Discover the Microbes Within! The Wolbachia Project

A Bugs Life: WolbachiaDonna C. Sullivan, PhDDivision of Infectious DiseasesFebruary 10, 2012Introduction Lecture - Dr. Seth Bordenstein1Base Pair Program

Ok. First let me explain how I got interested in Wolbachia. For the last 10-15 years or so, I have served as a mentor or co-investigator on a Howard Hughes Medical Institute grant to support the Base Pair program. This program was initiated by Dr. Norman Nelson when he basically cornered Dr. Rob Rockhold in an elevator and told him we needed to do some service project with the Jackson Public School system. By the time Rob was able to escape from the elevator, he found himself committed to a grant proposal to fund a project. The program as it has evolved involves the enrollment of approximately 15 high school students (split between juniors and seniors) from Murrah High School. Students are paired with mentors in various research labs here at UMC and spend an hour and a half, Monday-Friday, during the second semester of their junior year and their senior year. Introduction Lecture - Dr. Seth Bordenstein2They All Go to College, But Then What?M.D./M.D.-Ph.D. = 21Ph.D. = 6Masters (Science) = 22Law = 5Nurse = 5

TOTAL= 59 (of 89 college graduates)Introduction Lecture - Dr. Seth Bordenstein3O for a Muse of fire, that would ascend The brightest heaven of inventionHenry V, Act 1, PrologueWilliam Shakespeare

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http://www.editorialmanager.com/biolbull/account/mbl_logo_bb.jpg4Collaboration Partners: HHMI, UMMC, Woods Hole Marine Biological Laboratories and Princeton University

Discover the Microbes Within! The Wolbachia Project

Kathy McKone, RBI Lead Teacher and HHMI/Princeton Workshop LeaderIntroduction Lecture - Dr. Seth Bordenstein5Wolbachia RodeoSix high schools sent teams for a day long competition. Positive Wolbachia samples were sent to Woods Hole for sequencing

Introduction Lecture - Dr. Seth Bordenstein6Phylogeny of Wolbachia

NATURE REVIEWS | microbiology Vol OCT 2008, p741Phylogeny of Wolbachia. a | The phylogenetic relationships of Wolbachia relative to the closely related Rickettsiales order in the Anaplasmataceae family. b | An unrooted phylogenetic tree of the main supergroups of Wolbachia. Also shown are the dominant patterns of mutualism and reproductive parasitism across the supergroups. For some supergroups, functional effects of Wolbachia have not yet been determined. The G supergroup has been removed because its status is currently unclear8. The pattern suggests that the main supergroups of Wolbachia participate in either mutualism or reproductive parasitism. Rooting of the Wolbachia phylogeny, which could help resolve whether mutualism or reproductive parasitism is ancestral, is problematic owing to long-branch attraction to out-groups. Resolution requires genome-sequence information for additional taxa. Triangle size represents described diversity within each lineage. Circles represent a lineage based on a single Wolbachia strain. Part a reproduced from REF. 98. Part b reproduced, with permission, from REF. 99 (2007) Society for General Microbiology.Introduction Lecture - Dr. Seth Bordenstein7

R. Stouthamer, DAPI stained wasp embryoWolbachia pipientis1924: Hertig and WolbachHost range: Arthropoda and Nematoda; reproductive tissues Maternally transmitted w/in speciesHorizontally transmitted between speciesObligate intracellular lifestyleImportant to invertebrate evolution (speciation, sex determination, sexual selection)Potential tool for pest bio-control and curing human filariasisConsidered relatively obscure until the 90s

Introduction Lecture - Dr. Seth Bordenstein8

Number Of Scientific Papers With Wolbachia In TitleNobel prize awarded for PCR PCR detection of Wolbachia in ~20% of all insectsIntroduction Lecture - Dr. Seth Bordenstein9

The Wolbachia Pandemic At least 920,000 described species 85% of all known animal species 10 quintillion insects alive at any given time Up to 30 million estimated species (~22.5 million infected species!)Wolbachia = 1 of Lifes great pandemics

