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The Biomedical Relevance The Biomedical Relevance of Microbial Catabolic of Microbial Catabolic Diversity Diversity John Archer John Archer Department of Genetics Department of Genetics University of Cambridge University of Cambridge [email protected] [email protected]

The Biomedical Relevance of Microbial Catabolic Diversity

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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 - PowerPoint PPT Presentation

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Page 1: The Biomedical Relevance of Microbial Catabolic Diversity

The Biomedical Relevance of The Biomedical Relevance of Microbial Catabolic DiversityMicrobial Catabolic Diversity

John ArcherJohn Archer

Department of GeneticsDepartment of Genetics

University of CambridgeUniversity of Cambridge

[email protected]@gen.cam.ac.uk

Page 2: The Biomedical Relevance of Microbial Catabolic Diversity

Free Radical Theory of AgingFree Radical Theory of Aging

Harman, 1956Harman, 1956

Auto-oxidative damage ultimately impairs Auto-oxidative damage ultimately impairs metabolic efficiencymetabolic efficiency

Prediction: promotion of oxidative reactions will Prediction: promotion of oxidative reactions will correlate with reduced longevitycorrelate with reduced longevity

Genetic factors may promote oxidative stressGenetic factors may promote oxidative stress

Page 3: The Biomedical Relevance of Microbial Catabolic Diversity

MetabolismMetabolism

Cells+nutrient+OCells+nutrient+O22-> more cells+CO-> more cells+CO22+H+H22OO

Energy metabolism: derive high energy compounds Energy metabolism: derive high energy compounds from carbon-energy sourcefrom carbon-energy source

Anabolism: complexity of carbon-containing Anabolism: complexity of carbon-containing compounds increasescompounds increases

Catabolism: complexity of carbon-containing Catabolism: complexity of carbon-containing compounds decreasescompounds decreases

Enzyme-catalysed catabolism is highly sensitive to Enzyme-catalysed catabolism is highly sensitive to oxidative modification of substrate because modified oxidative modification of substrate because modified substrates may not bind their cognate enzymesubstrates may not bind their cognate enzyme

Page 4: The Biomedical Relevance of Microbial Catabolic Diversity

Degenerative Molecular Markers: CharacteristicsDegenerative Molecular Markers: Characteristics

Marker often formed by reactive oxygen speciesMarker often formed by reactive oxygen species

Marker concentration should increase with ageMarker concentration should increase with age

Rate of accumulation of the marker should be inversely Rate of accumulation of the marker should be inversely related to longevity of the organismrelated to longevity of the organism

Genetic factors influence rate of accumulationGenetic factors influence rate of accumulation

Aberrant accumulation of marker associated with Aberrant accumulation of marker associated with pathologypathology

Page 5: The Biomedical Relevance of Microbial Catabolic Diversity

Degenerative Molecular Markers: CandidatesDegenerative Molecular Markers: Candidates

LipofuscinLipofuscin

Ceroid-lipofuscinCeroid-lipofuscin

Modified lipids (especially cholesterol) in foam cells Modified lipids (especially cholesterol) in foam cells leading to atherosclerosisleading to atherosclerosis

N-retinyl-N-retinylidene ethanolamine (A2E) in retinal N-retinyl-N-retinylidene ethanolamine (A2E) in retinal pigment epithelial cellspigment epithelial cells

Page 6: The Biomedical Relevance of Microbial Catabolic Diversity

Degenerative Markers or Causative Agent?Degenerative Markers or Causative Agent?

Lipofuscin may not be direct cause of aging. At moderate Lipofuscin may not be direct cause of aging. At moderate levels it has no effect on RER in neurons, but in high levels it has no effect on RER in neurons, but in high levels (75% of pericarion) is deleterious to neuronal levels (75% of pericarion) is deleterious to neuronal adaptability. LSD are strongly linked to ceroid lipofuscin adaptability. LSD are strongly linked to ceroid lipofuscin accumulation.accumulation.

Atheroma is correlated with coronary disease and is a clear Atheroma is correlated with coronary disease and is a clear causative agent.causative agent.

