Transcript Wolbachia - University of Mississippi Medical Center

A Bug’s Life: Wolbachia
Donna C. Sullivan, PhD
Division of Infectious Diseases
February 10, 2012
Base Pair Program
They All Go to College, But Then
What?
M.D./M.D.-Ph.D. = 21
 Ph.D. = 6
 Masters (Science) = 22
 Law = 5
 Nurse = 5
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TOTAL= 59 (of 89 college graduates)
“O for a Muse of fire,
that would ascend The
brightest heaven of
invention…”
Henry V, Act 1, Prologue
William Shakespeare
Collaboration 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 Leader
Wolbachia Rodeo
Six high schools
sent teams for a
day long
competition.
 Positive
Wolbachia
samples were
sent to Woods
Hole for
sequencing

Phylogeny of Wolbachia
NATURE REVIEWS | microbiology Vol OCT 2008, p741
Wolbachia pipientis
1924: Hertig and Wolbach
 Host range: Arthropoda and Nematoda;
reproductive tissues
 Maternally transmitted w/in species

◦ Horizontally transmitted between species
Obligate intracellular lifestyle
 Important to invertebrate evolution (speciation,
sex determination, sexual selection)
 Potential tool for pest bio-control and curing
human filariasis
 Considered relatively obscure until the 90’s

R. Stouthamer, DAPI stained wasp embryo
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Number Of Scientific Papers With
Wolbachia In Title
120
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PCR detection of Wolbachia in ~20% of all insects
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Nobel prize awarded for PCR
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The Wolbachia Pandemic
Phylum Arthropoda
Phylum Nematoda
Insects: up to 67% of species
Isopods: 35% of terrestrial isopods
Mites: 43% of mites
Lice: ~100% of species
Purvis and Hector (2000)
Insecta
Filarial nematodes: 90% of species
Nonfilarial: ~0%
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 Life’s great pandemics
Ways That Vertically Transmitted
Microbes Can Increase In Frequency

Increase host survival & reproduction
(mutualism)
◦ Very common

Most famous cases are the lineages leading
to organelles
◦ Mitochondria evolved from the alphaProteobacteria about 2 billion years ago
◦ Chloroplasts evolved from cyanobacteria about 1
billion years ago

But Wolbachia aren’t mitochondria….they
are widow makers
Imagine…
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…physicians across the United States…documenting 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?"
By JOHN TRAVIS
Undesirable Sex Partners
Bacteria manipulate reproduction of insects and other species
Maternal Inheritance Is Imperfect
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There 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).
Wolbachia are Infectious Widow
makers!
Male-Killing
Feminization
Parthenogenesis
Reproductive Parasitism:
each of these reproductive
distortions leads to more
infected females in the insect
host species
Wolbachia Induced Phenotypes
NATURE REVIEWS | microbiology OCT 2008, p741
Wolbachia Strains and Their
Characteristics
How Wolbachia Might Be Used
As a vector for the expression of genes of
interest.
 As a tool to drive desirable genotypes
into arthropod populations.
 To directly suppress arthropod
populations.

