PATHOGEN AND HOST TARGET DISCOVERY IN BRUCELLA ABORTUS INFECTED MACROPHAGES

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PATHOGEN AND HOST TARGET DISCOVERY IN BRUCELLA
ABORTUS INFECTED MACROPHAGES NIAID Contract
HHSN26620040056C Eustache Paramithiotis PhD 10
June 2008
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BRUCELLA ABORTUS

• Brucella genus
• Isolated by David Bruce in 1887
• Gram-negative, non-motile, non-spore forming,
  aerobic cocobacilli
• Intracellular environment is primary niche
• Brucellosis
• Originally described by Hippocrates
• Chronic infection of domestic and wild mammals,
  human zoonosis
• Inhalation of aerosols, ingestion of infected
  materials
• Endemic in many developing nations
• Counter measures
• Antibiotic cocktail available treatment lengthy
  eradication difficult
• Human vaccine not available
• B. melitensis, B. suis, B. abortus genomes
  sequenced

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BRUCELLA INTRACELLULAR LIFECYCLE
Replication begins 6 hrs after infection
Adapted from Roy Trends Microbiol. 10416, 2002
Mouse macrophage 48hrs post infection E. Moreno
personal communication
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• Program Summary

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EXPERIMENTAL SYSTEM GOALS

• Brucella abortus infection of mouse macrophages
• Infection timecourse using virulent and
  attenuated bacteria

Aim 3 Secreted proteins
Aim 3 Plasma membrane changes
Macrophage
Aim 4 MHC peptide presentation
Aim 1 Phagosome composition changes
Aim 2 Extracellular virulence factors
Aim 2 Intracellular virulence factors
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WHERE WE ARE
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4
5
2
1
Year
1. System setup methods development
1.
2. Target discovery
2.
3.
3. Early target validation
June 2008
1. SYSTEM
Access to Select Agents Infection
conditions Bacterial purification Organelle
purification Sample processing Analytical tools
2. TARGET DISCOVERY
Extracellular bacteria completed Intracellular
bacteria completed Infected phagosomes
completed Infected protein secretion
completed Infected PM in progress Infected MHC I
II in progress
3. EARLY VALIDATION
Assay development RNAi Gene deletion Infectivit
y assays ELISA Confocal microscopy
COMPLETED
IN PROGRESS
IN PROGRESS
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ACCOMPLISHMENTS TO DATE

• Operations
• New Select Agent use approval
• Isolation methods for highly purified PM, MHC
  peptides early stage phagosomes
• Isolation methods for highly purified intact
  intracellular bacteria
• Multiple large scale sample production analysis
  runs
• Identified host pathogen antimicrobial target
  candidates
• Currently 5 under budget
• Deliveries
• 11 detailed sample preparation processing
  protocols
• 6 datasets encompassing 800 pathogen 2800
  host proteins
• Publications
• Lamontagne et al. Extensive cell envelope
  modulation is associated with virulence in
  Brucella abortus. J. Proteome Res. 61519, 2007.
• Lekpor et al. An evaluation of multidimensional
  fingerprinting in the context of clinical
  proteomics. Proteomics Clin. Appl. 1457, 2007.
• Kearney et al. Protein identification and peptide
  expression resolver harmonizing protein
  identification with protein expression data. J.
  Proteome Res. 7234, 2008.

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PLANNED YEAR 5 ACTIVITIES
Aim 3 host plasma membrane changes

• PM Sample preparation currently 50 completed
• Proteomics analysis pending

• Discovery

Aim 4 MHC peptide presentation

• Sample preparation currently 30 completed
• Proteomics analysis pending

• RNAi mediated inhibition of 100 phagosome
  targets currently 75 completed
• Secondary assays for selected targets pending

Aim 1 Phagosome composition changes
Early validation
Aim 2 Bacterial virulence factors

• Generation of mutants for 34 candidates
  infectivity assessment currently 25 completed

• Recombinant MHC I binding assay for 3 alleles
  currently 80 completed
• Binding assay of peptides and novel motifs pending

Aim 4 MHC peptide presentation

• Remaining Aim 1 datasets, Aim 3 Aim 4 datasets
• Remaining protocols

Resource center
Data materials
BEI

• Bacterial strains

• Aim 1 Aim 2 manuscripts currently being
  assembled additional manuscripts pending

Publications
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ANTICIPATED DELIVERY TIMELINES

• Datasets by Dec. 2008
• Infected phagosomes timecourse dataset (Aim 1)
• Mini (1-4hrs) intracellular bacterial timecourse
  (Aim 2)
• Secreted protein dataset (Aim 3)
• MHC I motif analysis dataset (Aim 4)
• Datasets in 2009
• Plasma membrane timecourse dataset (Aim 3)
• MHC presentation in infected cells dataset (Aim
  4)
• Protocols
• Additional 15 protocol submission anticipated.
• Reagents
• Brucella mutant strains submitted to BEI probably
  in 2009

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• Collaborations data use

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CURRENT COLLABORATIONS
Caprion Edgardo Moreno University of Costa Rica
Ignacio Moriyon University of Navarra, Spain
Brucella metabolism
Oswald Crasta Virginia Bioinformatics Institute
Brucella protein ID
Bruno Sobral Virginia Bioinformatics Institute
Rhizobium
Ignacio Moriyon University of Navarra,
Spain Jean-Jacques Letesson University of Namur,
Belgium
Brucella meta -omics

• Observations
• Field is generally aware of the Resource Center
  and of the Caprion PRC
• Current collaborations driven by scientists very
  familiar with similar type data (early adopter
  phenotype?)
• Early adopters key to getting momentum going
• Publications would increase momentum

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SUBMITTED DATA USE

• Experiment
• Sent 4 Caprion scientists to the Resource Center
  website between May 5 27.
• Only instruction find and use the Brucella data
• Provide feedback on experience, suggestions

• Feedback suggestions
• Brucella home page
• Master data page
• General

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FEEDBACK SUGGESTIONS

• Brucella home page
• Place prominently a table describing the
  experiments contained in the site
• Need to understand experimental design, upfront
• Link experiments to available protocols, upfront
• Need ability to view data by experiment
• Current home page is misleading 4440 proteins
  are listed under Brucella, but that includes
  pathogen proteins many host proteins. Host is
  not indicated until you get to the master sheet
• Master sheet
• Need ability to sort data by experiment, and by
  species
• Need to visit several pages to collect the
  experimental data the protein info data for
  each protein. Can we put all this together in one
  place?
• Links to view peptide sequences not obvious (PRC
  ID to Experiment list to Details view)
• AA sequence displayed in the protein info page
  why not overlay the identified peptides there?
• Why not use the BAB nomenclature the website
  specific protein ID limits usefulness

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FEEDBACK SUGGESTIONS

• General
• Some of the datasets submitted identify only
  differentially expressed, condition-specific
  proteins. Other datasets list all detectable
  proteins in the sample. The condition-specific
  differential expression information is absent,
  misleading the reader to assume that all
  experiments are catalogues of all detectable
  proteins.
• Image of 2D gel present. Nice picture, but
  misleading. We dont use 2D gels
• Company name is current in some pages, not so in
  others
• 3 of 4 searchers did not find the link to the
  contact person