N-retinyl-N-retinylidene ethanolamine (A2E) in retinal N-retinyl-N-retinylidene ethanolamine (A2E) in retinal pigment epithelial cells may have a role age-related pigment epithelial cells may have a role age-related macular degenerationmacular degeneration

Page 7: The Biomedical Relevance of Microbial Catabolic Diversity

Enzyme Addition TherapyEnzyme Addition Therapy

Degenerative marker compounds accumulate because they Degenerative marker compounds accumulate because they are not substrates for normal lysosomal enzymesare not substrates for normal lysosomal enzymes

Degenerative markers do not accumulate in the Degenerative markers do not accumulate in the environment – there must be enzymes which can process environment – there must be enzymes which can process these moleculesthese molecules

Can one identify enzymes from other living systems that Can one identify enzymes from other living systems that can recognise degenerative marker compounds?can recognise degenerative marker compounds?

Brady et al., mannose-terminal glucocerebrosidase Brady et al., mannose-terminal glucocerebrosidase treatment for Gaucher's Disease treatment for Gaucher's Disease

Page 8: The Biomedical Relevance of Microbial Catabolic Diversity

The Substrate LipofuscinThe Substrate Lipofuscin

30-70% protein (standard amino acids)30-70% protein (standard amino acids)

20-50% lipid (triglycerides, fatty acids, cholesterol, 20-50% lipid (triglycerides, fatty acids, cholesterol, phospholipids, dolichol, phosphorylated dolichol)phospholipids, dolichol, phosphorylated dolichol)

Fe, and other heavy metalsFe, and other heavy metals

Autofluorescent compounds 1,4-dihydropyridines, 2-Autofluorescent compounds 1,4-dihydropyridines, 2-hydroxy-1,2-dihydropyrrol-3-ones?hydroxy-1,2-dihydropyrrol-3-ones?

Resistant to lysosomal enzymesResistant to lysosomal enzymes

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RhodococcusRhodococcus Metabolic Diversity Metabolic Diversity

Rhodococcus harmless, Gram-positive Rhodococcus harmless, Gram-positive Actinomycete mycolic acid bacteriumActinomycete mycolic acid bacterium

Genome is sequenced >7 MbGenome is sequenced >7 Mb

Thousands of catabolic genes, specific for a vast Thousands of catabolic genes, specific for a vast range of carbon-energy sourcesrange of carbon-energy sources

Aliphatic, halogenated hydrocarbons, halogenated Aliphatic, halogenated hydrocarbons, halogenated aromatics (pentachlorophenol), BTEX, PAH, aromatics (pentachlorophenol), BTEX, PAH, Nitroaromatics, Lignin-related, alkoxy aromatics, Nitroaromatics, Lignin-related, alkoxy aromatics, terephthalates, heteroaromatics, steroids, dioxane, terephthalates, heteroaromatics, steroids, dioxane, tetrahydrofuran etc. etc..tetrahydrofuran etc. etc..

Page 10: The Biomedical Relevance of Microbial Catabolic Diversity

Isolation ProtocolIsolation Protocol

RhodococcusRhodococcus is an oligotrophic bacterium, highly is an oligotrophic bacterium, highly adapted to catabolise complex, recalcitrant mixtures of adapted to catabolise complex, recalcitrant mixtures of substrates simultaneously (no catabolic repression)substrates simultaneously (no catabolic repression)

Provide 80-100 microMolar lipofuscin as sole carbon-Provide 80-100 microMolar lipofuscin as sole carbon-energy source to energy source to Rhodococcus strains. Incubate and Rhodococcus strains. Incubate and score.score.

