Oct 112010

ResearchBlogging.orgClostridium difficile is an intestinal pathogen that causes diarrhea in hospitals and other healthcare settings (including nursing homes). Present as a commensal bacterium in a significant fraction of the population, C. difficile is usually rather harmless, its numbers suppressed by competition with the intestinal flora. When its competitors are decimated by antibiotics, however, C. difficile flourishes, releasing toxins that cause inflammation and diarrhea, which can be dangerous because the individuals suffering these effects are often already ill. There has been conflicting information, however, as to which of C. difficile‘s toxins are necessary to cause disease. A paper in the recent Nature (1) aims to resolve the question.

The two best-characterized C. difficile toxins (TcdA and TcdB) have the same general arrangement and function (and ~45% identical AA sequence). An N-terminal glucosylating domain attacks the cytoskeleton of host cells by inactivating Rho GTPases, a C-terminal domain mediates binding and uptake by the host cells, and a protease domain in the middle releases the glucosylating domain to do its work. Since these proteins appear to serve redundant functions, one might expect that both would support virulence. However, preceding work in the field has variously identified TcdA or TcdB as a key virulence factor (2,3). Differences in methodology and materials have contributed to the confusion, in part because different kinds of cells seem to be more or less susceptible to particular toxins, and different strains of C. difficile might have different behaviors.

Kuehne et al. aim to relieve some of the confusion by removing a subset of these confounding factors. In a single strain of C. difficile they inactivated the genes for either TcdA, TcdB, or both by inserting introns into them. An intron would be no problem for a eukaryote, but bacteria can’t handle them, so this has the effect of eliminating the expression of the gene. They then tested the toxin mixtures shed by the bacteria against cultured human and monkey cells. As expected, A-B- bacteria (with both toxins knocked out) showed no toxicity towards the cells, but A-B+ and B+A- variants were toxic towards both kinds of cells to roughly the same degree. This suggests that both toxins are sufficient for virulence.

This implication was largely backed up by a subsequent experiment in hamsters. The animals were dosed with an antibiotic and then infected with C. difficile spores of a single strain. Colonization occurred (in every case but one) within three days. The hamsters infected with A-B- C. difficile remained asymptomatic until the end of the experiment, but the recipients of the other strains all perished within a week. The A+B- group survived somewhat longer, but not dramatically so; again, this supports the interpretation that both proteins are sufficient for virulence.

This contrasts with an earlier study published in Nature (2) where it was shown that deletion of the B toxin protected hamsters from C. difficile-associated disease, using very similar protocols. Kuehne et al. attribute the differences in their results to the hamsters or genetic variation in the C. difficile strains used. While the virulence of the B- strain in this experiment was slightly attenuated, all colonized hamsters still died in relatively short order, and in human beings the situation might well be reversed, since cultured human cells are more vulnerable to toxin A.

The results of Kuehne et al. largely agree with earlier experiments (3) and with what one would naturally expect of two very similar toxins being released by the same organism. While susceptibility to a particular toxin may vary with characteristics of the host species or cell type, it seems likely that both toxins are capable of supporting virulence. While it is to be hoped that additional research will clarify the reasons for the discrepancy between these two experiments, efforts to treat C. difficile-associated disease by attacking the toxins should proceed with the assumption that both must inactivated. Thanks to their functional and sequence similarity this will hopefully not be too much of a complication.

1. Kuehne, S., Cartman, S., Heap, J., Kelly, M., Cockayne, A., & Minton, N. (2010). The role of toxin A and toxin B in Clostridium difficile infection Nature, 467 (7316), 711-713 DOI: 10.1038/nature09397

2. Lyras, D., O’Connor, J., Howarth, P., Sambol, S., Carter, G., Phumoonna, T., Poon, R., Adams, V., Vedantam, G., Johnson, S., Gerding, D., & Rood, J. (2009). Toxin B is essential for virulence of Clostridium difficile Nature, 458 (7242), 1176-1179 PMCID: PMC2679968 OPEN ACCESS

3. Voth, D., & Ballard, J. (2005). Clostridium difficile Toxins: Mechanism of Action and Role in Disease Clinical Microbiology Reviews, 18 (2), 247-263 PMCID: PMC1082799 OPEN ACCESS

Feb 292008
ResearchBlogging.orgA bunch of really great stuff came out today, so much that I’m not sure I’ll be able to get to all of it. We can start with the shortest article of interest, a paper in Science that’s sure to interest mothers whose children stand around with their tongues out in a flurry. Brent Christner and colleagues have discovered that snow from pretty much anywhere contains bacteria (1). Well sure, you probably suspected that, but these bacteria didn’t move up from the ground… they fell down from the sky.

Crystals generally form more readily when they have a nucleation point. Protein crystallographers are well aware that seeding their trays with microcrystals from a partially successful screen can significantly improve results. Non-scientists can see this as well, in the growth of ice crystals on certain surfaces or the popular home experiment of crystallizing sugar onto a string. The very rough string surface provides many more nucleation points than the smoother bowl surface, or the liquid, and so crystal formation on the string is preferred. A similar principle is at work in the formation of ice crystals in clouds. Any small particle can conceivably serve as a nucleation point: aerosols, dust, and even living things. And of course, once they form, those ice crystals can return to earth as snow or rain.

The authors aseptically collected fresh snow samples from various locations (France, Montana, the Yukon, and Antarctica) and melted them. They filtered the snowmelt and resuspended the filtrate in a smaller volume. They then assayed the freezing temperature between -2 and -9 °C. To prove that the ice nuclei (IN) were biological in origin they attempted to inactivate them using heat (to denature proteins) and lysozyme (to break up bacterial membranes). The experiment is pretty simple—in fact, except for the parts that explicitly prove bacteria are involved you could mimic this at home. The next part isn’t so easy to try yourself: they used flow cytometry to identify particles in the snow containing DNA.

DNA-containing particles were found at all locations, as were IN that could be inactivated by heat. Several locations also had IN that could be inactivated by lysozyme. The authors chalk up the apparent discrepancy to incomplete hydrolysis or lysozyme resistance. The authors don’t explicitly identify the bacteria in the article, but some of the points they make suggest that they may be species that are commensal or pathogenic towards plants. Of course there’s no guarantee that the bacteria that land this way are still alive: the authors didn’t assay motility or attempt to culture from the isolated IN. That lysozyme had an effect, however, suggests that the cells did not crack just from the conditions, and everyone who works in the biological sciences knows that even humdrum everyday bacteria like E. coli can survive freezing. The obvious implication is that it will be very difficult to control the spread of any species that can pull off the same trick. All they have to do is get blown up high enough and come down in the next rainstorm.

In spite of this news, it’s probably not dangerous for your kids to eat a few snowflakes from the sky. This finding isn’t so much a revelation as it is a reminder: bacteria are all around us and coat essentially everything we touch, eat, or drink. And that’s true even if you don’t drink anything but whisky and rainwater.

1. Christner, B.C., Morris, C.E., Foreman, C.M., Cai, R., Sands, D.C. (2008). Ubiquity of Biological Ice Nucleators in Snowfall. Science, 319(5867), 1214-1214. DOI: 10.1126/science.1149757

POSTSCRIPT: Tara Smith has good post on this, with a forum image for all you 4channers. Ed Yong also has a great post.

 Posted by at 1:34 PM