Field of Science

Cells that vomit fungus and other issues of science papers

This weeks
journal club was on Cryptococcus neoformans and an odd way it may get of out macrophage some of the time, at least in vitro, maybe. The paper in question is: 

The Human Fungal Pathogen Cryptococcus neoformans Escapes Macrophages by a Phagosome Emptying Mechanism That Is Inhibited by Arp2/3 Complex-Mediated Actin Polymerisation by Simon A. Johnston, Robin C. May. PLoS Pathogens 6(8) e1001041.

This work follows up a really cool observation published by two groups in back to back articles in Current Biology back in 2006. Basically Cryptococcus neoformans gets into your lungs and can cause pulmonary infections. Macrophage are defensive cells of your body that are essentially cellular Pac-People that go around scarffing up foreign organisms that get into you, like C. neoformans. When I say scarf up, what I mean is the macrophage take up the microbe into a large vesicle called a phagosome, this is like the garbage disposal of the cell. What's important to remember is that the phagosome is separate from the cytoplasm of the macrophage and is effectively the surrounding environment. Generally macrophage either kill the invader or, in some cases, are killed by it, although a few disease-causing microbes can survive and grow just fine within the macrophage. C. neoformans was one of those microbes that either killed or were killed by macrophage (it is a competition, if things go well the C. neoformans is killed, if things go poorly, the macrophage is killed), or sometimes lived inside macrophage. However, in 2006 two groups published something completely different that is better seen than described.
(link in case it doesn't work for you)
What you are looking at is a big macrophage cell that contains 6-7 small C. neoformans cells within phagosomes. Around minute ~350 you'll notice the number of C. neoformans cells increases (the C. neoformans cells are dividing), and then around minute 540 something subtle happens. You may need to watch it several times to see it. Yep, all the freaking C. neoformans cells are vomited from the macrophage!!! Importantly the macrophage didn't lyse (explode) in the process. In fact, the macrophage is still alive, it divides at minute 740! That is the coolest shit eveh!

I&I 2000 paper Figure 9C. This is part of a single macrophage,
all the white glop is capsule. (The edge of the macrophage
can be seen near the bottom of the figure as the thin curvy white
area. The black box is irrelevant here.) 
Unfortunately, I was not as thrilled by this follow up paper. One big issue is that while the original observation was filled with awesomeness, there was the issue of relevance. This issue can be overlooked in the initial analysis because of the shear awesomeness. The phenomenon may be amazing to see in the laboratory under artificial conditions, but does it matter in real life, in the human host? There is a large body of literature demonstrating the ability of C. neoformans to kill macrophage. This happens by the production of massive amounts of capsule which gums up the macrophage (see figure). So while the initial observation was amazing and worth the publicity, now the question of relevance needs to be addressed.

Sadly, today's paper essentially ignored the question and went right on into determining some of the macrophage requirements for this vomiting phenomenon. I want to discuss a number of concerns that came out of our departmental journal club. (I can not take credit for all of these concerns, but I do agree with them and appreciated the discussions that arose because of them.) You should read the paper for the details as I gloss over many of them below. Plus, there is a tremendous amount of cellular biology in the paper if you are interested in cell biology. 

Concern 1. So what?
From Lab Rat
L. monocytogenes
swimming on actin
I have already noted the relevance issue regarding the phenomenon in question. This paper demonstrates that the macrophage Arp2/3 complex is important for this phenomenon as well. The authors show that actin filaments (one of the internal skeletons of a eukaryotic cell) surround phagosomes containing C. neoformans and that this requires Arp2/3. The Arp2/3 complex is required for formation of virtually all actin filaments in a cell, the notable exception being actin filaments that occur during cytokinesis. Once Arp2/3 was shown to be involved, that basically ends the story. There was no need to kill the horse it rode in on and then start beating it. Don't get me wrong it was important to show Arp2/3 was required. If Arp2/3 was not required, then that would have been interesting in its own right, as that would have been similar to what is seen for Listeria monocytogenesL. monocytogenes, an intracellular bacterial pathogen, uses host cell actin to move around and shoot itself into neighboring cells. (While much of  L. monocytogenes motility is Arp2/3-dependent, there is Arp2/3-independent motility as well.)

