229 replies to this topic

[John Schloendorn]

Update:
Earlier than I had dared to hope, we have obtained two new cultures that efficiently degrade one of our major targets, 7-ketocholesterol. They reproducibly deplete it from the medium within 10 days, which is more rapid than anything we have seen before, and utilize it as a growth substrate. Now the fun will start to isolate the bugs from the mixed cultures and take them apart. One of the cultures originated from a mixed inoculum containing the early samples submitted by QJones and Mind and a few other donated garden samples. We cannot tell in which submission precisely they originated, since we pool several samples for efficiency. (you have to submit a couple of them to get your own exclusive pool ;-) The other culture came from cemetary samples collected by ourselves. Both cultures seem equally efficient, look alike and it could be the same organism.

Eternal thanks to our soil donors who made this wonderful result possible, together.

We have also gratefully received all other submissions announced in this thread, and more. These samples are now targeting A2E, for which we expect results in the coming weeks.

[Bruce Klein]

Great... thanks for the updates!

[olaf.larsson]

Congratulations!!!
Could you post a picture of this beautifull culture?

[John Schloendorn]

Heh, sorry, it is actually not beautiful at all [sfty]

Attached Files

•  culture.jpg   35.76KB   0 downloads

[JonesGuy]

This really made my day!

Are you saying you couldn't get speedy decomposition before, when you had tried other types of soil samples? Or was there success on the first batch of dirt?

Anyway, it's nice to see my name in lights!

[olaf.larsson]

he he..I assumed it they were on agar plates. What is the koncentration of 7KC in this solution?

[John Schloendorn]

Well well... please keep in mind that failure can come in many ways. The organisms might not grow on plates, compromising isolation, the genetics might not work out as we hope, the enzyme may be toxic and so on...

QJones, there were five other 7KC cultures that did not grow or degrade 7KC. It is possible that we previously had one that degraded it more slowly. So it seems that 7KC degraders are so abundant in nature that a medium-throughput experiment like this can reliably detect them. We did not know this before, so I would call it luck.

Wolfram, there used to be 0.1%, but now very little is left (perhaps some 0.001%), approaching the lower limit of detectability. The residual flocks you see in the bottle are probably soil, clumped bacteria, and perhaps to a small degree insoluble intermediate products.

[Mind]

It is soooo beautiful!

[eternaltraveler]

It's a beautiful sight to behold to me as well

[jmmathieu]

It's good to see that you've made so much progress. I found it easy to isolate 7KC degraders as well, but I didn't get anything from the soil as I expected. My own colonies were from an oil refinery's wastewater. DGGE shows that I've isolated at least two pure strains, but the others are mixed. It will be interesting to see what you get when you attempt A2E degradation.

[John Schloendorn]

Hey Jacques, great you're here, I just wrote you an email about this ;-) We need to make better plans on how to "divide and conquer" this field.

[jrhall]

Hey John,

Do you think the bacteria that are breaking down the 7KC already had some 7KC degrading protein/enzyme, or do you think they may have modified an existing protein/enzyme to do the job?

[John Schloendorn]

Jeff, I would guess that 7KC catabolism is almost always to be seen as a side-effect of cholesterol catabolism, because they are structurally very similar and cholesterol is way more abundant in nature. Cholesterol oxidase, which catalyzes the the first step in cholesterol breakdown comes in several variations among different microbes, and some of these variations are known to accept other compounds, such as steroid hormones, which bear sufficient similarity to cholesterol.
This would leave 7KC degrading bacteria with two basic strategies: Modify all (10-20) enzymes in the cholesterol pathway such that they also work on 7KC, or have a single dedicated enzyme that would reduce 7KC back to cholesterol, so that it can then be chased down the cholesterol pathway, or any mixture of the two (i.e. reducing an intermediate product). From a therapeutic viewpoint I think 7KC reduction would be most worth going for, but we have to take what the microbes give us. To pin down the best enzymatic systems, it is particularly great to have access to the large diversity of samples that is made possible by this soil donor program.

[guybryant]

John,
I have just mailed you two samples for water/silt from two different underwater cave systems in north Florida. Hope you get them soon.
Guy

[jrhall]

An interesting experiment might be to first isolate and sequence the enzyme that is breaking down the 7KC, assuming it has a dedicated 7KC enzyme. Then let the bacteria 'fight it out' for a few hundred (thousand?) generations on a diet of only 7KC and see if the enzyme sequence has changed as the bacteria adapts. Maybe this would give you an enzyme better targeted to 7KC and less likely to be toxic in humans.

[John Schloendorn]

Guy, great many thanks!

Jeff, yes, such experiments are under consideration. One project (I'm not involved in) might even seek to mutagenize existing cholesterol oxidase into accepting 7KC. The method of these mutagenesis experiments is slightly different, though. You would use some sort of mobile genetic element (transposon) to deliberately shuffle parts of the gene around, to arrive at a library of mutant genes. The library would then be transfected back into the host, or a suitable surrogate host, and screened for 7KC degradation just like any environmental sample (saving the thousands of generations -- and who would pay all the 7-keto ;-).

Another idea is to take 7KC processing cholesterol oxidase and mutagenize it to no longer accept plain cholesterol as a substrate with a similar system.

