Wow, it's finally out!

It took so long but here is my work on gut #phages. Inspired by @implosian I'm going to try this paper thread thing below. Please read it all!! https://twitter.com/biorxivpreprint/status/1285417872815271938

Previously, we worked on #phages against bacteremia-causing pathogens.

ExPEC are very bad bugs that cause a lot of bad extraintestinal infections. They are #MDR, there are no #vaccines against it and they colonize your gut asymptomatically https://www.nature.com/articles/srep46151

Knowing that they are in the #gut, we set out to try to eliminate this reservoir to prevent infection and spread of MDR.

But the phages (PHI) we used previously that killed ExPEC in blood did not kill in a #gut model using .

But antibiotics (ABX) worked

But ABX cause changes to the microbiome. Decreases diversity.

Diversity = good for the #gut, #health.

So to figure out what's going on we tried to move this ex vivo by using cecal (large intestine) contents to test bacteria and phage interaction.

It's like a cecal slurry medium (or media??)

We see the same trend with our slurry. Only with cecal and not fecal pellet slurry.

The phage don't kill bacteria in cecal. Why??

So, what was inhibiting the #phage? Well, yadda, yadda, yadda, it was mucin!!

We centrifuged cecal (CM) and resuspended the pellet with NAC (mucolytic drug).

To the supernatant (has less or no mucin) we added mucin.

Voila! Here is the data.

Now what? Well, we decided to screen for phages in just mucin. If we find phages that kill bacteria in mucin then they should kill in the gut (hypothesis alert ).

One did!! ES17.

ES17 is podovirus but with a big head! My husband said it looks like a corn dog. Cute, huh?! It's a Kuravirus (species).

ES17 really loves mucin btw!

We coated bacteria with mucin and performed an adsorption assays with ES17.

Adsorption went from 0% phage adsorbed to 98% in 10 minutes with mucin-coated bacteria. Holy crap!

Almost like mucin is a receptor for phage ES17.

This phage is also our best biofilm killer.

This is an MTT assay. Decrease in absorbance (I spelled it wrong on the fig ) usually means less biofilm-forming bacteria.

Are these two things related? Mucin and biofilm? Both are made up of #sugars!

Phage ES17 also makes these nice halos around the plaques indicative of exopolysaccharide-degrading enzymes or sugar-degrading enzymes.

This is an MTT assay with diff concentrations of ES17.
Misspelled again absorbance again

ES17 has a tail fiber (receptor) with a putative pectinesterase domain.

These are domains found on enzymes that break down sugars from biofilms.

I cloned, purified the tail fiber but it didn't break down biofilms or mucin. So..maybe it binds to mucin?

Like a bridge that brings the phage closer to the bacteria so it can infect.

@jrclark_unc did some bioinformatics for us. He found this receptor was very similar to the tail spike of another phage, K5.

Yellow are identical residues. Guess what K5 binds?

K5 binds K5 capsule also known as Heparosan. Why is it called Heparosan? bc it is identical to heparan-sulfate precursors.

Heparan sulfate is one of the most ubiquitous ANIMAL SUGARS found in the body including the gut!

Our phage loves SUGARS!!

Glycan array showed that this phage protein bound heparan sulfate out of 900 glycans.

We tested our #phage in human intestinal enteroids since these are better models of the gut.

Enteroids are mini-guts derived from human biopsy cells. This is with help from @organoidsnooper

Wow! ES17 loves the gut! The green is phage. The red is mucus and blue are cells. Look how ES17 coats the gut compared to another phage HP3.

We added an enzyme to cut heparan S where we think ES17 may bind. Top is without enzyme and bottom is with. Phage are green again.

Looks like these phages bind HS and by doing so coat the gut. Does this move the phage closer to pathogens in the gut?

We tested this using EAEC. A very, very bad diarrhea-causing bug.

EAEC colonizes these gut enteroids making these aggregative chains (red arrows) on intestinal cells (large pink/purple circles).

@organoidsnooper images are better but I tried.

But when we coat the enteroids with ES17, wash, then add EAEC we see a reduction in adhered bacteria to cells.

This phage is a gut shield!!

And this phage kills really well in a gut by the way. This time we added 10X more phage than the last experiment.

Only 1 mouse had ExPEC in their gut when treated with ES17.

Here is our model. ES17 loves intestinal sugars! We think this is why it works so well in gut models. Maybe we need to get more phages like ES17 if we want to treat the #gut.

I hope you all enjoyed reading this, I enjoyed writing it!

I also love sugar by the way!