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? “

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.
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 readershipSo 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.

Crosspost from microBEnet: Collection of papers on "The Science of Science Communication"

Crossposting this from microBEnet 

 Just got pointed to this by Sharon Strauss, the chair of the Evolution and Ecology department here at UC Davis: The Science of Science Communication II Sackler Colloquium.  This is a collection of papers from a colloquium held in Septment 2013.  Slides and videos of the talks are available online. The papers and links (copied from the PNAS site) are listed below.  There are many papers here of relevance to work done at microBEnet and are also likely of general interest to many:

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New paper from some in the Eisen lab: phylogeny driven sequencing of cyanobacteria

(Cross post from my lab blog)

Quick post here.  This paper came out a few months ago but it was not freely available so I did not write about it (it is in PNAS but was not published with the PNAS Open Option — not my choice – lead author did not choose that option and I was not really in the loop when that choice was made).

Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. [Proc Natl Acad Sci U S A. 2013] – PubMed – NCBI.
Anyway – it is now in Pubmed Central and at least freely available so I felt OK posting about it now.  It is in a way a follow up to the “A phylogeny driven genomic encyclopedia of bacteria and archaea” paper (AKA GEBA) from 2009 with this paper a zooming in on the cyanobacteria.

Story behind the paper: from Jeremy Barr on "Bacteriophage and mucus. Two unlikely entities, or an exceptional symbiosis? "

I am pleased to have a guest post in my “Story behind the paper” series.  This one is from Jeremy Barr in Forest Rohwer’s lab about a new PNAS paper. 
Bacteriophage and mucus. Two unlikely entities, or an exceptional symbiosis?

By Jeremy J. Barr

Our recent research at The Rohwer Lab at San Diego State University investigates a new symbiosis formed between bacteriophage, viruses that only infect and kill bacteria, and mucus, that slimy stuff coating your mouth, nose, lungs and gut.

Bacteriophage, or phage for short are ubiquitous throughout nature. They are found everywhere. So it shouldn’t surprise you to learn that these phage are also found within mucus. In fact, if you actually sat down and thought about the best place you would look for phage, you might have picked mucus as a great starting point. Mucus is loaded with bacteria, and like phage, is found everywhere. Almost every animal uses mucus, or a mucus-like substance, to protect its environmentally exposed epithelium from the surrounding environment. Phage in mucus is nothing novel.

But what if there were more phage in mucus? What if the phage, immotile though they may be, were actually sticking within it?

It turns out that there are more phage in mucus, over four times more phage, and this appears true across extremely divergent animal mucosa. But this apparent increase in phage could very simply be explained by increased replication due to access to increased bacterial hosts residing within mucus layers. But this assumption alone doesn’t hold up. Applying phage T4 to sterile tissue culture cells resulted in significantly more phage sticking to the cell lines that produced a mucus layer, compared to those that did not. There were no bacterial hosts for phage replication in these experiments. Yet still, more phage accumulated in mucus.

Surely the law of mass-action could explain this apparent accumulation. The more phage we apply to an aqueous external environment, the more phage will diffuse into and enter the mucus layer, being slowed in the process due to the gel-like properties, and eventually resulting in an apparent accumulation of phage in mucus. But when we removed mass-action from the equation, and simply coated mucus-macromolecules onto a surface, still more phage stuck. Our assumptions were too simple.
Phage are ingenious. They have evolved, traded, and disseminated biological solutions to almost every biological problem, whether we are aware of it or not. So in order to solve the phage-mucus quandary, we needed to look to one of the most ubiquitous and populous families of proteins found in nature: the immunoglobulin superfamily. This protein fold is so ubiquitous that it appears in almost every form of life. Within our own bodies, it is the protein that affords us immunological protection. Bacteria utilize the protein fold to adhere to each other, to surfaces, and as a form of communication. And as it would turn out, phage make an innovative use of the same protein fold to stick to mucus.