But these are for the future and should depend on some yet unknown properties of these enzyme systems.

[John Schloendorn]

Update: Soil donors are now acknowledged at the Methuselah Foundation's research supporters website.

[olaf.larsson]

The idea of injecting cholesterol eating bacteria sound appealing to me, the technology is in the lower complexity range, compared with genetheraphy, which means that it is doable with compairably little resources and expertise. There are arteosclerotic mice aviable to do the testing on, once this bacteria would have been developed. Would it be a problem if this bacteria included normal cholesterol in their diet?

[JonesGuy]

That probably depends out how voracious they are for cholesterol.

But, I'm quite sure they're not actually looking to inject bacteria, but isolate an enzyme that could help, instead.

[John Schloendorn]

Indeed. Non-pathogenic bacteria are decorated with lost of stuff that would immediately trigger an inflammatory response, much stronger than an enzyme alone. Since atherosclerosis is to a large degree an inflammatory disorder, this would probably be an exceptionally bad thing to do in the lesion. And for the same reasons, whole bacteria would also face much more rapid clearance by the immune system than isolated enzymes.

Gene therapy is another issue. We would need to gene-engineer at least the macrophages, because they are the major cell type involved in atherosclerosis. And here we're really in luck because macrophages come from the bone-marrow. So a gene/cell therapy approach similar to ones already in wide clinical use for non-malignant blood cell disorders would seem feasible. If done right, this should also tolerize the recipient immune system to the foreign enzymes and you could deliver entire pathways, rather than just single genes via an artificial chromosome. I would tend to think this is the preferable route of treatment for atherosclerosis.

Would it be a problem if this bacteria included normal cholesterol in their diet?

If you use only cholesterol oxidase, almost certainly yes, because the product cholest-4-ene-3-one is mildly toxic and can be pro-atherogenic. Cholesterol oxidase is in fact a virulence factor for some animal pathogens (e.g R. equi) that exploit this to damage host immune cells. Even if the enzyme has only a low affinity for cholesterol, this could be a problem, because there is much more cholesterol in the cell than 7KC. Ways to get enzymes that accept 7KC but no cholesterol include looking for them (I'm doing that) or mutagenizing ones that can do both to lose their affinity for cholesterol (someone else is going to do that).
If you use a couple of steps, or a complete degradation pathway, confined to macrophage lysosomes, then probably no, and actively removing cholesterol from the lesion by such a route should in fact be a considerable additional benefit.
So exploring the whole degradation pathway has a rather high priority for me.

Edited by John Schloendorn, 18 January 2006 - 07:17 PM.

[olaf.larsson]

Thank you John for your answers. I see that you have done some heavy reading in the cholesterol area.
Included in the idea of cholesterol eating should be that the bacteria could be coated with some biological non
antigenic material to lower or nullify any immune response.

[caver]

Very creative, and very ambitious. Keep up the hard work Mr. Schloendorn et al.

As for soil samples, lot's of work there. I'm wondering if you couldn't "farm" some of the needed microbes from cadaver tissues. Depositing these in places where such suspect microbes may be present. Like in graveyards! [:o]

[olaf.larsson]

If you have some old meat in the fridge you could burry it in various places and harvest the bacteria after 2 weeks.

[Mind]

As for soil samples, lot's of work there. I'm wondering if you couldn't "farm" some of the needed microbes from cadaver tissues. Depositing these in places where such suspect microbes may be present. Like in graveyards!

We already discussed and turned down the idea of any association with graveyards. From a science standpoint it makes some sense, but it would be terrible publicity.

[John Schloendorn]

Caver: Thanks for the encouragement!

All: Update: After 14 days of culture with over 50 independent soil samples, we have no convincing evidence of A2E degradation. If you can, please continue to contribute, this time with special focus on A2E.

The class of compounds most relevantly similar to A2E are probably retinoids. They are found in fruit, flowers, other colored natural compouds, and are also enriched in some carnivorous vertebrates. (I found no A2E degraders in kitten feces, but maybe a tiger would do. Game anyone?)
Any place associated to the regular presence and/or decomposition of those should be a good place to check out, but please also be creative. Nature is huge and incomprehensibly complex, so seeking out any type of bizarre habitat is in principle a great idea. Retionids aren't exactly infrequent. (I'm really looking forward to giving Guy's latest deep sea ones a shot!)

[Bruce Klein]

John, for reference, do you know how many of the samples were sent via those watching this forum topic?

[Da55id]

I wonder if a slaughterhouse is a good bet? Or a fishery where heads are cut off and discarded?

[John Schloendorn]

Bruce, my guess is about half of the 50 independent samples we had.

Dave, sounds good to me, especially if they have been rotting in the place for years so that all the juicy stuff is already gone. There's only one way to find out.

[Da55id]

well - I live in DC. The only heads that roll around here are political :-) Anybody in Chicago wanna go digging in the old stockyards?

[caver]

AHAHA...Mouse

I have seen large guano piles benieth bat roosts in caves wherein I saw numbers of dead bats in different stages of decomposition. I wonder if this wouldn't be a place to obtain a sample for you guys.