The story behind the paper by @JeremyJBarr on phage using mucus to hunt prey

This is a guest post by Jeremy Barr about a new paper of his. Also see his previous post from 2013: Story behind the paper: from Jeremy Barr on "Bacteriophage and mucus. Two unlikely entities, or an exceptional symbiosis? "

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The story behind the paper “Subdiffusive motion of bacteriophage in mucosal surfaces increases the frequency of bacterial encounters”

Here’s the story behind our recent publication on the subdiffusive motion of bacteriophage in mucus published in PNAS – a manuscript that builds on our Bacteriophage Adherence to Mucus (BAM) model of phage-derived immunity. You can also find a recent write up on the work by San Diego State University (SDSU) News Center here.

Inception

In early 2013, I attended my first Keystone Symposia conference on “Emerging Topics in Immune System Plasticity” in Santa Fe, New Mexico. Apart from the excellent snow conditions, I was beginning to question my decision to attend an immunity conference as an experimental microbiologist, but one of the last presentations at the conference, given by Christopher Hunter from UPenn, stuck with me. The Hunter lab was investigating the ability of CD8⁺ T cells to control the parasite Toxoplasma gondii in the brains of mice. Using a powerful microscopy system, they were able to watch T cell movement in real time while they were searching the brain for the sparsely distributed parasites. They found the T cells moved in a specific pattern, characterized by many short-distance movements interspersed with occasional longer-distance flight to a new area. This search strategy is known as a Lévy flight, and it allowed the T cells to more effectively search an area of the brain for hiding Toxoplasma than if they searched by directed or random motion (see paper here). Once I saw this talk, the idea behind our paper was planted. I knew that by adhering to mucus, bacteriophage could also use this strategy to hunt bacteria, but it wasn’t until a couple of years later that I was able to test this hypothesis.

The makings of a microfluidic mucus layer.

During this time, I had been reading a number of papers that were reconstituting organ-level functions on microfluidic devices, making simulated lung or gut environments.

Recognizing the potential of these systems, I began working with Samuel Kassegne and his Masters student Nicholas Sam-Soon in the Department of Mechanical Engineering at San Diego State University (SDSU) to develop our own microfluidic ‘chip’ aimed to simulate a mucus layer with fluid flow and secretion dynamics. I had no idea how difficult this endeavor would be. Our first chip was as close to a complete failure as one could get. The device leaked, it was dirty, and I had the bright idea that we could simple poke a syringe into the chip to set up fluid flow.

But we persevered. We continually solved problem after problem, with every solution leading to new problems, be it leaks, growths, or cracks in the chip. Two years and a Masters thesis later, the system was finally working at a useful throughput for us to experimentally test. We could now run up to nine chips simultaneously and immediately set out to recapitulate our prior results – that mucus-adherent phage protected mucosal epithelium from bacterial infections.

What we found from these experiments was quite surprising. Firstly, I should explain that the model system we were using was phage T4, a strictly lytic phage that infects and kills Escherichia coli that we previously showed was capable of adhering to mucus, and a T4∆hoc phage that is equally capable of killing E. coli but lacks the capsid proteins required to adhere to mucus. When we infected the chips with E. coli bacterium and the non-mucus adherent T4∆hoc phage, we found that these phage-treated chips were no better at reducing bacterial abundance in the mucus layer compared to control chips where no phage had been added at all. Meanwhile, the mucus-adherent T4 phage was capable of reducing bacterial colonization in the mucus by over 4000-fold. We next investigated whether differences in phage accumulation or persistence in the mucus could explain this stark difference, but we found no effect. The question remained, why were the mucus-adherent phage better suited at finding and reducing bacteria in mucus than the same phage that could not stick?

Weekly math meetings to the rescue

For the last four and a half years I have been extremely fortunate to have the opportunity to work as both a post-doc and now an adjunct faculty in Forest Rohwer’s lab at SDSU. During that time, one of Forest’s many punishments for me was compulsory, weekly Bio-Math meetings, which are still being run here at SDSU. These meetings were something that I initially rebelled against – what good could math do me? But as I unwillingly persisted, I came to realize the value in using math to describe biological systems. This is especially true for phages that play the game of life at a speed and scale that is at times incomprehensible.

