Medical Bioremediation

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Medical bioremediation is the technique of applying microbial xenoenzymes in human therapy. The process involves screening for enzymes capable of catabolizing the target pathogenic substrate, engineering microbes to express sufficient quantities of the enzyme and finally delivering the enzyme to the appropriate tissue and cell types.

Introduction

Bioremediation

Bioremediation is the technique of using organisms to catabolize toxic waste such as oil spills or industrial runoff. The most commonly used organisms are microbes, though phytoremediation is also used. ¹ Wild-type microbes have proven capable of digesting highly toxic and stable compounds, but organisms can be genetically engineered to augment their ability. For example, Deinococcus radiodurans, the most radio-resistant organism known, has been modified to digest toluene and ionic mercury. ²

Microbes are the source of approximately 22,500 bioactive drug compounds. Of these, 17% were from unicellular bacteria (mainly Pseudomonas and Bacillus), 45% from filamentous bacteria (actinomycetes) and 38% from fungi. ³ Microbes are the predominant source of manufactured protein, ever since microbial human insulin production began 25 years ago. There are presently more than 130 protein therapeutics used worldwide and many more undergoing clinical trials. ⁴

Organic, energy-rich molecules introduced to the environment are potential microbial nutrients. The “microbial infallibility hypothesis,” coined by Ernest Gayle in 1952, ⁵ states that the buildup of compounds initially resistant to biodegradation exerts a strong selective pressure on nearby microbes to evolve to consume them.

Strategies for Engineered Negligible Senescence (SENS)

In 2002, Cambridge biogerontologist Aubrey de Grey theorized that the principles of bioremediation and Gayle’s hypothesis could be applied to human pathology as a part of his seven-part longevity protocol, Strategies for Engineered Negligible Senescence (SENS): ⁶

1. Cell loss or atrophy (without replacement) ⁷
2. Oncogenic nuclear mutations and epimutations ⁸
3. Cell senescence (death-resistant cells) ⁹
4. Mitochondrial mutations ¹⁰
5. Intracellular junk (particularly lysosomal aggregates) ¹¹
6. Extracellular aggregates ⁹
7. Random extracellular cross-linking (Advanced glycation end products) ¹²

Medical bioremediation is applicable to protocols five, six and seven, termed “LysoSENS,” “AmyloSENS” and “GlycoSENS.” The SENS Foundation is funding research in these areas at their headquarters in Mountain View, California and externally, led by researchers such as David Spiegel at Yale, Chris Lowe at Cambridge, Sudhir Paul at the University of Texas-Houston Medical School, Brian O’Nuallain at Harvard, John Schloendorn at Arizona State University and Jacques Mathieu at Rice University.

Recognizing that bone is all that remains of the deceased in graveyards, de Grey made it obvious that there must be soil microbes capable of catabolizing all of the organic compounds comprising the human body. Certain such compounds are pathogenic, including amyloids, neurofibriliary tangles, cholesterol, lipofuscin, huntingtin, alpha-synuclein and many others. The rapid selective pressure on microbes in the presence of these pathogenic compounds as food sources may yield therapeutic new enzymes. De Grey coined the term “medical bioremediation” to describe this emerging therapy.

The Machinery of Cellular Catabolism

Cellular components must constantly be recycled due to damage and poor construction. Proteasomes are responsible for the simpler catabolic jobs, but major evisceration is carried out by the lysosome. Lysosomes were discovered in 1949 by Christian de Duve, for which we won the 1974 Nobel Prize in Physiology or Medicine. ¹³ Lysosomes are highly acidic (pH 4.8) vesicular structures that serve as the cell’s primary waste disposal system. Sometimes called “suicide-bags,” these globules fuse with and release hydrolytic enzymes into the autophagic vacuolues which engulf malfunctioning cellular structures.

diagram

Certain pathogenic molecules build up at a rate that indicates they are impervious or highly resistant to catabolism. Some enzymes are rate-limiting – for example, when deconstructing a car, one needs many different tools in sequence. The lack of any one of them can halt the process. This fact is a double-edged sword, however: although numerous enzymes are involved in any given metabolic pathway, it may be the case that few will be required to transform the problem substance into a compound that the mammalian metabolism can already handle. It is a matter of tipping the scale in the patient’s favor, not guiding the entire reaction from start to finish.

Lysosomes are fallable. Certain molecules, like lipofuscin or “age pigment” builds up within clogged lysosomes called residual bodies. This process progresses at a set rate during “normal” aging – some medical conditions arise due to a faster rate of aggregation.

Lysosomal Storage Disorders

Lysosomal malfunction can lead to lysosomal storage diseases (LSDs), which are a group of genetically recessive disorders resulting from deficiencies of acid hydrolases. They have a prevalence of 1 per 7700 live births and constitute a significant burden on society. These include disorders such as Gaucher’s, Pompe’s, Krabbe’s and Febry’s diseases, which are treated with intravenous enzyme replacement therapy (ERT). ¹⁴ In the future, these conditions may be treated with gene therapy. LSDs are relevant to medical bioremediation for other conditions as a proof-of-concept for enzyme supplementation.

