Synopsis: A blurb about a recent paper from my lab [1], on our discovery that the bacterium that causes cholera can manipulate the mechanical environment of an animal gut to expel resident bacteria.
Introduction: cholera’s tools, and ours
Cholera killed more people in the 19th century than any other epidemic disease, and it continues to kill around 100,000 people every year [2]. For over a hundred years we’ve known that the disease is caused by the bacterium Vibrio cholerae, which colonizes the intestine and induces severe diarrhea. We’ve uncovered a great deal about the molecular biology of this intensely studied microbe. We don’t, however, understand how it manages to colonize the human gut, a space densely occupied by tens of trillions of resident microbes.
Many bacteria, Vibrio cholerae among them, wield a weapon known as the Type VI Secretion System (T6SS), a syringe-like machine with which the bacterium stabs adjacent cells and injects toxins:
At a conference organized by the Research Corporation for Scientific Advancement and the the Gordon and Betty Moore Foundation, I met Joao Xavier, a mostly computational biologist at Memorial Sloan Kettering Cancer Center, and Brian Hammer, a microbiologist at Georgia Tech who has been studying cholera and the T6SS for years. We realized that the combination of Brian’s genetic tools for manipulating the Type VI apparatus, Joao’s insights into the T6SS from microbiome sequencing data, and my lab’s tools for quantitative 3D microscopy of bacterial communities inside the intestines of live zebrafish [link] could be combined into a really unique set of experiments to explore Vibrio cholerae’s capabilities.
What we learned (part 1)
After establishing that Vibrio cholerae can, in fact, colonize the intestines of larval zebrafish, and looking a bit at inter-Vibrio competition, we tackled the interesting setup of Vibrio cholera versus a native species. A key reason that zebrafish are a great model for studying the gut microbiome is that one can prepare them initially devoid of gut bacteria, i.e. “germ-free,” and then introduce particular species, enabling controlled experiments. (The gut microbiome is both complex and highly variable, and so this control is invaluable.) We let an abundant native microbe, Aeromonas veronii colonize initially germ-free zebrafish larvae for 24 hours, and then introduced one of our Vibrio cholerae strains into the water, from which it could potentially invade the fish.
Here’s what Aeromonas looks like in the gut, over a span of 12 hours starting 8 hours after the introduction of Vibrio choleraethat lacks the Type VI Secretion System:
The Aeromonas is doing fine! As is the case when it’s on its own, Aeromonas populations take the form of large aggregates that experience rapid growth punctuated by collapses driven by expulsion from the gut.
Here, in contrast, is Aeromonas after the introduction of Vibrio cholerae with a functional and always active Type VI Secretion System:
The Aeromonas is annilihated! On average, the Aeromonaspopulation is reduced by over 100x, with complete extinction in many fish. (You can see graphs in the paper that quantify all these dynamics.)
We were surprised to see that the effect of Vibrio’s T6SS has such a large effect on a native species, but the real surprise was still to come.
What we learned (part 2)
What is Vibrio’s Type VI Secretion System doing? Killing the native bacteria, right? That’s what we thought, but then we looked further and imaged the gut itself. Here’s what a normal gut, or the gut of a fish colonized with the Type-VI-defective Vibrio cholera, looks like:
Here’s what the gut of a fish colonized with the Type-VI-active Vibrio cholera looks like:
The bacterial T6SS induces a huge increase in the strength of intestinal contractions! (Again, this is quantified in the paper.) We were able to figure out exactly what part of the stabbing apparatus is responsible for this stimulation, remove it, and look at the impact on both the animal and the resident microbes — I’ll leave it to you to (i) guess what happened, and (ii) look at the paper to see if you’re correct.
Our observations revealed a previously unimagined role for the T6SS in the gut microbiome, and one that points to a previously undiscovered path by which microbes can influence the physical environment of the gut and the physiology of animals, which is exciting!
How we learned it
This project was made possible by several remarkable people and some powerful techniques. I’ll just point here to the people in my own lab: undergraduate Drew Shields, and graduate student Savannah Logan. Savannah, who did everything related to the setup, execution, and analyis of live imaging experiments, and is the first author of our paper, is shown here in her native habitat:
Brian Hammer’s group, especially postdoc Jacob Thomas, engineered the Vibrio cholerae variants that we used, all of which are fluorescent: the “wild type” bacterium that has the T6SS but might not always be expressing it, one in which the T6SS is always “on” and the bacteria are always ready to stab their neighbors, and two different sorts of Type VI defective strains. They also, quite quickly, made the strain I hinted at above, that’s incapable of stimulating gut activity despite having an active Type VI system.
Being able to actually image gut bacteria in a live animal was crucial to this project, and all our imaging is done with the technique of light sheet fluorescence microscopy, using our home-built microscope. I continue to be amazed that there’s so little light sheet imaging of microbial communities, as I’ve written before, and that ours is the only high resolution live imaging of the gut microbiota.
Things we still don’t know
A lot of questions, and potentially exciting research projects, remain regarding the T6SS and the gut microbiome. For example:
We’ve found that the Type VI Secretion System can manipulate intestinal mechanics, but how exactly are the bacteria communicating with the animal? Which of the animal’s cells are involved? There are a lot of candidates: the epithelial cells lining the gut, secretory cells also at this border, immune cells that can sense and take up bacteria, neurons surrounding the gut, and more.
In normal bacteria, the Type VI Secretion System isn’t always expressed, but rather is activated by various cues. For Vibrio cholerae in the wild, for example in the oceans where it’s abundant, these cues include materials like the chitin that makes up crustacean shells. What activates the T6SS in the gut?
Could understanding Vibrio cholerae’s intestinal abilities help cure cholera? The standard answer to give to questions like this is ‘yes,’ and papers are filled with exaggerated claims of the health relevance of various findings. To be honest, though, the answer is almost certainly ‘no.’ We already have a cure for cholerae: clean water with salts in it. (Really.) The fact that 100,000 people each year die of cholera is a sad testament to the poor state of civic infrastructure in much of the world. Addressing this doesn’t need biophysics or microbiology.
Could understanding Vibrio cholerae’s intestinal abilities help us manipulate the gut microbiome? Yes! (At least, I think so…) This, in fact, was one of our original motivations in exploring the T6SS: can we use Vibrio’s tricks to engineer ways to alter resident gut microbial communities, for example turning disease-associated consortia into ones characteristic of health? Will this work? We’ll see…
Today’s illustration
My illustration of Vibrio cholerae poking the gut. It’s a bit garish, but I like it. I suggested it, unsuccessfully, as a cover illustration for PNAS.
— Raghuveer Parthasarathy, May 31, 2018
References
[1] S. L. Logan, J. Thomas, J. Yan, R. P. Baker, D. S. Shields, J. B. Xavier, B. K. Hammer, and R. Parthasarathy, “The Vibrio cholerae Type VI Secretion System Can Modulate Host Intestinal Mechanics to Displace Commensal Gut Bacteria.” Proc. Natl. Acad. Sci. 115: E3779-E3787 (2018). [link] Preprint:[Link] — the bioRxiv preprint, which preceded reviewer comments, is very similar to the final published version.
[2] Ali M, Nelson AR, Lopez AL, Sack DA (2015) Updated Global Burden of Cholera in Endemic Countries. PLoS Negl Trop Dis 9(6): e0003832. Link
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