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Treatment of Filarial Disease

Still putting the pieces together

1st sequenced genome of a parasitic nematode

  Brugia malayi   95 megabase - ~19,000 genes   Project initiated in 1994 (WHO)

  Representative nematode   Availability of material from all life

cycles stages   Goals

  Construction of genomic libraries - mapping of genome

  Identify novel genes (different life cycle stages)

  Implement globally accessible database and resources

  Dissemination of filiarial genome data and materials

  NIAID/TIGR 2001 - turning point

Steve Williams Mark Blaxter

Univ. of Edinburgh Sanger Center

Smith College TIGR

New England Biolabs

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  The Filariasis Repository Service was first funded by the NIAID/NIH in 1968.   The Repository has served as a unique supply source of filarial research

materials and technical information for investigators   unable to cyclically maintain parasites due to expense,   lack of expertise   lack of space   backup facility for investigators who experienced difficulties with their own

colonies.   Molecular and Biological Resources

  Long term storage - cryopreservation   Distributed freely - only have to pay for shipping cost

  Those in endemic countries do not even pay for shipping!

Brugia malayi Annotation Jamboree May 2005: TIGR, Rockville, MD, USA

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Intriguing Observations!

  1975 - W.J. Kozek (Electron microscopist)   High resolution imaging revealed intracellular bacteria in filarial worms but not

in other nematodes (non-quantitative - observational)

  Clinicians believed that antibiotics were effective against acute symptoms of filariasis (India) - treated patients

  Klei and McCall independently found that a broad-spectrum antibiotic resulted in the failure of parasites to grow in infected animals.

Wolbachia (B. malayi) genome project   Filarial Genome Project

  Williams/Blaxter found many sequences resembling bacterial rDNA - contaminants?

  Rediscovery of Kozek, Keil and McCall data

  Completed March, 2004

  Comparative genome analysis with other endosymbionts March, 2005

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Wolbachia   Gram-negative bacterial endosymbiont   form intracellular inherited infections in many

invertebrates.   In reproductive tissues   Infects arthropods (insects, crustaceans, spiders and

mites) and nematodes - approx 20% of insects species are infected

  Does not infect vertebrates (mammals and humans)!   Host range of Wolbachia is not fully appreciated   Parasitism or mutualism?

  Host gives bacteria nutrients and protection   Bacteria gives host? Manipulates sex life of host

Wolbachia

  Related to Rickettsia bacteria   Alpha-proteobacterium

  Much of the success of Wolbachia can be attributed to the diverse phenotypes that result from infection:

  Wolbachia can   override chromosomal sex determination   induce parthenogenesis (typically all female)   selectively kill males   influence sperm competition   generate cytoplasmic incompatibility in early embryos

  Research interest ranges from the evolutionary implications of infection to the use of this agent for pest and disease control.

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Wolbachia genomes

Trends Parasitol. 2006 22:60-65 Wolbachia genomes: revealing the biology of parasitism and mutualism

Para

sitic

Mut

ualis

tic

Primarily CI

Wolbachia Wolbachia

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Unidirectional Cytoplasmic Incompatibility

Red - infected Black - uninfected Infected females produce more offspring

Distribution of Wolbachia

Microfilaria: Wolbachia are detected with

anti-WSP

Mosquito derived L3 Bacteria were rarely seen

L3: 9 days pi lateral cord localization

L3: 9 days pi Enlargement

Dividing cell

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Distribution of Wolbachia L4 - large numbers in lateral cords

Adult female - lateral cords, mf

Low Wolbachia numbers

High Wolbachia numbers

Distribution of Wolbachia

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Wolbachia association

  A: Demonstration of Wolbachia in hypodermis (anti-WSP staining)

  B: Staining for Wolbachia (inset) and neutrophils stained for defensin protein

  C: Patient treated with doxycyline. No anti-WSP staining in worm, substantial decrease in defensin staining.

Wolbachia of filarial worms

  Obligate endosymbionts! - mutualistic relationship   Depletion of Wolbachia results in filaria that fail to molt to adult stage   “ “ fail to mate   “ “ fail to produce progeny

  STERILIZING effect!

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Host response to filarial worms   Interpretation of host responses

  Crude extract of filarial antigens - what did these contain?   Mimicked Host response to acute infections/ adult killing

  Wolbachia antigen preparations could account for pathology in a mouse model of Onchocerca volvulus.   Combined treatment with anti-filarial and tetracycline

  Clearing of microfilaria - decrease in total number   Pretreatment with tetracycline

  Decreased number of wolbachia, as well as the # and severity of adverse reactions

Filarial Disease Control   New tool for eradication?

  Deplete endosymbiont   Worms develop distinct

phenotype   Decrease # of microfilaria   Embryogenesis is blocked   Uteri of adult females contain

degenerated embryos   Some studies induced killing of

adults

New measure for control?

+ Mass drug treatment