24. Journal Club

Sustained in situ protein production
and release in the mammalian gut
by an engineered bacteriophage:
What is the point of this experiment

To use phage infected resident gut bacteria to deliver therapeutic agents into the GI tract without having to take daily medication

Hypothesis of the experiment

the lytic phage T4 could help release the GFP from inside the bacteria into the surrounding environment by causing the bacteria to burst open (lyse)

Rationale of the experiment

By using the phage to lyse the bacteria, they could potentially deliver therapeutic proteins (like GFP) produced inside bacteria, to the gut more effectively

Experimental: With phage

significantly increases GFP fluorescence in the supernatant over time, indicating effective release of GFP due to phage-induced lysis, while GFP fluorescence in the cell pellet decreases as the bacteria lyse

Control: Without phage

GFP fluorescence remains high in the cell pellet and low in the supernatant, indicating that GFP is contained within the bacterial cells without phage-induced lysis

AU/OD is arbitrary units/optical density. Why did they divide AU by OD?

the data reflect the amount of protein released per cell, rather than being influenced by the total number of cells

Transcription from phage promoters occurs in waves, first “early” promoters are turned on, then “middle”, then “late” promoters.

What class/function of genes are commonly transcribed at each stage?

Early promoters transcribe genes for DNA replication and host modification, middle promoters for DNA replication and recombination, and late promoters for structural proteins and lysis enzymes

How did they determine which promoter should encode the inserted foreign gene in the phage?

They tried promoters in the early, middle, and late stage of phage reproduction and chose the one with the maximum gene expression but lowest phage propagation impact

Given the chosen parameters (maximal sfGFP levels and minimal impact on phage titer) which promoter would you use and why?

gp22 is an early promoter that enables high expression of sfGFP while minimizing the effect on phage propagation.

Conclusion of experiment 1

Engineered T4 phage can orchestrate the production and release of heterologous proteins in the mammalian gut during the phage infection process, leveraging its co-existence with its bacterial host for a sustained in situ effect

Chimeric infective particles expand species boundaries in phage inducible chromosomal island mobilization

capsid-forming PICIs (cf-PICIs) only require what from helper phages

They only need tail proteins because they encode their own capsid

What is the main function of phage tail proteins

mediate the attachment and infection of host cells, facilitating the transfer of genetic material during the infection process.

SaPI vs cf-PICIs
Which are only found in S. aureus and have very narrow host genera?

SaPIs

SaPI vs cf-PICIs
Which are highly abundant and have a diverse group of hosts across species and genera?

cf-PICIs

cf-PICIs are highly abundant, and
often identical in sequence, across different
species and genera. What does this mean

suggest that these elements are highly efficient at horizontal gene transfer, allowing them to spread virulence and antibiotic resistance genes widely and rapidly

Tail-less cf-PICIs were isolated from the supernatant of induced Kp DSM30104. In addition, supernatants were isolated from induced E. coli isolates containing capsid mutant prophage (either HK106 or HK022, which have different tails). Why capsid mutants?

To make sure the prophage can only donate a tail

What was the purpose of experiment 2

To see if tail from one species specific phage will impact the ability of a different prophage to infect its host.

What was the purpose of experiment 3:
Natural inter-species cf-PICI transfer

transfer of the cf-PICI can occur naturally in mixed bacterial populations, but it requires a compatible helper phage (like HK022) to provide the necessary tails for the cf-PICI capsids

You hypothesize that the gene encoding a therapeutic protein could be encoded into a lytic phage so that it is co-expressed with phage genes during the infection of a patient’s resident gut bacterium, and that phage–bacterial co-existence would lead to the sustained production of your protein.  Which of the options below would be best if the goal is to treat your patient?

High concentrations of your protein and of phage particles are released from each lysed bacterial cell.