Tuberculosis: New Insights from Molecular Epidemiology

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Microarray-based screening for BCG deletions
TB H37Rv
BCG
Both strains
16 genomic deletions from BCG vaccines
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Polygenic regions deleted in BCG strains
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Genomic deletions of BCG

• At sequence level, 99 homology
• Dozen regions of M. tuberculosis not in BCG
  IN/DELS
• Most are coding regions
• Truncated open reading frames in BCG
• Mutations in genes for cell wall, regulation,
  antigenic proteins, nutrition

TB
BCG
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Targeted analysis of RD1

• One region (RD1) absent from all BCG strains,
  present in all virulent strains
• RD1 region disrupted in part in M. microti,
  (alternative vaccine), per Brosch 2002
• Function unknown
• 9.5 kB in length
• 9 open reading frames, unknown function
• 2 antigenic proteins ESAT-6, CFP-10
• Goal
• Delete RD1 from H37Rv by allelic exchange
• Study the mutant

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Growth in macrophages
Bacteria / well (x 10(3))
Alamar blue reducing activity ( of day 0)
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Growth in C57BL6 mice
TB TB?RD1 BCG
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Current thoughts on H37Rv?RD1

• Attenuation mutant re-created
• Mutant makes less antigens, yet growth impaired
• The bacteria that makes more antigenic proteins
  grows better in the host!
• Why this happens unclear
• Put RNA from mutant on a microarray and genes
  outside region up/down
• Regulatory mutation

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M. tuberculosis Life cycle
Establish infection
Survive intracellular life
Convert lung into atomizer
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M. tuberculosis Skills needed
Establish infection
Survive intracellular life
Convert lung into atomizer
Get into cell Evade innate defence
Survive immune activation and nutritional
limitations
Induce immunopathology Survive trip through air
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BCG in vitro Skills lost
Establish infection
Survive intracellular life
Convert lung into atomizer
Get into cell Evade innate defence
Survive immune activation and nutritional
limitations
Induce immunopathology Survive trip through air
Altered cell wall
Altered nutritional needs
Loss of antigenic proteins
Loss of regulators
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How about the successful isolates?

• Array/chip based study of virulent isolates of M.
  tuberculosis
• Study of deletions in 19 clinical clones
• Association of genetic deletions with clinical /
  epidemiologic variables

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Deletion hunting with Genechips
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Virulent M. tuberculosis (Contd)

• Average clone missing 2.9 deletions containing
  17.2 ORFs
• On average, clones lack 0.3 of genome (up to
  99.7 genome shared among clinical isolates)

Kato-Maeda M et al. Genome Res. 2001, 11 547-554
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Predicted significance of M. tuberculosis
deletions

• Phages, insertion elements over-represented
• Information pathways underrepresented
• More or less opposite of BCG vaccines
• Clones with most deleted DNA had lowest amount of
  patients with cavitation on chest radiograph

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Phenotype for M. tuberculosis deletions?
cavitation on x-ray
Kato-Maeda M et al. Genome Res. 2001, 11 547-554
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Bacterial work - synopsis

• Can brand the clones
• Can determine important genes in bacterium
• Increasingly finding that
• Phenotype is hard part
• The action is at interaction with host

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How does the host see things?

• Host 90 of infections do not result in disease
• Therefore, disease is anomaly
• Bacteria infection without disease is dead end
• Therefore, disease is necessity
• This does not set up well for compromise
• Best case scenario, have disease in minority

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Determining host factors

• Classic epidemiologic studies
• Genetic epidemiologic studies
• Candidate gene approach
• Agnostic search (whole genome scan)
• Rare genetic diseases
• Animal models mouse genetics
• Acquired immunedeficiency
• Natural experiments, e.g. HIV

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Host factors Epidemiology

• Extremes of age (infants, elderly)
• More likely to develop active TB
• Male sex
• Men outnumber women throughout world
• HIV infection
• Stongest risk factor in causing reactivation of
  previous latent infection
• Also accelerates rate from new infection to rapid
  primary disease
• Smoking, Drinking, Drugs
• Appear to increase risk of TB, but marker of
  exposure or risk factor?

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Host factors Genetic Epi

• Candidate genes from animal models
• Nramp1 a player in the Gambia, Northern Alberta
• Stronger effect in primary disease
• Vitamin D receptor mutations
• Mannose binding protein
• Unidentified gene on X chromosome
• Note all these studies empowered by bacterial
  fingerprinting
• Dont want to lump together reactivation TB with
  primary progressive TB

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Host factors Rare genetic defects

• Occasional families observed with rapidly
  disseminating disease
• Can be disease due to BCG vaccine
• Can include other non-pathogenic Mycobacteria
• Also, occasionally Listeria, Salmonella
• Lesions
• Gamma-interferon receptor
• IL-12
• IL-12 receptor
• Can confirm with e.g. gamma KO mouse

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Host vs. pathogen Key Steps in TB Pathogenesis
revisited

• Bacteria get into the cell
• Is the host phagocytosing or does the bacteria
  actually want to make the macrophage its home?
  Bacteria survive in phagocytes
• Bacteria survive in the cell
• Why is the bacteria so good at this? Is its
  natural home an intracellular niche, such as
  free-living amoebae?

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Host vs. pathogen Key Steps in TB Pathogenesis
- II

• Latency
• Effective immune response by the host, or a great
  way for the bacteria to infect on one continent
  and get a free trip over the pond?
• Tissue destruction
• Whos fault is this? Is the host trying to kill
  bacteria, or is the bacteria trying to get out?

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Concluding thoughts - TB

• TB is a disease involving
• A bacterium
• A host
• An environment
• TB is not the inevitable consequence of these
  getting together but rather results from an
  interplay of all three
• Rational study of problem will require
  interdisciplinary perspective

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Concluding thoughts Infectious Diseases in
general

• When I went to medical school
• Infectious diseases due to agents
• Bug x causes disease X
• Drug D kills bug x
• We should treat the disease not the bacterium
• In future, more attention to role of host in both
  cause of disease and treatment of condition

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Concluding speculation Infectious Diseases

• Dont be surprised in future to learn that many
  infectious diseases are genetic diseases
• E.g. Septic shock from E. coli, Typhoid fever
  from Salmonella
• Converesely, dont be surprised if certain
  genetic diseases have infectious component
• E.g. Crohns disease

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Questions?

• Marcel Behr
• marcel.behr_at_mcgill.ca