Medical Applications

Pathology

Atherosclerosis

Atherosclerosis is the condition of arterial hardening due to plaque buildup. This process may result in the formation of a thrombus or clot that occludes blood flow (stroke or heart attack). Beginning in childhood, ¹⁵ atherosclerotic plaques slowly form on the endothelium of blood vessels. These plaques are composed of cholesterol, triglycerides and foam cells. Cholesterol and triglycerides are bound to and deposited by low-density lipoproteins (LDL) and are carried away by high-density lipoproteins (HDL). The “unstable” plaques most prone to rupture are inflamed and composed of macrophages and foam cells rather than the “stable” plaques composed of more extracellular matrix and smooth muscle cells. The LDL molecules in the atheroma become toxic after being oxidized by free radicals, triggering immune cells to migrate to the site. Macrophages and T-lymphocytes are unable to catabolize the LDL and become lipid-loaded foam cells. The lysosomes of these foam cells are precisely what fill with lipids. ^{16, 17} Lysosomes require a low pH for enzymatic function; this is established by vacuolar ATPases (proton pump). The over abundance of free cholesterol within the foam cells may actually inhibit the proton pumps, generating a positive feedback loop. ¹⁸ Lipase enzymes are responsible for triglyceride degradation and could be applied in enzyme replacement therapy. ¹⁹

Macular Degeneration

Photons are absorbed by retinal, which transmits the signal into electrical impulses carried by the optic nerve and then to the visual cortex. The retinal itself stereoisomerises from 11-cis-retinal to all-trans-retinal, then to all-trans-retinol to 11-cis-retinol and back to 11-cis-retinal. Occassionally, all-trans-retinal reacts with the membrane phospholipid phosphatidylethanolamine for form a compound called A2E that builds up in the photoreceptor cells and retinal pigmented epithelial cells (which conduct lysosomal recycling). A2E cannot be catabolized by any enzyme and builds up in the RPE cells, constituting 20% of cellular mass by old age,²⁰ leading to vision issues. Engineering microbes to produce an enzyme capable of catabolizing A2E may attenuate the costly effects of macular degeneration, and some peroxidases have proven capable of A2E degradation. ²¹

Neurodegeneration

Unwanted molecules also build up in diseases of the nervous system. Alzheimer’s dementia, an emerging epidemic, is characterized by the presence of two culprits: a 40-43 amino acid extracellular amyloid-β peptide and the 352-441 amino acid hyperphosphorylated, microtubule-associated tau proteins which form neurofibrillary tangles.²² α-synuclein is another peptide putatively involved with both dementia and Parkinson’s disease. Huntingtin is a peptide aggregate involved with Huntington’s disease. Various neural pathologies are caused or exacerbated by the buildup of particular peptide molecules into aggrosomes. ²³ It has been demonstrated aggrosomes can be degraded by that glial cells called Schwann cells, ²⁴ furthermore lysosomal turnover can be pharmacologically stimulated to reduce aggregate accumulation and ameliorate neurological deficits in mice.²⁵ Proteolytic enzyme supplementation could again be used to treat neurological conditions caused by unwanted peptide aggregation.

Microbial Engineering Advancements

Microbial Degradation of 7-Ketocholesterol

7-ketocholesterol (7KC) is an oxidized derivative of cholesterol, a plasma membrane stabilizer and hormone precursor. 7KC is suspected of involvement in both atherosclerosis,²⁶ macular degeneration ²⁷and Alzheimer’s ²⁸ as a pro-oxidant and pro-inflammatory. ²⁹ 7KC is the most commonly consumed dietary oxysterol has been shown to accumulate in human plasma. ³⁰ Mammalian enzymes are capable of degrading 7KC, but they’re not found in the lysosome.

Although bacteria do not have cholesterol, they haves structurally similar “surrogate” compounds called hopanoids, which confer membrane stability in a similar fashion. ³¹ Despite this absence, Mathieu et al. (2008) demonstrated that several microbial species are capable of degrading cholesterol as a sole carbon source, including γ-Proteobacterium Y-134, Sphingomonas sp. JEM-1, Nocardia nova, Rhodococcus sp. RHA1 and Pseduomonas aeruginosa. ^{32, 33} Nocardia nova was most effective, degrading nearly 100% of the 7KC substrate in 25 days (30°C, pH 7). The runners up, γ – Proteobacterium and Sphingomonas were only able to catabolize about 80% at 25 days. This discrepancy may be due to Nocardia’s ability to produce biosurfactants ³⁴ that dissolve the hydrophobic 7KC and increase the surface area available for enzymatic binding.

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These microbes possess oxygenase enzymes that bind molecular oxygen with hydrogen and attach the hydroxyl groups onto highly reduced aromatics. As these new hydroxyl groups are attached, the molecule becomes susceptible to dehydrogenase and hydroxylase attacks. ³⁵ This may tip the scale and enable mammalian enzymes to act on the sterol substrate.