Phylum ArthropodaInsects: up to 67% of species Isopods: 35% of terrestrial isopodsMites: 43% of mites Lice: ~100% of species

Phylum NematodaFilarial nematodes: 90% of speciesNonfilarial: ~0%

Purvis and Hector (2000)InsectaIntroduction Lecture - Dr. Seth Bordenstein10Ways That Vertically Transmitted Microbes Can Increase In FrequencyIncrease host survival & reproduction (mutualism) Very commonMost famous cases are the lineages leading to organelles Mitochondria evolved from the alpha-Proteobacteria about 2 billion years agoChloroplasts evolved from cyanobacteria about 1 billion years agoBut Wolbachia arent mitochondria.they are widow makers

Introduction Lecture - Dr. Seth Bordenstein11By JOHN TRAVISUndesirable Sex PartnersBacteria manipulate reproduction of insects and other speciesImaginephysicians across the United Statesdocumenting similar events. In each case, an unfertilized egg in a woman had spontaneously begun to develop, ultimately producing a healthy female baby.One young researcher, who had analyzed the timing and locales of the virgin births, suggested a spreading infection might be causing the incidents. The Federal Centers for Disease Control and Prevention in Atlanta quickly dismissed the idea, calling it "ridiculous."Several months later came a well-publicized report in the Journal of the American Medical Association concluding that the number of infertile couples was rising rapidly worldwide. The international uproar intensified when physicians began to observe another reproductive curiosity: Some newborns that were genetically male appeared to be female. One week, the New England Journal of Medicine and the National Enquirer ran articles with the headline, "Is this the end of mankind, or just men?"Introduction Lecture - Dr. Seth Bordenstein12Science fiction? Definitely. For many insect species and other arthropods, however, the truth can be as strange as fiction when bacteria known as Wolbachia are around. Maternal Inheritance Is ImperfectThere is a direct physiological cost to infection.Infections can only be maintained where they increase either the survival or production of female hosts.Symbionts become parasites (manipulate reproduction of hosts to enhance their own transmission).Distort the sex ratio of the host towards females.Some become mutualists, increasing the survival or reproductive success of their hosts Essential for the host to survive and reproduce (primary symbionts).Non-essential facultative roles and typically only infect a subset of the population (secondary symbionts).Introduction Lecture - Dr. Seth Bordenstein13Wolbachia are Infectious Widow makers!

Parthenogenesis

Feminization

Male-KillingReproductive Parasitism: each of these reproductive distortions leads to more infected females in the insect host speciesSeveral species are so dependent on Wolbachia that they are unable to reproduce effectively without the bacteria in their bodies.[9]Wolbachia are present in mature eggs, but not mature sperm. Only infected females pass the infection on to their offspring. One study on infected woodlice showed that the broods of infected organisms had a higher proportion of females than their uninfected counterparts.[10] It is thought that Wolbachia, especially Wolbachia-caused cytoplasmic incompatibility, may be important in promoting speciation. Wolbachia strains that distort the sex ratio may alter their host's pattern of sexual selection in nature, and also engender strong selection to prevent their action, leading to some of the fastest examples of natural selection in natural populations[16]

Introduction Lecture - Dr. Seth Bordenstein14Wolbachia Induced Phenotypes

NATURE REVIEWS | microbiology OCT 2008, p741Wolbachia- induced phenotypes. Wolbachia cause four distinct reproductive phenotypes in a range of arthropod orders (top). Male killing: males are killed during larval development, which increase the rate of born females.[6] Feminization: infected males develop as females or infertile pseudo-females. Parthenogenesis: reproduction of infected females without males. Some scientists have suggested that parthenogenesis may always be attributable to the effects of Wolbachia.[7] An example of a parthenogenic species is the Trichogramma wasp, which has evolved to procreate without males with the help of Wolbachia. Males are rare in this tiny species of insect, possibly because many have been killed by that very same strain of Wolbachia.] Cytoplasmic incompatibility: the inability of Wolbachia-infected males to successfully reproduce with uninfected females or females infected with another Wolbachia strain.