Ok, once we know Arp2/3 is required, so what? It is well known that actin filaments require Arp2/3. Actin filaments also require the degradation of previously generated filament (to recycle the actin monomers) and even translation of the mRNA encoding actin. What have we actually learned here? What's the new important finding?

Concern 2. Please deal with contradictions.
In the first paragraph of the results, if you say "Long term time lapse imaging over 18 hours revealed that phagosomes containing cryptococci showed rapid, transient increases in actin-GFP fluorescence...that appeared similar to actin flashes previously macrophages loaded with latex beads [16]. " then please remember it four sentences later when you say "Notably, flashes were extremely rare on phagosomes containing latex beads (either unopsonised or IgG-opsonised) (Figure 1D)." Doesn't this seem like a discrepancy that needs to be addressed? 

Also, the results suggest that the more actin "flashes" that occur the more likely vomiting will occur (Figure 3A and B). Based on this, the prediction I would make is that drugs that promote filament formation would promote vomiting; drugs that inhibit filament formation would inhibit vomiting. Well the results of the drug experiments (Figure 4) reveal completely different data. Not a big deal, but I would expect an explanation, which was absent. (If the authors had different predictions that these results agreed with, it was not apparent to me.)

Finally, the idea the authors propose is that the phagosomal membrane gets leaky, which is why actin filaments form there. This is tested using dextran blue, a large molecular polymer of glucose linked to a blue dye. Dextran blue is taken up by macrophage along with  C. neoformans and located within the phagosome with the C. neoformans. If the phagosome becomes leaky, one expects the dextran blue to be in the cytoplasm. Well, the dextran blue does separate from the C. neoformans but it is not diffuse in the cytoplasm. The dextran blue is found in punctate spots (Figure 5), which I think could be vesicles that have separated from the major phagosome. If someone has better insights than myself, post them in the comments.

Concern 3. Philosophy of Science.
Science, at least the biological sciences, is usually based in hypothesis testing although there is a fair bit of observational science as well. When we discuss of findings, like in the discussion part of the paper, we put out our favorite ideas for what is happening (favorite ≠ best, necessarily). We sell our stuff to make it interesting to the largest audience possible. But, we also consider the limitations to our favorite ideas and even consider other possible explanations, even if we discount them shortly thereafter. We do this in writing. We do this as part of the process of being critical thinkers. I saw no evidence of this in the paper. The discussion flew right down the road of we think this, and it means that, yadda, yadda, yadda until we reach the Methods and Materials section. Part of this lack may have been due to point #1 So What? The authors may have been trying to avoid this issue being raised.

Concern 4. Conflict of interests.
Personally, I thought this work, which was labor intensive, was still quite preliminary. The data presented was generally well done, but there were a fair number of holes that, in my opinion, needed to be filled in to justify the conclusions being made. That is not to say that this paper needed any additional ducks in a row to be published. However, this paper was published in PLoS Pathogens, one of the premier journals dealing with pathogens and disease, so more ducks would be helpful. I was a little surprised...and then I saw this "One of the authors (RCM) is an editor for PLoS Pathogens". On the positive side, this is laid out clearly at the beginning of the paper as a potential conflict of interest. But I cannot help but wonder if there is some unconscious bias when one of the editors for a journal submits their paper to said journal. Yes, the manuscript was handled by a different journal editor, but these people do work together to strengthen the journal and make it successful. Do you really want to shit on a colleague who's a member of your team? I'll point out that I would not be eligible to even review a paper submitted to PLoS Pathogens if the author were also a faculty member at my university (note there are ~1500 faculty members at my university). 

Overall the paper is dealing with an interesting phenomenon, but I do think its time to determine if the phenomenon is biologically relevant. It may be possible to identify C. neoformans mutants that get vomited more or less often. If such mutants can be identified, you can see how these mutants behave in the host, which may clue you in on if/how this vomiting phenomenon contributes to colonization/infection.

(For the record, I have had a couple of papers rejected from PLoS Pathogens, which constitutes my own potential bias.)

Johnston SA, & May RC (2010). The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation. PLoS pathogens, 6 (8) PMID: 20714349

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