Human Health: Wolbachia May Help Control
The Transmission Of Arthropod-borne Diseases
Does Not
Transmit
Disease
Transmits
Disease
Reinfect vector
with transgenic
symbiont
Isolate and
culture
symbiont
Transform
symbiont
Anti-pathogen
gene
(Malaria, Dengue fever, Filariasis, Trypanosomiasis, West Nile, Chagas)
Human Health: Wolbachia May Help Control
The Transmission Of Arthropod-borne
Diseases
Old Females
Transmit
Disease
Females Die Too
Early to Transmit
Disease
Earlymortality
Wolbachia
strain
Reinfect
vector with
transgenic
symbiont
(Malaria, Dengue fever, Filariasis, Trypanosomiasis, West Nile, Chagas)
Drive via Cytoplasmic
Incompatibility
Male
Female
X
X
X
X
Progeny
None
(Incompatible)
Population Replacement
Transmits Disease
Does Not Transmit Disease
Wolbachia as Means for Insect Pest
Control: Medfly
Ratios of uninfected females to uninfected
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Introduced Wolbachia
into medfly Ceratitis
capitata population using
cherry fruit fly Rhagoletis
cerasi as donor
Induced complete
cytoplasmic
incompatibility
Results encouraged idea
of introducing Wolbachia
into vector species
males to trans-infected males (cannot
mate with uninfected females).
Zabalou et al., 2004. PNAS 101:15042-15045
Successful Establishment Of Wolbachia In Aedes
Populations To Suppress Dengue Transmission
wMel Wolbachia strain
from Drosophila
melanogaster stably
introduced into A. aegypti
 Introduction through
open releases of
mosquitoes with the
wMel Wolbachia strain
into wild Australian A.
aegypti populations

Hoffman et al., 2011. Nature 476: 454-259
Occurrence Of Aedes aegypti And Wolbachia
Infection In Ovitraps Outside Release Areas

Successfully invaded two natural A. aegypti
populations in Australia, reaching near-fixation in
a few months following releases of wMel
infected A. aegypti adults.
Wolbachia and Nematodes
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The 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.
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Human Health: Bill and Melinda
Gates Foundation
Wolbachia are Chemotherapy Targets for Curing
River Blindness & Elephantiasis Caused By
Filarial Nematodes
Onchocerca volvlulus
Untreated
11 months post-treatment
Horeauf et al, 2003
Tissue Nematode (Roundworm):
Onchocerca volvulus and River Blindness
Transmitted by biting black
flies
 Larvae develop into adults in
subcutaneous tissues
 Adult females migrate via the
blood to the eyes, provoking
inflammatory reactions
 Females produce millions of
microfilariae that migrate
through the body and cause a
manifestation of symptoms
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Skin pathology
Ocular pathology
Subclinical or intermittent
dermatitis
Hyperpigmentation or
depigmentation (leopard skin)
Lichenified onchodermatitis
with pruritic hyperpigmented/
hyperkeratotic plaques
(asymetrical, localized) with
local lymphadenopathy
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Visual impairment (500,000
cases)
Blindness (270,000 cases)
Varies between geographical
locations, more common in
savannah areas of West/
Central Africa, Latin America
Involves cornea (punctate
keratitis, sclerosing keratitis)
Anterior chamber where
dead microfilaria cause uveitis
Onchocerciasis: Cutaneous and
Ocular Pathology
Factors Affecting Visual Pathology
Localization of nodules in upper part of
the body
 Vector species
 Microfilarial burdens
 Parasite strain
 Higher Wolbachia load in more virulent
savannah strain

Wolbachia-infected Onchocerca
volvulus

All individual worms and all life cycle
stages contain the endosymbiont
Wolbachia
◦ Inhabit lateral chords of adult worms and
reproductive system of females

Clearance of endosymbionts by antibiotic
treatment causes inhibition of worm
development, blocks embryogenesis and
fertility, reduces worm viability
Immunopathology Of
Onchocerciasis
Obviously, host inflammatory responses
to microfilariae, including autoimmune
processes driven by cross-reaction
between retinal and parasite protein
 Wolbachia and Wolbachia-derived
molecules released from worms elicit
immune responses

