipientis phage may impact public health by increasing knowledge on how to employ the phage as (i) a conduit for spreading transgenic traits into arthropod-borne vectors and (ii) as a therapeutic tool for removing filarial Wolbachia and associated human pathologies ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Genetic Variation and Evolution Study Section (GVE)
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Eckstrand, Irene A
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Vanderbilt University Medical Center
Schools of Arts and Sciences
United States
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Bordenstein, Sarah R; Bordenstein, Seth R (2016) Eukaryotic association module in phage WO genomes from Wolbachia. Nat Commun 7:13155
Metcalf, Jason A; Funkhouser-Jones, Lisa J; Brileya, Kristen et al. (2014) Antibacterial gene transfer across the tree of life. Elife 3:
Stilling, Roman M; Bordenstein, Seth R; Dinan, Timothy G et al. (2014) Friends with social benefits: host-microbe interactions as a driver of brain evolution and development? Front Cell Infect Microbiol 4:147
Bordenstein, Seth R (2014) Genomic and cellular complexity from symbiotic simplicity. Cell 158:1236-1237
LePage, Daniel; Bordenstein, Seth R (2013) Wolbachia: Can we save lives with a great pandemic? Trends Parasitol 29:385-93
Duncan, Stacy S; Valk, Pieter L; McClain, Mark S et al. (2013) Comparative genomic analysis of East Asian and non-Asian Helicobacter pylori strains identifies rapidly evolving genes. PLoS One 8:e55120
Funkhouser, Lisa J; Bordenstein, Seth R (2013) Mom knows best: the universality of maternal microbial transmission. PLoS Biol 11:e1001631
Duncan, Stacy S; Valk, Pieter L; Shaffer, Carrie L et al. (2012) J-Western forms of Helicobacter pylori cagA constitute a distinct phylogenetic group with a widespread geographic distribution. J Bacteriol 194:1593-604
Metcalf, Jason A; Bordenstein, Seth R (2012) The complexity of virus systems: the case of endosymbionts. Curr Opin Microbiol 15:546-52
Kent, Bethany N; Funkhouser, Lisa J; Setia, Shefali et al. (2011) Evolutionary genomics of a temperate bacteriophage in an obligate intracellular bacteria (Wolbachia). PLoS One 6:e24984

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