The Biomedical Relevance of Microbial Catabolic Diversity

1
The Biomedical Relevance of Microbial Catabolic
Diversity

• John Archer
• Department of Genetics
• University of Cambridge
• j.archer_at_gen.cam.ac.uk

2
Free Radical Theory of Aging

• Harman, 1956
• Auto-oxidative damage ultimately impairs
  metabolic efficiency
• Prediction promotion of oxidative reactions will
  correlate with reduced longevity
• Genetic factors may promote oxidative stress

3
Metabolism

• CellsnutrientO2-gt more cellsCO2H2O
• Energy metabolism derive high energy compounds
  from carbon-energy source
• Anabolism complexity of carbon-containing
  compounds increases
• Catabolism complexity of carbon-containing
  compounds decreases
• Enzyme-catalysed catabolism is highly sensitive
  to oxidative modification of substrate because
  modified substrates may not bind their cognate
  enzyme

4
Degenerative Molecular Markers Characteristics

• Marker often formed by reactive oxygen species
• Marker concentration should increase with age
• Rate of accumulation of the marker should be
  inversely related to longevity of the organism
• Genetic factors influence rate of accumulation
• Aberrant accumulation of marker associated with
  pathology

5
Degenerative Molecular Markers Candidates

• Lipofuscin
• Ceroid-lipofuscin
• Modified lipids (especially cholesterol) in foam
  cells leading to atherosclerosis
• N-retinyl-N-retinylidene ethanolamine (A2E) in
  retinal pigment epithelial cells

6
Degenerative Markers or Causative Agent?

• Lipofuscin may not be direct cause of aging. At
  moderate levels it has no effect on RER in
  neurons, but in high levels (75 of pericarion)
  is deleterious to neuronal adaptability. LSD are
  strongly linked to ceroid lipofuscin
  accumulation.
• Atheroma is correlated with coronary disease and
  is a clear causative agent.
• N-retinyl-N-retinylidene ethanolamine (A2E) in
  retinal pigment epithelial cells may have a role
  age-related macular degeneration

7
Enzyme Addition Therapy

• Degenerative marker compounds accumulate because
  they are not substrates for normal lysosomal
  enzymes
• Degenerative markers do not accumulate in the
  environment there must be enzymes which can
  process these molecules
• Can one identify enzymes from other living
  systems that can recognise degenerative marker
  compounds?
• Brady et al., mannose-terminal glucocerebrosidase
  treatment for Gaucher's Disease

8
The Substrate Lipofuscin

• 30-70 protein (standard amino acids)
• 20-50 lipid (triglycerides, fatty acids,
  cholesterol, phospholipids, dolichol,
  phosphorylated dolichol)
• Fe, and other heavy metals
• Autofluorescent compounds 1,4-dihydropyridines,
  2-hydroxy-1,2-dihydropyrrol-3-ones?
• Resistant to lysosomal enzymes

9
Rhodococcus Metabolic Diversity

• Rhodococcus harmless, Gram-positive Actinomycete
  mycolic acid bacterium
• Genome is sequenced gt7 Mb
• Thousands of catabolic genes, specific for a vast
  range of carbon-energy sources
• Aliphatic, halogenated hydrocarbons, halogenated
  aromatics (pentachlorophenol), BTEX, PAH,
  Nitroaromatics, Lignin-related, alkoxy aromatics,
  terephthalates, heteroaromatics, steroids,
  dioxane, tetrahydrofuran etc. etc..

10
Isolation Protocol

• Rhodococcus is an oligotrophic bacterium, highly
  adapted to catabolise complex, recalcitrant
  mixtures of substrates simultaneously (no
  catabolic repression)
• Provide 80-100 microMolar lipofuscin as sole
  carbon-energy source to Rhodococcus strains.
  Incubate and score.

11
Rhodococcus Catabolism of Lipofuscin

• Demonstrated Rhodococcus could utilise
  lipofuscin, or components of lipofuscin, as a
  carbon-energy source
• Rhodococcus is a fungal-like bacterium, possesses
  membrane bound vesicles in which substrates are
  degraded by membrane associated enzyme complexes
• It is very probable that the entire spectrum of
  lipofuscin can be metabolised by Rhodococcus
• We propose that Rhodococcus can act as a source
  of xeno-enzymes to augment human metabolism

12
Atheroma

• Macrophages enter artery wall to recycle modified
  lipoproteins entrapped
• Recalcitrant modified lipoprotein products
  accumulate in foam cell lysosome
• Lysosomal function impaired
• Additional macrophage are recruited
• Aberrant proliferative response by vascular
  smooth muscle cells
• Formation of atherosclerotic plaque

13
Rhodococcus and Atherosclerosis

• Rhodococcus can utilise cholesterol as a sole
  carbon-energy source
• Both extracellular and intracellular membrane
  bound cholesterol oxidases are characterised
• Reaction catalysed by cholesterol oxidase-
• Cholesterol ---gt 4-cholesten-3-one
• We propose that Rhodococcus can act as a source
  of xeno-enzymes to augment catabolism of
  atherosclerotic plaque

14
Supporting Indications

• Cross-talk Problems
• Substrate specificity of the bacterial
  xeno-enzyme will restrict the level of cross-talk
  between the bacterial enzyme and the human
  metabolism
• Delivery to lysosomal compartment
• Mannose-terminal glucocerebrosidase treatment of
  Gaucher's Disease
• Lysosomal targeting by glycosylation
• Acid pH of lysosomal compartment
• Enzyme properties can be engineered in vitro
• Immune response
• Small sample data, but promising so far

15
Steps to Biomedical Application of Xenohydrolases

• Isolate competent enzymes using a genomics
  approach
• Engineer the recombinant protein for lysosomal
  targeting
• Partner
• Competence assay in cell system
• Murine tests
• Assay competence in disease models

16
Conclusions

• Lipofuscin, a degenerative molecular marker and
  component of several lysosomal storage diseases
  can be catabolised completely or partially by
  enzyme(s) encoded by the bacterium Rhodococcus
• Rhodococcus can catabolise several components of
  atheroma
• It is highly likely that recalcitrant lysosomal
  components can be removed by xeno-enzyme treatment