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@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 • 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

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 >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..

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 ---> 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