Bacteriophage Defense Mechanisms

Flashcards about bacteriophage defense mechanisms against CRISPR and restriction enzymes.

What mechanisms do bacteriophages use to avoid host immune pathways?

Inhibition or evasion of immune pathways that target nucleic acids, such as CRISPR-Cas and restriction-modification systems.

How does jumbo phage ΦKZ segregate its DNA from host immunity nucleases?

By constructing a proteinaceous nucleus-like compartment.

What immunity mechanisms is ΦKZ resistant to?

Many DNA-targeting mechanisms, including subtypes of CRISPR-Cas3, Cas9, Cas12a, and restriction enzymes HsdRMS and EcoRI.

What CRISPR-Cas enzyme is ΦKZ sensitive to, and why?

Cas13a, because it targets RNA, and phage mRNA localizes to the cytoplasm.

What is the proposed mechanism by which Pseudomonas jumbo phages evade DNA-targeting nucleases?

Assembly of a protein barrier around their genome.

How do some P. aeruginosa phages avoid CRISPR destruction?

By encoding anti-CRISPR proteins that inhibit type I-E and I-F CRISPR-Cas systems.

What happened when ΦKZ was challenged with different type I-C crRNAs?

The ΦKZ titre did not decrease, showing resistance to type I-C CRISPR-Cas.

Does the ΦKZ genome contain homologues of known anti-CRISPR (Acr) genes?

No, the ΦKZ genome has no homologues of known Acr genes.

What was used to determine the mechanism by which ΦKZ evades CRISPR-mediated destruction?

Type II-A CRISPR-Cas9 from Streptococcus pyogenes (SpyCas9).

How did SpyCas9 affect control phage JBD30 versus phage ΦKZ?

SpyCas9 robustly targeted control phage JBD30, but phage replication and lysis of host cells were unaffected during phage ΦKZ infection.

What does the ability of ΦKZ to evade unrelated CRISPR systems suggest?

That ΦKZ might be generally resistant to CRISPR-Cas immunity, instead of relying on specific inhibitor proteins, which is called ‘pan-CRISPR’ resistance.

What are restriction-modification (R-M) systems?

The most common form of bacterial immunity in nature.

How did ΦKZ behave when subjected to type I and type II R-M systems?

No restriction was observed for ΦKZ, it was refractory to both.

Where does the replication of phage DNA take place in ΦKZ and ΦKZ-like phages?

In an elaborate proteinaceous nucleus-like compartment.

What role does PhuZ play in ΦKZ phage infection?

A phage-encoded homologue of tubulin, PhuZ, that centers the compartment within the host cell.

What is the localization of proteins involved in DNA replication, transcription and recombination in ΦKZ?

They localize inside the shell, whereas mRNA and proteins that mediate translation localize in the cytoplasmic space.

What happens to immune enzymes during ΦKZ phage infection?

The immune enzymes are excluded from the shell during phage infection.

Where is the phage DNA located, and what happens to the host genome during ΦKZ infection?

The phage DNA is located inside the shell, whereas the host genome is degraded.

What governs protein internalization in the ΦKZ phage shell?

Each protein of known function that localizes inside the shell interacts with DNA.

What is the adaptive function of the ΦKZ phage shell?

Exclusion of DNA-binding Cas and restriction proteins.

What happened when EcoRI and Cas9 were assayed in vitro on ΦKZ DNA?

Both enzymes cleaved the DNA, showing that the ΦKZ phage genome could be a substrate for DNA cleavage if accessed.

What did the experiment fusing Cas9 to ORF152 show?

The fusion redirected Cas9 from its previously diffuse state but the large Cas9 protein was unable to traverse the shell border.

What happened when EcoRI was fused to Cherry-tagged ORF152?

This fusion resulted in a notable decrease in the ΦKZ titre and markedly reduced plaque sizes.

What was the effect of a catalytic mutant EcoRI(E111G) fused to Cherry–ORF152?

It exhibited no immune activity against ΦKZ.

What phenotype was observed as a result of expressing EcoRI-Cherry-ORF152?

EcoRI–Cherry–ORF152 impaired the ability of infected cells to form full shells and proceed through the infection process.

What resistance is conferred by the shell of jumbo phage ΦKZ?

Cause of the resistance to immunity that is shown by jumbo phage ΦKZ.

What immune system may exist in vivo to evade the ΦKZ phage defense mechanism?

A ribonuclease that could provide anti-ΦKZ immunity.

How was Cas13a used to target ΦKZ?

Adapted the type VI-A CRISPR RNA-guided RNA nuclease Cas13a from Listeria seeligeri (LseCas13a) for phage targeting in P. aeruginosa.

What was the effect of LseCas13a on ΦKZ plaquing efficiency?

Three LseCas13a spacers decreased the efficiency of ΦKZ plaquing by more than 106 fold.

What happens to the injected phage genome before shell assembly?

The injected phage genome may be protected before shell assembly by injected phage proteins or pre-existing host factors.