Over time, I came to have my own weekly math meetings with a group of SDSU mathematicians, statisticians, and physicists. I owe a big thanks to Peter Salamon, Arlette Baljon, Jim Nulton, and Ben Felts, who all took countless hours out of their days to meet with me and discuss the complexities of diffusion. During these meetings we analyzed hundreds of thousands of data points detailing phage diffusivity in mucus, and eventually we answered the question as to why mucus-adherent phage were better at reducing bacterial numbers – the phage were employing a search strategy to hunt bacteria in mucus. But this search strategy was not the same as the Lévy flights I had seen the T cells use at the conference talk years earlier. This was something different, something that no predator had been shown to utilize before. Our phage were using a type of motion know as subdiffusion.

Phage are like ticks in a grass field

We found that phage that adhere weakly to mucus, through reversible binding interactions to one or more mucin strand, exhibit subdiffusive motion, not normal diffusion, in mucosal surfaces. The question now was what that means for the phages. What benefit could subdiffusive motion provide?

Subdiffusion is a very abstract concept that is difficult to explain without mathematical formula, and we spent many hours discussing the possible biological implications. Subdiffusive particles move slower and slower over time, remaining in their original positions longer, and in certain models the chance of finding a nearby target is significantly increased. Using similar logic, we hypothesized that mucus-adherent phage moved slower in specific regions of the mucus layer, remained nearby sites of productive bacterial infections, and concentrated in regions of the mucus that overlapped the niche of their bacterial host – all resulting in a greater chance for the phage to encounter a bacterium. Now we just had to prove it.

One of the beautiful things about phage biology is the detailed and expansive literature published over the last 100 years. Going back through these papers, we found a classical phage experiment that was first published in 1932 by Martin Schlesinger. This experiment measured the adsorption rate of a specific phage to its bacterial host. Using this assay, we showed that phage adsorption rate was increased in mucin solutions at low, but not high, bacterial concentrations. The logic here is that when bacterial hosts are abundant, the chance of a random phage-host encounter is high, and any improvement in the search strategy employed doesn’t provide a noticeable benefit. But when bacterial abundance is low and chance phage-host encounters are comparatively low, performing a more efficient search can greatly improve the chances of a successful infection.

The implications here become apparent when we consider that phages are typically quite specific and that mucosal surfaces harbor a large diversity of bacterial hosts – dynamics that reduce the chance of any successful phage-host encounter. From the perspective of the phage subdiffusing within a mucus layer, the world is a three-dimensional web, and like ticks in a grass field, the phage are holding onto the mucus network, awaiting a bacterial host.

The publication process

I presented this work at another Keystone Symposia on “Gut Microbiota Modulation of Host Physiology” earlier this year. During one of the conference dinners, an editor for Science happened to join the table where I was seated. We started speaking and they suggested that I submit the work for review at Science. At the time, I was reading Steven Pinker’s The Sense of Style and wanted to write the paper in ‘Classic Style’ to simply explain phage subdiffusion and appeal to a broader audience. I was very fortunate to be able to work once again with Merry Youle. We wrote a very stylized paper for Science, but after a two-week internal review we were told that although the work would likely be of great interest to the field, it was not broad enough for their general readership. So we quickly edited the paper and sent it to PNAS for review.

Our reviewers from PNAS were very helpful and suggested a number of experiments that strengthened the work, but they all hated the writing style and asked us to cut out many of the phage anthropomorphisms we had used (e.g., phage hunting bacteria). We spent another three months collecting and analyzing additional data and rewriting the paper, now with a more serious tone (e.g., search strategies instead of hunting). Overall, I felt our resubmitted paper was much stronger scientifically, even though it lost some readability. But the paper was still not accepted, and we had to go through a third revision. The final reviewer insisted on us including in vivo experiments (not something we could easily do for this paper, but we’re working on it) and continued to argue that the use of ‘search strategy’ obfuscated phage subdiffusion in mucus. Although we disagreed with this final point, the thought of going through another review was enough for us to concede, and we removed the use of this term from the paper. The rest of the editorial process was handled extremely well and we were in press at PNAS just three weeks later.