A cross section of a male filarial nematode, Onchocerca ochengi, that contains Wolbachia is shown (bottom left), in which Wolbachia are falsely colored yellow and fill three of the four syncytial lateral cord cells. Wolbachia (yellow) are also shown within the ovaries of a female Drosophila simulans (bottom right). The image on the bottom left is courtesy of M. Taylor, Liverpool School of Tropical Medicine, UK. The image on the bottom right is courtesy of M. Clark, University of Rochester, New York, USA.Introduction Lecture - Dr. Seth Bordenstein15

Wolbachia Strains and Their CharacteristicsWe've now sequenced the genomes of a number of strains of Wolbachia, only to find that there isn't a lot that's special about them. The genomes tend to be small, in the area of one to two million base pairs, but aren't as small as some dedicated parasites. They also seem to have an unusual amount of junk compared to most bacteria, with about 15 percent of the genome being repeated elements like transposons and viruses. They also have a high number of duplicated genes compared to other bacteria. All of these would suggest both that the genome is in a regular state of flux, and that it has plenty of raw materials for the evolution of new functions.

There are some unusual things about the proteins, however. Many are short peptides, which could act as hormones or signaling molecules that manipulate their hosts. There are also lots of cell surface proteins that may help the bacteria interact with host systems. Finally, there are a number of proteins carrying sequences that help them interact with other proteins, which may help Wolbachia manipulate systems like the host's cell division machinery.

Still, none of this explains precisely how the bacteria get the hosts to do so many remarkably specific things.

Introduction Lecture - Dr. Seth Bordenstein16How Wolbachia Might Be UsedAs a vector for the expression of genes of interest. As a tool to drive desirable genotypes into arthropod populations. To directly suppress arthropod populations.Introduction Lecture - Dr. Seth Bordenstein17Human Health: Wolbachia May Help Control The Transmission Of Arthropod-borne Diseases(Malaria, Dengue fever, Filariasis, Trypanosomiasis, West Nile, Chagas)

Isolate and culture symbiontReinfect vector with transgenic symbiontTransform symbiontTransmits Disease Does Not Transmit DiseaseAnti-pathogen gene Introduction Lecture - Dr. Seth Bordenstein18

Human Health: Wolbachia May Help Control The Transmission Of Arthropod-borne Diseases(Malaria, Dengue fever, Filariasis, Trypanosomiasis, West Nile, Chagas)

Early-mortality Wolbachia strainReinfect vector with transgenic symbiontOld Females Transmit Disease Females Die Too Early to Transmit DiseaseIntroduction Lecture - Dr. Seth Bordenstein19Drive via Cytoplasmic Incompatibility

XMaleFemaleProgeny

X

X

XNone (Incompatible)Introduction Lecture - Dr. Seth Bordenstein20Population Replacement

Transmits Disease Does Not Transmit Disease

Introduction Lecture - Dr. Seth Bordenstein21Wolbachia as Means for Insect Pest Control: MedflyIntroduced Wolbachia into medfly Ceratitis capitata population using cherry fruit fly Rhagoletis cerasi as donorInduced complete cytoplasmic incompatibilityResults encouraged idea of introducing Wolbachia into vector speciesZabalou et al., 2004. PNAS 101:15042-15045

Ratios of uninfected females to uninfected males to trans-infected males (cannot mate with uninfected females).Introduction Lecture - Dr. Seth Bordenstein22Successful Establishment Of Wolbachia In Aedes Populations To Suppress Dengue TransmissionwMel Wolbachia strain from Drosophila melanogaster stably introduced into A. aegyptiIntroduction through open releases of mosquitoes with the wMel Wolbachia strain into wild Australian A. aegypti populations

Hoffman et al., 2011. Nature 476: 454-259Introduction Lecture - Dr. Seth Bordenstein23Occurrence Of Aedes aegypti And Wolbachia Infection In Ovitraps Outside Release AreasSuccessfully invaded two natural A. aegypti populations in Australia, reaching near-fixation in a few months following releases of wMel infected A. aegypti adults.

