Transcript The Biomedical Relevance of Microbial Catabolic Diversity

The Biomedical Relevance of
Microbial Catabolic Diversity
John Archer
Department of Genetics
University of Cambridge
[email protected]
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
Metabolism
Cells+nutrient+O2-> more cells+CO2+H2O
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
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
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
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
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
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, 2hydroxy-1,2-dihydropyrrol-3-ones?
Resistant to lysosomal enzymes
Rhodococcus Metabolic Diversity
Rhodococcus harmless, Gram-positive
Actinomycete mycolic acid bacterium
Genome is sequenced >7 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..
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 carbonenergy source to Rhodococcus strains. Incubate and
score.
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
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
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 ---> 4-cholesten-3-one
We propose that Rhodococcus can act as a source of xenoenzymes to augment catabolism of atherosclerotic plaque
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
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
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