Wolbachia-induced Responses Of
Specific Cell Types: In vitro Studies
Clin. Microbiol. Rev. 2011, 24(3):459.
Murine Model of Onchocercal
Keratitis
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O. 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.
Role Of Wolbachia In River
Blindness
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Wolbachia 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 receptor
• MyD88, myeloid differentiation
primary response gene (88)
• TNF-, tumor necrosis factor alpha
• DEC, diethylcarbamazine
Predicted Sequence Of Events In O.
volvulus/Wolbachia-induced Keratitis
Clin. Microbiol. Rev. 2011, 24(3):459.
KC, keratinocyte-derived chemokine;
MIP, macrophage-inflammatory protein 2
Microfilaridermia (Mf) After Treatment With
Doxycycline Plus Ivermectin In Comparison
To Ivermectin Alone
Hoerauf et al. 2001. Lancet 357:1415.
Tissue Nematode (Roundworm):
Wuchereria bancrofti
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Tropical infection spread by
mosquitoes
Vector deposits larvae
which move into lymphatics
and develop
Chronic infection causes
blockage of lymphatic
circulation and elephantitis,
massive swelling in the
extremities
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Tissue Nematode (Roundworm):
Filariasis due to W. bancrofti
Endemic in central Africa, Mediterranean
coast, parts of Asia (China, Korea, Japan, the
Philippines)
 Blood specimens may show microfilariae
 Acute symptoms include: fever,
lymphangitis/lymphadenitis
 Result of inflammatory response to molting
adolescent worms and dead adults in
lymphatic vessels
 May involve any part of body, blocking
lymphatic system
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Tissue Nematode (Roundworm):
Elephantitis
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Elephantiasis: Wucheria bancrofti
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Lancet 2005; 365: 2116–21
Wolbachia and Virus Protection in Insects
(The “Not In My House” Idea)
Wolbachia infection of D. melanogaster has
been shown to increase the fly’s
resistance to some viral infections,
resulting in infections with lower virus
titers and less associated pathology.
 The resistance phenotype appears to be
limited to RNA viruses, with the strength
of resistance varying substantially among
the viruses tested thus far.

Mosquitos With and Without
Wolbachia
Wolbachia transfected:
inbred PGYP1 line
Wild Type Cairns
Wolbachia transfected backcrossed with
Mosquito: Wolbachia
wild type Cairns mosquito (outbred):
Negative
PGYP1.out line
Treat with
Tetracycline (kill
the Wolbachia)
PGYP1.tet line
PGYP1.out.tet line
DENV-2 In Whole Mosquito Bodies
(ELISA Assay)
Mosquitoes were fed an artificial blood meal spiked with
DENV-2
 No Wolbachia-infected mosquitoes (PGYP1.out) tested positive
for DENV-2
 DENV-2 infection rates in Wolbachia-uninfected mosquitoes
(PGYP1.out.tet and Cairns3) ranged from 30%–100%

Cell 139, 1268–1278, December 24, 2009
Quantitative PCR Analysis of
Dengue Virus in Mosquitoes
Cell 139, 1268–1278, December 24, 2009
Localization of Wolbachia and Dengue Virus
in A. aegypti Mosquitoes
PGYP1.tet (- Wolb)
PGYP1. (+ Wolb)
Cell 139, 1268–1278, December 24, 2009
Sections were probed
simultaneously with polyclonal
anti-wsp antibody (Wolbachia)
and monoclonal anti-DENV
antibody
 Wolbachia-uninfected control
mosquitoes showed DENV-2
infection predominantly in
mosquito fat body, ommatidia
and nervous system.
 DENV-2 was not detected in any
of these tissues in Wolbachiainfected mosquitoes.
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Does This Phenomenon Extend to
Other Viruses?
Day 14 infection rates were 87% and 79% for the
PGYP1.out.tet and Cairns3 controls (Wolbachia negative) and
17% for the Wolbachia infected PGYP1.out line (p < 0.001, chisquare).
 Viral interference effect appeared robust for two unrelated
arboviruses