Immunoglobulin, phage and mucus, are all pervasive throughout environments. The interaction between these three entities forms a new symbiosis between phage and their animal hosts. This symbiosis contributes a previously unrecognized immune system that reduces bacterial numbers in mucus, and protects the animal host from attack. We call this symbiosis/immunity, Bacteriophage Adherence to Mucus, or BAM for short.

Our work is open access and available through PNAS .

If you would like to read further about BAM and its implications see these two commentaries by Carl Zimmer at National Geographic  and by Ed Yong at Nature News

No need to oversell the human microbiome with studies like these …

I know I complain all the time about news stories and people “overselling the microbiome“.  Mind you, I believe microbial communities are likely to be found to have very very important roles in the biology of the plants and animals and other organisms on which they live, but I worry about overhyping the possibilities.  But thankfully, there are some good researchers at work out there documenting just what the microbiome can and does do.  And the results continue to be promising.

Here is the one that caught my eye most recently: BBC News – ‘Weight loss gut bacterium’ found about this PNAS paper.  While the study is in mice and it is what one could call “limited” in some ways, it is really fascinating and has much promise.  Basically, they isolated a new bacterium (with the awkward name of Akkermansia muciniphila, and did a series of experiments in mice looking at the role this bacterium can play in many mouse gut properties.  But most interesting, treatment of mice with this bacterium (and only when the bacterium was alive) led to a reduction in high fat induced metabolic disorders and obesity.  I am still re-reading the paper but the result seems solid.  And exciting.

So – there is no need to oversell the microbiome when the results coming in sell themselves …

UPDATE 30 minutes after posting

Of course, I should have checked to see if Ed Yong wrote anything about this.  And he did: The Mucus-Lover that Stops Mice from Getting Fat.  Read his post.  It is excellent.  With ALL sorts of links and background and other detail.

Cool new paper from DeLong lab: Pattern and synchrony of gene expression among sympatric marine microbial populations

Definitely worth looking at this paper if you are interested in uncultured microbes: Pattern and synchrony of gene expression among sympatric marine microbial populations.  From Ed Delong and team, it is published under the “Open” pathway in PNAS.

Also see press release here: Scientists track ocean microbe populations in their natural habitat to …

Powerpoint slides from Nature and others do not give full credit to the sources of images

Well, I browsed around the Nature web site and did some searches for terms like “Reprinted with permission” and then looked at how they handled Figures that were reprinted from other places. I found some additional examples where the Figure image did not seem to do a complete job of crediting the source of the material. But without a doubt the most disturbing thing I found is that you can download powerpoint slides of the figures and all the ones I looked at only had “Copyright Nature” or something like that and no information crediting the actual source of the material. This is basically because they do not include the Figure legends on the ppt slides. I am not sure if technically they are allowed to do this in some cases (my gut feeling is there is something wrong with what they are doing) but it could not be that hard to include the Figure legend on the ppt slides, even in small font. They certainly are able to inlcude their “Copyright Nature” in large font. But even if technically they are allowed to do this, they should not.

Some examples:

  • Download the powerpoint slide of Figure 1 or Figure 4 from a paper on Listeria in Nature Reviews Microbiology if you have access to it here.
  • Or look at Figure 1 of a paper in Nature Reviews Genetics here.

I guess I could load up here on other examples, but it might be more interesting for people to find their own.

Here is how I found these.

  1. Go to the Nature Advanced Search page here
  2. Search for terms like “Reproduced with permission” — I used the “The exact phrase” option.
  3. Browse away

I assume that this is not a purposeful thing they do, but it certainly seems pervasive there.
Unfortunately, it seems common in other places. For example, PNAS provides powerpoint slides but does not include Figure legends with them either. Look here. So – not trying to single out Nature here but that was where I looked first. It seems that many publishers are trying to hard to provide material (e.g., powerpoint slides) without being careful enough attributing the original sources.