Monday, June 25, 2018

BacterioFiles 344 - Bacteriophages Bypass Body Barriers

Cell culture setup
Nguyen et al, 2017,
mBio 8:e01874-17
This episode: Phages may be passing through the barriers in our body all the time!

Thanks to Dr. Jeremy Barr for his contribution!
Download Episode (13.2 MB, 14.5 minutes)

Show notes:
Microbe of the episode: Rhodobacter virus RcCronus

News item

Journal Paper:
Nguyen S, Baker K, Padman BS, Patwa R, Dunstan RA, Weston TA, Schlosser K, Bailey B, Lithgow T, Lazarou M, Luque A, Rohwer F, Blumberg RS, Barr JJ. 2017. Bacteriophage Transcytosis Provides a Mechanism To Cross Epithelial Cell Layers. mBio 8:e01874-17.

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    Episode outline:
    • Background: Speaking of phages, they’re everywhere
      • All over environment and in our bodies
    • Affect microbial communities in various ways
      • Turnover, genetic modification
    • Don’t infect our cells though, of course
      • But seem to be able to get around the body freely in various ways
      • Statement 1: That was Dr. Jeremy Barr, final author on today’s study.
    • What’s new: Now, Dr. Barr and coauthors Sophie Nguyen, Kristi Baker, Benjamin Padman, Ruzeen Patwa, Rhys Dunstan, Thomas Weston, Kyle Schlosser, Barbara Bailey, Trevor Lithgow, Michael Lazarou, Antoni Luque, Forest Rohwer, and Richard Blumberg, publishing in mBio, have discovered how phages get transported across the wall of the gut into the rest of the body!
    • Methods: Used T4 phage to study
      • Iconic alien-looking spidery virus with icosahedral head and many legs
    • And cell culture of canine kidney cell line growing in surface
      • Suspended on membrane with different media on each side; imitates epithelial surface
      • Some with one side on top and some with other, to see which way phages go
      • Put phages on top and observed how many transferred to other side
    • Saw ~4000-70000 phages per ml moving from “outside” to “inside”
      • Only up to ~800 moving the other way
      • So big selective effect on direction
    • Tried other cell cultures from different organs too, to see about organ effects
      • Gut, lung, liver, brain
      • All showed same directionality
    • Tested to see if toxins from bacteria present in phage preparation had effect
      • Endotoxin, LPS from bacterial surface, causes inflammation and such
      • Removing it didn’t affect the transfer of phages through cells
    • Then tested other phages of other types, from various bacterial hosts
      • All showed some transcytosis/transfer, but different amounts by type
    • Then labeled phages with fluorescent probes and used microscopy
      • See where phages were going in cells, how getting through
      • Combined images from fluorescence with electron microscopy
        • Saw virus-like particles inside in membrane-bound compartments
        • So seem to be moving through system of membrane compartments within cells
    • Also broke cells up and split into different parts to see how many phages in each
      • Counted by plating on bacterial host, see how many plaques
      • Found phages in all different places in cell
      • But especially in places associated with Golgi
    • Tried to stop transportation with diff chemicals
      • Only effect from Brefeldin A, inhibits movement between ER and Golgi
      • No effect related to receptors or microtubules or endosome acidification
      • So seems Golgi-related, but chemical effects not specific enough to know for sure
    • Finally estimated how many phages getting into our body
      • ~2 trillion phages in large intestine
      • Based on observed rate here, ~30 billion get into body per day
      • Implication: for so many to get thru a “leaky gut”, would require massive intestinal damage
        • But such levels of phages observed in people without gut problems
    • Summary: statement 2
    • Applications and implications: Possible that body is bringing in phages for defense?
      • Prevent any gut microbes from invading out of their bounds
      • Could also help infants by going through breastmilk to populate gut
    • Clarifications if necessary: Not all details yet clear
      • Also not in actual animals
    • What do I think: statement 3
    • Lots of interesting things to study
      • Why/how doesn’t body generate antiphage responses to get rid of them?
        • Or cause massive inflammation?
      • Could be having some effect, hard to detect cos always happening
        • Do some study with germ-free mice!
      • Also possible that abnormal transport does cause subtle symptoms
        • Allergy, inflammation
    • Even more speculative: possibility of phage genes expressed within cells
    • Just a lot of unknowns, but pretty exciting

    Author Transcript:
    The motivation for this work really stems from the observation of phages within sterile regions of the body. And researchers have seen, for decades now, phages within the blood, serum, urine, or even organs of the human body. Yet we haven't ever really had a good mechanism for how phages were getting into these areas.

    So we used a relatively simplified in vitro epithelial cell model, where we obtained and grew cells isolated from diverse regions of the body, and we showed that these cells were capable of endocytosing and trafficking phages throughout the cellular structure. We then used a range of microscopy and cell biology approaches to show that phages were capable of dispersing and trafficking throughout the entire eukaryotic epithelial cell.

    in my opinion, this work has a lot of exciting biological implications, because it provides a direct mechanism for phages to interact and influence the eukaryotic cell. And we're following up on this research and we're looking at a number of naturally occurring environments, such as the gut, where phages may modulate biological effects with these epithelial cell layers and surfaces.

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