Posted 9:08 PM by Paul

Challenges in Microbiology
Here's a little thought experiment for you. You've set yourself up a nice little system for examining the genome of an environmental isolate for genes involved in some important phenotype. Now, there are lots of ways to do such a thing, but lets keep it simple. You use a mutagenic technique (and there are many) to introduce random changes in the genome. You then use your powers of observation to identify colonies (or growth in some other form) that is unusual. When you find a wierd colony, you isolate it for further study.

Is this science?

It is a real question. Many rigorous science theorists (Kuhn, I'm looking at you) would say "no". After all, at this point, you don't have a "falsifiable hypothesis", unless you would suppose "Mutagenesis causes changes in phenotypes" is a falsifiable hypothesis, but that's a bit trivial. However, I'd say "bollocks!" to that (or at least, if I was English I'd say that).

This is the start of science. It's not a full experiment, but rather a way to build hypotheses. And, if you want my opinion, this is the most underappreciated part of the scientific method. After all, hypotheses don't fall out of the sky like apples. We don't just wake up in the middle of the night thinking "maybe Scar-2 is encodes a phospholipase C like protein" or something like that. A huge part of the process is recognizing something interesting when you see it, and then following it up with efforts to gather supporting evidence.

Why am I belaboring this? Well, I think it's worth thinking about generally, first of all, but also because it gets at something I've been thinking about a lot lately, which is "how do you know when you've found something?"

This is particularly important when studying previously uncharacterized bacteria. If you go out to a patch of uncultivated land, and dig in the dirt, you will find countless bacteria. Simple technques can allow you to culture many of them (not percentage-wise, but numerically, thousands of unique strains); alternatively, you can use molecular methods to catalogue all the different types of organisms in that sample. However, when we look in the literature, we find people don't explore all this diversity. Rather, they pick things of well understood importance, and probe them deeper. So, instead of finding a brand new never cultured previously organism from the soil, they find a new nuance to the pathogenesis of E. coli gastroenteritis. One reason for this is obviously that it is much easier to figure out what's important with E. coli (or some other well known pathogen) because the parameters have already been hashed out historically. Another reason is funding (there's much more money in studying disease).

Here's an underappreciated reason. It's tough to study previously unstudied bugs. How do you know that you've identified a mutant, and not a variable phenotype. If it has a complex lifestyle, how many person-years can you devote to figuring it out? How do you attack the basic questions of function without clear guideposts. How many things can you assume are the same as the ones known in E. coli (depends!). Will you be able to use genetic tools? How hard are you willing to try to find out? Finally, how will you know when to publish it? (that's a big one!) And how will you persuade the editors to accept it? (another big one!)

Comments? Thoughts? It's not quite the usual animalcules fare, but it explains a lot about why we know so much about so few organisms, and so little about the vast, vast majority.

Posted 9:08 PM by Paul

Connecting environmental microbiology to the "important stuff"

I was perusing the wonderful blogs hosted by Seed Magazine (all of 'em are great, but Frinktank is clearly a rising star!) when I saw a new microbiology paper(subscription req'd) noted by Afarensis. The paper is about MRSA (Methicillin Resistant S. aureus) being shown to persist inside amoeba. I argued, hopefully justifiably, that you can artificially create a situation like this in many systems, and we shouldn't all run for the hills yet. Unfortunately, I haven't had a chance to read the paper (never stopped me before!), but I have to suspect that they would acknowledge my simple, naive criticism, and either deal with it experimentally, or simply admit that it needs to be studied further. Either is a very reasonable tack. I suspect someone smarter, more capable, and with better institutional access will do the thorough once over of this paper, so I'll tackle a larger question. Why would bacteria want to be able to do this, and why would it be important to us?

Because I am just not up to a long exposition, I'll tease you a bit with this, and hopefully get some comments. We know that many pathogens are highly adapted to life in the animal, plant or human host. We also know that one of the major obstacles to persistence and infection is the innate immune system, consisting of humoral responses (complement) and cellular responses (neutrophils and macrophages, cells that eat bacteria). Note, for the immunologists, that there are two analogous branches of adaptive immunity, and based on evolutionary analysis, it is thought that the innate system developed much earlier, and the adaptive system piggybacks on it (I'm simplifying here!!).

There are a lot of complex things going on in pathogenesis, some of which I've alluded to before. A major part of the process is resisting the immune response, both innate and adaptive. We'll leave the adaptive system for now, and focus on the innate system. The big danger to any invading bug is that a phagocytic cell (like I mentioned above) will eat it. So, many mechanisms for evading this response have evolved. A big one is the ability to persist in the endocytotic vesicle (depending on the cell, the endosome or the phagosome). The phagocytic cell has a vesicle called a lysosome that fuses with the phagosome to make, unsurprisingly, a phagolysosome. This fused vesicle contains acid and lytic enzymes to break the bacterium down. Defenses against this fusion, resistance to these enzymes, and acid tolerance are all great ways for bacteria to avoid this fate. In pathogenic systems, some classic examples of these mechanisms are found in Mycobacterium tuberculosis, Listeria monocytogenes, and Legionella pneumophila.

So what? Well, it turns out that each one of these bacteria have close relatives that live in natural environments inside amoeba and other phagocytic protozoa. A good review (that I just found and should read!) by Jorge Galan gives info about this and other commonalities between environmental persistence and infection. So, here's the $64,000 question (and I pose it honestly as well as rhetorically); Did bacteria already well adapted to life in soil, battling for nutrients and fighting off eukaryotic predators (protozoans, fungi) evolve all of their mechanisms in that environment, and then adapt them to colonizing and infecting eukaryotes? It's a reasonable hypothesis, and the rough time frames of the rise of various types of eukaryotes and bacterial divergences are reasonably congruent with it (note, I'm no expert on the time scales of branching events in microbial phylogeny; any knowledgeable parties should please comment).

So, here I'll leave you with the big challenge. How do we gather evidence to support or invalidate this hypothesis? Can you plan out an experimental scheme to understand this system?

Obligatory anti-ID snark; no fair just saying "God did it"!
Second anti-ID snark; there is simply no rational way to understand how different pathogenic bacteria attack many different hosts in fundamentally similar ways without acknowledging the role of evolution by variation and natural selection)