Cell 139, 1268–1278, December 24, 2009
Plasmodium gallinaceum Detection in Aedes
spp. Mosquitoes
Fed mosquitos on infected
chickens
 Reduced the frequency of
mosquitoes in the population
infected with malaria
 Reduced number potentially
able to transmit Plasmodium
from 74% (tet controls) down
to 42%.
 Plasmodium genomic DNA
was 26-fold less abundant
Wolbachia infected cells
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Cell 139, 1268–1278, December 24, 2009
Immune Gene Regulation in
Response to Wolbachia Infection
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The expression levels of eleven immune pathway genes in the
Wolbachia wMelPop-CLA infected PGYP1.out and its uninfected
control line were compared for two independently reared
cohorts of mosquitoes.
Four genes encoding representatives of immune effector
molecules were significantly up-regulated in the presence of
Wolbachia.
Cell 139, 1268–1278, December 24, 2009
Conclusions

wMelPop-CLA Wolbachia infection reduces
the ability of two distantly related
arboviruses and a malaria parasite from
establishing productive infections in the
mosquito
◦ Shows that the pathogen interference effect is
general and fundamental.

Mechanism unclear although data suggest
that immune effector genes are up-regulated
in the mosquito in the presence of the
Wolbachia wMelPop-CLA strain and are
likely to play a role.
Bottom Line
Wolbachia-infected mosquitoes display
priming of immune system genes, possibly
underpinning viral inhibition.
 Arboviral infection in mosquitoes and
some mosquito cell lines induces
antimicrobial immune pathways such as
Toll, JAK/STAT and Imd/JNK as well as
RNAi based defenses.

How do YOU
discover
the Wolbachia
within?
Integrated Set of Lab Exercises
From Organisms to Molecules and Back!
Lab 1 - Insect Identification (Biodiversity)
Lab 2 - Isolation of Insect and Wolbachia DNA (Molecular Biology)
Lab 3 - PCR of Wolbachia 16S rDNA (Molecular Biology)
Lab 4 - Presence/Absence of Amplicon (Molecular Biology)
Lab 5 - Evolutionary Sequence Analysis (Bioinformatics)
Features of These Labs

Original Research in Lab Exercises
• YOU can make new discoveries
• Integrates across Science
Biodiversity -> Molecular -> Evolution
• Integrates Science and Technology
• Covers Emerging Fields
Microbial diversity, Bioinformatics,
Molecular Phylogeny
Discover the Microbes Within: The Wolbachia Project
Lab 1: Insect Identification
Coleoptera
Collembola
Diptera
Hemiptera
Lepidoptera
Orthoptera
beetles; 370,000
species
spring tails; 6,000
species
flies; 120,000
species
aphids; 67,500
species
butterflies, moths,
skippers; 140,000
species
grasshoppers,
crickets,
katydids; 17,000
species
Hymenoptera
Isoptera
Odonata
bees, wasps, ants;
108,000 species
termites; 1,900
species
dragonflies,
damselflies;
5,000 species
Dermaptera
Dictypotera
Ephemeroptera
earwigs; 1,200
species
cockroaches and
mantids; 6,000
species
mayflies; 2,000
species
Discover the Microbes Within: The Wolbachia Project
Lab 2: DNA Extraction
EXTRACT DNA FROM
YOUR SAMPLES
WHY?
Infected sample (+):
QuickTi me™ a nd a
TIFF (Uncompre ssed ) decomp resso r
are need ed to se e th is p icture.
Uninfected Sample (-)
Insect
Insect
Mitochondria
Mitochondria
WOLBACHIA
-
Discover the Microbes Within: The Wolbachia Project
Lab 3: Polymerase Chain Reaction
Discover the Microbes Within: The Wolbachia Project
Lab 4: Gel Electrophoresis
Discover the Microbes Within: The Wolbachia Project
Lab 4: Gel Electrophoresis
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Discover the Microbes Within: The Wolbachia Project
Lab 5: Sequence Analysis
• NCBI
• BLAST
• No programming
skills required!
When It all comes together, it looks like this!
High School
MBL
ATGCGC
Primers, Insect controls,
Where Does Your Data Go?
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To…
Class reports
Peers, family, teachers,
research scientists
Online database
(website repository
for your data)
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Consequences
New discoveries
Collaborations
between research
scientists and high
schools
Summer
“envisionships”
Professional meetings
Professional societies
Journal publications