Page 11: The Biomedical Relevance of Microbial Catabolic Diversity

RhodococcusRhodococcus Catabolism of Lipofuscin Catabolism of Lipofuscin

Demonstrated Demonstrated RhodococcusRhodococcus could utilise lipofuscin, or could utilise lipofuscin, or components of lipofuscin, as a carbon-energy sourcecomponents of lipofuscin, as a carbon-energy source

RhodococcusRhodococcus is a fungal-like bacterium, possesses membrane is a fungal-like bacterium, possesses membrane bound vesicles in which substrates are degraded by membrane bound vesicles in which substrates are degraded by membrane associated enzyme complexesassociated enzyme complexes

It is very probable that the entire spectrum of lipofuscin can be It is very probable that the entire spectrum of lipofuscin can be metabolised by metabolised by RhodococcusRhodococcus

We propose that We propose that Rhodococcus can act as a source of xeno-Rhodococcus can act as a source of xeno-enzymes to augment human metabolismenzymes to augment human metabolism

Page 12: The Biomedical Relevance of Microbial Catabolic Diversity

AtheromaAtheroma

Macrophages enter artery wall to recycle modified Macrophages enter artery wall to recycle modified lipoproteins entrappedlipoproteins entrapped

Recalcitrant modified lipoprotein products accumulate in Recalcitrant modified lipoprotein products accumulate in foam cell lysosomefoam cell lysosome

Lysosomal function impairedLysosomal function impaired

Additional macrophage are recruitedAdditional macrophage are recruited

Aberrant proliferative response by vascular smooth Aberrant proliferative response by vascular smooth muscle cellsmuscle cells

Formation of atherosclerotic plaqueFormation of atherosclerotic plaque

Page 13: The Biomedical Relevance of Microbial Catabolic Diversity

Rhodococcus and AtherosclerosisRhodococcus and Atherosclerosis

Rhodococcus can utilise cholesterol as a sole carbon-Rhodococcus can utilise cholesterol as a sole carbon-energy sourceenergy source

Both extracellular and intracellular membrane bound Both extracellular and intracellular membrane bound cholesterol oxidases are characterisedcholesterol oxidases are characterised

Reaction catalysed by cholesterol oxidase:-Reaction catalysed by cholesterol oxidase:-

Cholesterol ---> 4-cholesten-3-oneCholesterol ---> 4-cholesten-3-one

We propose that Rhodococcus can act as a source of xeno-We propose that Rhodococcus can act as a source of xeno-enzymes to augment catabolism of atherosclerotic plaqueenzymes to augment catabolism of atherosclerotic plaque

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Supporting IndicationsSupporting Indications

Cross-talk ProblemsCross-talk ProblemsSubstrate specificity of the bacterial xeno-enzyme will restrict the Substrate specificity of the bacterial xeno-enzyme will restrict the level of cross-talk between the bacterial enzyme and the human level of cross-talk between the bacterial enzyme and the human metabolismmetabolism

Delivery to lysosomal compartmentDelivery to lysosomal compartmentMannose-terminal glucocerebrosidase treatment of Gaucher's Mannose-terminal glucocerebrosidase treatment of Gaucher's DiseaseDisease

Lysosomal targeting by glycosylationLysosomal targeting by glycosylation

Acid pH of lysosomal compartmentAcid pH of lysosomal compartmentEnzyme properties can be engineered Enzyme properties can be engineered in vitroin vitro

Immune responseImmune responseSmall sample data, but promising so farSmall sample data, but promising so far

Page 15: The Biomedical Relevance of Microbial Catabolic Diversity

Steps to Biomedical Application of Steps to Biomedical Application of XenohydrolasesXenohydrolases

Isolate competent enzymes using a genomics approachIsolate competent enzymes using a genomics approach

Engineer the recombinant protein for lysosomal targetingEngineer the recombinant protein for lysosomal targeting

PartnerPartner

Competence assay in cell systemCompetence assay in cell system

Murine testsMurine tests

Assay competence in disease modelsAssay competence in disease models

Page 16: The Biomedical Relevance of Microbial Catabolic Diversity

ConclusionsConclusions

Lipofuscin, a degenerative molecular marker and Lipofuscin, a degenerative molecular marker and component of several lysosomal storage diseases can be component of several lysosomal storage diseases can be catabolised completely or partially by enzyme(s) catabolised completely or partially by enzyme(s) encoded by the bacterium encoded by the bacterium RhodococcusRhodococcus

Rhodococcus can catabolise several components of Rhodococcus can catabolise several components of atheromaatheroma

It is highly likely that recalcitrant lysosomal components It is highly likely that recalcitrant lysosomal components can be removed by xeno-enzyme treatmentcan be removed by xeno-enzyme treatment