Introduction Lecture - Dr. Seth Bordenstein24Wolbachia and NematodesThe relationship between Wolbachia and filarial nematodes is different. If Wolbachia is removed from host nematodes, the host organism loses its fertility and/or dies. Wolbachia are housed in the lateral chords and oocytes of their filarial nematode hosts. 25

2525

Human Health: Bill and Melinda Gates FoundationIntroduction Lecture - Dr. Seth Bordenstein26Wolbachia are Chemotherapy Targets for Curing River Blindness & Elephantiasis Caused By Filarial Nematodes

Horeauf et al, 200311 months post-treatmentUntreatedOnchocerca volvlulus

Introduction Lecture - Dr. Seth Bordenstein27Tissue Nematode (Roundworm): Onchocerca volvulus and River BlindnessTransmitted by biting black fliesLarvae develop into adults in subcutaneous tissuesAdult females migrate via the blood to the eyes, provoking inflammatory reactionsFemales produce millions of microfilariae that migrate through the body and cause a manifestation of symptoms

28

2828Onchocerciasis: Cutaneous and Ocular PathologySkin pathologyOcular pathologySubclinical or intermittent dermatitisHyperpigmentation or depigmentation (leopard skin)Lichenified onchodermatitis with pruritic hyperpigmented/ hyperkeratotic plaques (asymetrical, localized) with local lymphadenopathyVisual impairment (500,000 cases)Blindness (270,000 cases)Varies between geographical locations, more common in savannah areas of West/ Central Africa, Latin AmericaInvolves cornea (punctate keratitis, sclerosing keratitis)Anterior chamber where dead microfilaria cause uveitisIntroduction Lecture - Dr. Seth Bordenstein29Factors Affecting Visual PathologyLocalization of nodules in upper part of the bodyVector speciesMicrofilarial burdensParasite strainHigher Wolbachia load in more virulent savannah strainIntroduction Lecture - Dr. Seth Bordenstein30Wolbachia-infected Onchocerca volvulusAll individual worms and all life cycle stages contain the endosymbiont WolbachiaInhabit lateral chords of adult worms and reproductive system of femalesClearance of endosymbionts by antibiotic treatment causes inhibition of worm development, blocks embryogenesis and fertility, reduces worm viabilityIntroduction Lecture - Dr. Seth Bordenstein31Immunopathology Of OnchocerciasisObviously, host inflammatory responses to microfilariae, including autoimmune processes driven by cross-reaction between retinal and parasite proteinWolbachia and Wolbachia-derived molecules released from worms elicit immune responsesIntroduction Lecture - Dr. Seth Bordenstein32