7KC resistance in fibroblasts transfected with a lysosomally targeted Chromobacterium oxidase

In oxidized LDL-loaded macrophage foam cells, which are a primary component of atherosclerotic plaques, approximately 54% of the free 7KC is inside the lysosome. ³⁶

High levels of 7KC cause lysosomal membrane permeabilization (LMP), leading to leakage of protease enzymes (like cathepsins), apoptosis and necrosis. ³⁷

Mathieu et al. (2012) discovered that, unlike other enzymes capable of catabolizing 7KC, a novel cholesterol oxidase from Chromobacterium sp. DS1 was able to withstand the low pH, proteases and lack of cofactors that characterize the lysosome. ³⁸

The authors localized DS1 ChOx to the lysosome by fusing Lysosomal Associated Membrane Protein 1 (LAMP1) to both the C terminus and N terminus of the cholesterol oxidase peptide.

At all but the highest interval of 7KC exposure (0, 25, 37.5, 50 µM but not 100 µM) the ChOx-LAMP1 construct afforded significant protection (maximum of 160% increase in cell viability at µ37.5mM compared to control). Cytoplasmically targeted ChOx a statistically insignificant level of protection. The authors conclude that this study “strongly suggests that reducing lysosomal oxysterol concentrations is promising for mitigating oxysterol toxicity.”

Amyloid Beta Degradation by Mycoplasma hyorhinis

Amyloid beta is an extracellular aggregate protein putatively implicated in Alzheimer’s dementia. Zhao, H. et al. (2008) found that Mycoplasma hyorhinis is capable of degrading A-β. It is estimated that 5% to 35% of cell cultures in current use are infected with various mycoplasma species.³⁹ Mycoplasmas are the smallest and simplest known self-replicating bacteria and the consequences of infection range from no apparent effect to apoptosis induction. The researchers observed no accumulation of A-βin mycoplasma-positive cells transfected with A-β precursor protein, whereas eradication of M. hyorhinis using a quinolone-based antibiotics restored extracellular A-β accumulation. ⁴⁰

Microbial Enzyme Screening

Leveraging the microbial infallibility hypothesis to find candidate genes and hydrolytic enzymes is the truly novel concept in medical bioremediation and microbial ecology is the key discipline. Since the 1970s, bioremediation has become more sophisticated, largely due to the realization that microbes are syntrophic, digesting compounds as a chain of hydrolytic, oxidative and reductive reactions across multiple species. These organisms also show redundancy, in that different species carry out the same steps using similar enzymes.

To harvest the critical xenoenzymes, the microbe must be identified and isolated. Given that less than 1% of microbial species known and culturable, we must use techniques that allow both accuracy and speed. These techniques include molecular fingerprinting and DNA microarray analysis. Typically, 16S SSU rRNA is used to discern the phylogentic identity of a sample microbe. 16S rRNA is used because it is conserved across species and serves as an accurate molecular clock. ⁴¹

The DNA/RNA isolation process, in brief, is as follows:

1. Lysis: The sample microbe’s cell wall is lysed using a detergent (commonly sodium dodecyl sulfate) or sonication.
2. Filtration Extraction: One may employ a column-filtration system, but the centrifugal phenol-chloroform extraction is most commonly used. Proteases are added to separate the DNA and RNA from associated proteins. Depending on whether one wishes to extract DNA or RNA, the appropriate DNAse or RNAse must be added.
3. Isolation: The extraction solution is mixed with water-saturated phenol, chloroform and the denaturing agent guanidinium thiocyanate, resulting in a phase separation with an upper aqueous phase and a lower organic phase (phenol). Nucleic acids are partitioned into the aqueous phase and protein is partitioned into the organic phase. The size, charge and structure of the isolated nucleic acids can be identified using gel electrophoresis.

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Then, newly identified microbes demonstrating the desired catabolic ability would need to be sequenced using a DNA microarray. With the sequence in hand, loss-of-function knockout methods (like random transposon-mediated mutagenesis) can painstakingly illuminate DNA sequences that code for the target catabolic enzyme(s). Then, it may be possible to constitutively overexpress the enzyme-coding gene and recover the viable product.

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Conclusion

Overall paper length should be 3,000 words, with at least 3 figures.

Electron micrograph of the Ebola Zaire virus. This was the first photo ever taken of the virus, on 10/13/1976. By Dr. F.A. Murphy, now at U.C. Davis, then at the CDC.

At right is a sample image insertion. It works for any image uploaded anywhere to MicrobeWiki. The insertion code consists of:
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Legend/credit: Electron micrograph of the Ebola Zaire virus. This was the first photo ever taken of the virus, on 10/13/1976. By Dr. F.A. Murphy, now at U.C. Davis, then at the CDC.
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Subscript: H₂O
Superscript: Fe³⁺

References

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Edited by Sebastian Aguiar, a student of Nora Sullivan in BIOL187S (Microbial Life) in The Keck Science Department of the Claremont Colleges Spring 2013.