Bacteriophages and their Therapeutic Applications

Cluster representation is heterogeneous.
Cluster A: Over 850 members.
Cluster B: 458 members.
Clusters C, F, K are also well represented.
Several clusters have very small numbers of phages.
Six singletons exist.
Newly isolated phages mirror this profile, with Clusters A and B most represented.
New cluster AI emerged with two phages: Saint Augustine (isolated in 2023) and Causa (isolated in 2024).

Isolated by Nathan Simpson (University of North Georgia) and Hope College students.
Related but not closely related to other mycobacteria phages.
Large genomes: ~101 kilobases.
~180 ORFs and ~17 tRNAs.
Likely lytic, with no obvious lysogeny genes.
Suspected DNA modifications due to genomic features.
Long predicted tails, with long tape measure protein genes.
lots of repeated sequences
Capsids likely constructed from pentagons and hexameric subunits.

Arthrobacter phages: Two cluster FS phages popped up.
- Smaller, ~40 kb.
- Probably temperate, carrying integrase and putative repressors
Phroglets:
- Arthrobacter phage singleton with low GC content of 43.1%.
- Modest-sized genome of 47.2 kilobases.
- 50% orphams
- Likely a podophage with a short, stubby tail, likely lytic.
Lishka:
- Streptomyces phage in BC3 with high GC content of 72.6%.
- Relatively small, 36.2 kilobases.
- Suspected to be lytic.

Mycobacteria phage in Subcluster A1.
Encodes a well-studied serine integrase used for genome engineering.
Efforts to engineer BxB1 to display SARS-CoV-2 antigens on to the surface of the capsid revealed technical constraints.
Cryo-electron microscopy provided high-resolution structure (2.7-3 angstroms).
Revealed the capsid, tail, and tail tip structure that binds to the host cell surface.

Bottom-most ring of tail tube subunits.
Hub in the middle with a cage wrapping around it.
Aqua-colored spike protein in close contact with the bacterial host surface.
Intimate associations between proteins.
Potential for engineering phages by mixing and matching parts to create new specificities.

Cryo-electron topography used to study binding and interactions.
Cage rotates and flattens to form a landing pad on the mycobacterium cell wall.
Fibers flatten out.
Density stretches from the phage particle into the cell wall, but not to the cytoplasmic membrane.
Tape measure protein and DNA must traverse into cell cytoplasm.

Muddy: A purple-colored protein sits on the outer edge, encoded by a gene in a different part of the genome.
one gene is encoded within the tape measure protein gene.

Isolating mutants resistant to BPs or Muddy.
Transposon mutagenesis to identify genes required for phage infection.
Transposon insertions mapped to genes involved in the synthesis of trehalose polyphleates (TPPs).

Isolated Tn mutants resistant to BPs and Muddy in TPP Genes
These mutants have substitutions in the tail spike proteins.
TPS-independent mutants negate the possibility of phage-resistant mutants due to the TPS pathway.

Using the same transposon library to find mutants resistant to TPS-independent mutants.
Resistance is rare and involves other mechanisms.
Mutants resistant to one phage are no longer resistant to the other phage.

Identified phages with killing activity against Mycobacterium abscessus in a disseminated infection case.
Successful outcome.
Received over 550 clinical isolates of Mycobacterium abscessus.
Provided phages for treatment of about 45 patients on a compassionate use basis.
Reported a consecutive case series of 20 patients.

Mycobacterium abscessus grows as smooth or rough colonies, with different phage infection behaviors.
Isolates are streaked out and scored as either rough or smooth.
Tested against a panel of promising phages (about three dozen).

Plaque assay to look for phages with high efficiency of plucking.
Killing assays to see if phages efficiently kill bacteria:
- Mixing cells with phages in a checkerboard of combinations and growing in liquid culture.
- Mixing a large number of cells with phages and letting them sit in liquid culture.

Good outcome (75% of rough strains): One or more phages work well and completely kill bacteria.
For smooth strains: Harder time finding phages; no efficient plucking.

No data from five patients (patient died or appeals stopped).
No obvious clinical benefit from phage treatment after six months for four patients.
Favorable clinical or microbiological outcomes in 11 patients.
No resistance or therapy failure due to resistance in patients administered a single phage.
Some patients raise antibodies against phages.

Many Mycobacterium abscessus strains carry prophages integrated into their genomes.
Prophages can be released into the supernatant.
Combining cell lysates with other strains can identify phages that infect and form plaques.
50-100 new phages identified in this manner.
These phages can be engineered for therapeutic use to expand the repertoire of useful phages.