Clin. Microbiol. Rev. 2011, 24(3):459.Wolbachia-induced Responses Of Specific Cell Types: In vitro StudiesFIG. 1. Wolbachia-induced responses of specific cell types. Wolbachia-exposed dendritic cells are activated via the TLR2/6-MyD88-Mal pathway, as shown by enhanced expression of surface costimulatory molecules and produce proinflammatory cytokines, inducing a preferential type 1 (Th1) immune response (11, 77). Macrophages stimulated with Wolbachia or Wolbachia- containing but not Wolbachia-depleted filarial extracts enhance their surface expression of costimulatory molecules and produce proinflammatory cytokines and oxidative products. Macrophages can be homo- and heterotolerized by a subsequent stimulation, contributing to the immune downregulation characterizing the majority of filarial infections (29, 67, 76, 77). Neutrophils and corneal stromal cells are also able to interact with Wolbachia via the TLR2-MyD88 pathway, producing CXC chemokines and contributing to the inflammatory response to the parasite (2224). Mast cells are stimulated by Wolbachia via TLR2 to degranulate and increase vascular permeability to facilitate establishment of infection (63). Abbreviations: TLR, Toll-like receptor; CD, cluster of differentiation; MHCII, major histocompatibility complex class II; IL, interleukin; TNF-, tumor necrosis factor alpha; RANTES, regulated upon activation, normal T-cell expressed, and secreted; TGF-, transforming growth factor ; NO, nitric oxide; MyD88, myeloid differentiation primary response gene (88); Mal, MyD88 adaptor-like. Introduction Lecture - Dr. Seth Bordenstein33Murine Model of Onchocercal KeratitisO. volvulus depleted of Wolbachia by antibiotic treatment does not induce corneal inflammation.Related filarial species containing Wolbachia (Brugia malayi) induce keratitis while species lacking Wolbachia (Acanthocheilonemma viteae and Loa loa) do not.Isolated Wolbachia bacteria induce neutrophil recruitment to the corneal stroma.Introduction Lecture - Dr. Seth Bordenstein34Role Of Wolbachia In River BlindnessWolbachia release in the cornea causes corneal edema and opacity by inducing neutrophil and macrophage infiltration and activation in the corneal stroma.This induces cutaneous and systemic side effects such as fever, tachycardia, hypotension, lymphadenopathy, and pruritus. Adverse events correlate with microfilarial loads and are associated with Wolbachia DNA and whole bacterial levels in blood, proinflammatory cytokines, neutrophilia, and antibacterial peptides (calprotectin and calgranulin).

Clin. Microbiol. Rev. 2011, 24(3):459.TLR, Toll-like receptorMyD88, myeloid differentiation primary response gene (88)TNF-, tumor necrosis factor alphaDEC, diethylcarbamazineFIG. 2. Role of Wolbachia in river blindness. Wolbachia release after microfilaria death in the cornea causes corneal edema and opacity by inducing neutrophil and macrophage infiltration and activation in the corneal stroma that are dependent on TLR2-MyD88 activation and production of CXC chemokines. Keratocytes and bone marrow-derived cells in the corneal stroma can initiate this response, which is then perpetuated by inflammatory cells (2224, 29, 56, 77). When large loads of Wolbachia bacteria are released from microfilariae after microfilaricidal treatment, this induces cutaneous and systemic side effects such as fever, tachycardia, hypotension, lymphadenopathy, and pruritus. In the skin, neutrophils are the first cells to be recruited and activated, inducing dermal inflammation. At a systemic level, adverse events correlate with microfilarial loads and are associated with Wolbachia DNA and whole bacterial levels in blood, proinflammatory cytokines, neutrophilia, and antibacterial peptides (calprotectin and calgranulin) (25, 43, 52, 80). The presence of Wolbachia is associated with neutrophil infiltration in the cornea, skin, and onchocercomas (8, 22, 23). Abbreviations: TLR, Toll-like receptor; MyD88, myeloid differentiation primary response gene (88); TNF-, tumor necrosis factor alpha; DEC, diethylcarbamazine. Introduction Lecture - Dr. Seth Bordenstein35Predicted Sequence Of Events In O. volvulus/Wolbachia-induced Keratitis

Clin. Microbiol. Rev. 2011, 24(3):459.KC, keratinocyte-derived chemokine; MIP, macrophage-inflammatory protein 2FIG. 3. Predicted sequence of events in O. volvulus/Wolbachia-induced keratitis. Wolbachia released from dying microfilariae in the corneal stroma (1) activates resident cells, including fibroblasts, dendritic cells, and macrophages (2). These cells produce chemokines MIP-2 and predominantly KC (3). KC induces a CXCR2-dependent neutrophil migration to the corneal stroma, where neutrophils are also activated via TLR2/6 by Wolbachia (4). Neutrophils produce additional chemokines, predominantly MIP-2, inducing further neutrophil migration (5). Neutrophils degrade the corneal matrix, causing corneal haze and visual impairment (6). Abbreviations: TLR, Toll-like receptor; MyD88, myeloid differentiation primary response gene (88); Mal, MyD88 adaptor-like; KC, keratinocyte-derived chemokine; MIP, macrophage-inflammatory protein 2; TNF-, tumor necrosis factor alpha; DC, dendritic cell. Introduction Lecture - Dr. Seth Bordenstein36Microfilaridermia (Mf) After Treatment WithDoxycycline Plus Ivermectin In Comparison To Ivermectin Alone

Hoerauf et al. 2001. Lancet 357:1415.Ivermectin is the drug used for mass chemotherapy of onchocerciasis within the WHO African Programme for Onchocerciasis Control. This approach aims to eliminate the disease as a public health problem but using one dose per year may not completely interrupt transmission since it does not suppress microfilaridermia thoroughly enough. Here we show that additional treatment with doxycycline, previously shown to sterilize adult female worms for a few months by depletion of symbiotic wolbachia endobacteria, significantly enhances ivermectin-induced suppression of microfilaridermia, rendering anti-Wolbachia treatment a promising basis for blocking transmission by a drug-based approach.

Figure 1: Microfilaridermia (mf) after treatment with doxycycline plus ivermectin in comparison to ivermectin alone Mf load is shown as Williams means (one less than the geometric means of one plus the mf counts). 5 Numbers in brackets are the 10th and the 90th percentiles, respectively. Mann-Whitney U test (differences from control group) *: p Molecular -> EvolutionYOU can make new discoveriesCovers Emerging Fields Microbial diversity, Bioinformatics, Molecular PhylogenyIntegrates Science and TechnologyIntroduction Lecture - Dr. Seth Bordenstein57Dictypoteracockroaches and mantids; 6,000 species

Discover the Microbes Within: The Wolbachia Project Lab 1: Insect IdentificationOdonatadragonflies, damselflies; 5,000 species

Dermaptera earwigs; 1,200 species Coleopterabeetles; 370,000 species

Collembola spring tails; 6,000 species

Dipteraflies; 120,000 species

Ephemeropteramayflies; 2,000 species

Hemipteraaphids; 67,500 species Hymenopterabees, wasps, ants; 108,000 species Isopteratermites; 1,900 species

Lepidopterabutterflies, moths, skippers; 140,000 species Orthopteragrasshoppers, crickets, katydids; 17,000 species Introduction Lecture - Dr. Seth Bordenstein58

Discover the Microbes Within: The Wolbachia Project Lab 2: DNA ExtractionEXTRACT DNA FROM YOUR SAMPLES

WHY?

Infected sample (+): Uninfected Sample (-)Insect InsectMitochondria MitochondriaWOLBACHIA -Introduction Lecture - Dr. Seth Bordenstein59Discover the Microbes Within: The Wolbachia Project Lab 3: Polymerase Chain Reaction

Introduction Lecture - Dr. Seth Bordenstein60

Discover the Microbes Within: The Wolbachia Project Lab 4: Gel ElectrophoresisIntroduction Lecture - Dr. Seth Bordenstein61

+++++++++++++++++++++Discover the Microbes Within: The Wolbachia Project Lab 4: Gel ElectrophoresisIntroduction Lecture - Dr. Seth Bordenstein62

Introduction Lecture - Dr. Seth Bordenstein63

Discover the Microbes Within: The Wolbachia Project Lab 5: Sequence Analysis NCBI BLAST No programming skills required!Introduction Lecture - Dr. Seth Bordenstein64

When It all comes together, it looks like this!Introduction Lecture - Dr. Seth Bordenstein65MBLHigh School

ATGCGC

Primers, Insect controls,Introduction Lecture - Dr. Seth Bordenstein66

Where Does Your Data Go?

ToClass reportsPeers, family, teachers, research scientistsOnline database (website repository for your data)

ConsequencesNew discoveriesCollaborations between research scientists and high schoolsSummer envisionships Professional meetingsProfessional societiesJournal publicationsIntroduction Lecture - Dr. Seth Bordenstein67