Access to host receptors

• Adsorption = the specific interaction between the phage receptor-binding protein (RBP) + its bacterial cell surface receptor

• Bacterial receptors (biochemical families) = surface proteins, polysaccharides and lipopolysaccharides (LPSs)

Adapting to new receptors.

• Tailed phages are able to modify their RBPs to acquire novel receptor tropism

• phage λ attacks E.coli through cognate receptor LamB and interaction occurs between RBP protein J of phage and LamB of the host E.coli

but • EVOLUTION • phage targets new receptor, OmpF of E.coli B• When expression of LamB of host is reduced due to

mutation

• when a mutated form of a host receptor is expressed, phages can evolve to recognize the altered receptor structure.

Phage strategies to access host receptors

a. Evolving a new RBPb. Gaining access to masked receptorsc. Stochastic expression of RBPs

Phage strategies to access host receptors

Phages can adapt to receptor changes. Phage receptor-binding proteins (RBPs) recognize specific receptors on the host cell surface, but if the cognate receptor is modified through mutation or replaced by a different molecule, then the phage is unable to adsorb to the host. To infect this receptor-modified host, the phage can evolve to target a new receptor, by acquiring mutations in the genes encoding the RBP or tail fibres. For example, mutations in the gene encoding protein J of coliphage λ enables this RBP to recognize a new receptor, OmpF, in addition to the cognate receptor, LamB.

The expression of surface molecules (for example, capsule or exopolysaccharide (EPS)) at the receptor site can limit or prevent phage access. If the phage lacks the enzymes required to degrade the capsule or biofilm matrix, it cannot access the receptor. However, if the phage possesses a depolymerase, it can degrade these substances to unmask the receptor.

Some bacteria express phage receptors in a stochastic manner, either through phase variation or through physiological regulation (such as in response to growth phase). Phages can modify their RBPs in a manner that allows them to interact with a surface component that is expressed by the host at that time. This can be achieved by mutations in the gene encoding major tropism determinant (Mtd) for the Bordetella spp. phages, by proteolytic cleavage of tail fibres for Lactococcus lactis phages TP901 1 and Tuc2009 or by duplication of a ‑His box element in the coliphage T4 tail proteins.

Passive and active strategies to avoid restriction–modification

Some phages have few restriction sites in their genome, or these sites are too far apart to be recognized by the host restriction endonuclease (REase), thus preventing targeting.

The phage genome can be modified by the host methyltransferase (MTase). The modified phage is able to multiply, and the newly synthesized viral DNA will be protected against this specific REase when it infects other host cells containing the same restriction–modification system. Alternatively, the phage can encode its own MTase, and if the modification generated by this enzyme is compatible with the host REase, the phage DNA is protected at each replication cycle.

A phage can co inject proteins such as DarA and ‑DarB (of phage P1) with its genome to bind directly to the phage DNA and mask restriction sites.

A phage protein (such as Ocr of phage T7) mimics the target DNA and sequesters the restriction enzyme. Ocr binds to both the MTase and the REase of the type I restriction–modification system EcoKI and inhibits its activity.

A phage protein such as Ral of phage λ can activate the activity of the MTase and thereby accelerate protection of the phage DNA. The peptide Stp of phage T4 can also inhibit restriction by direct perturbation of the REase –MTase complex.

CRISPR–Cas systems These are the part of bacterial immune system which detects and recognize the foreign DNA and cleaves it.1. The CRISPR (clustered regularly interspaced short

palindromic repeats) loci and2. Cas (CRISPR-associated) proteins can target and cleave invading DNA in a sequence-specific manner.

Spacer=The direct repeats in a CRISPR locus are separated by short stretches of non-repetitive DNA called spacers that are typically derived from invading plasmid or phage DNA.

Protospacers = The nucleotide sequence of the spacer must be similar to a region in the phage genome called a protospacer in order to recognize and subsequently block phage replication.

CRISPR–Cas systems function in three general steps

1) adaptation or immunization (involving the acquisition of spacers

2) biogenesis of CRISPR RNA (crRNA; encoded by the repeat–spacer regions)

3) interference (cleavage of invading nucleic acid).

1. Adaptation The direct repeats of the CRISPR locus are separated by short stretches of non-repetitive DNA called spacers, which are acquired from the invading DNA of plasmids or viruses in a process known as adaptation.

2. biogenesis of CRISPR RNA

The CRISPR locus is transcribed as a long primary pre-crRNA transcript, which is processed to produce a collection of short crRNAs (a process referred to as biogenesis of crRNA. Each crRNA contains segments of a repeat and a full spacer and, in conjunction with a set of Cas proteins, forms the core of CRISPR–Cas complexes. These complexes act as a surveillance system and provide immunity against ensuing infections by phages or plasmids encoding DNA complementary to the crRNA. 3. Interference

On recognition of a matching target sequence, the plasmid or viral DNA is cleaved in a sequence-specific manner (known as interference). The nucleotide sequence of the spacer must be identical to a region of the viral genome or plasmid (known as the protospacer) for the CRISPR–Cas complex to block replication of the foreign element.

Phage strategies to by-pass CRISPR–Cas systems.

• Mutations in the phage protospacers or in the protospacer-adjacent motif (PAM) render the phage insensitive to the interference step of the CRISPR–Cas system, owing to the requirement for complementarity between the CRISPR RNA (crRNA) and the target DNA.

• The anti-CRISPR system of Pseudomonas aeruginosa lysogens. The phage-encoded anti-CRISPR protein blocks the interference step by preventing the formation or blocking the action of the CRISPR–Cas complexes.

The CRISPR–Cas system of Vibrio cholerae phages. After entry of the phage genome into the cell, the viral crRNAs and CRISPR–Cas complexes are expressed and target an uncharacterized antiphage system of the V. cholerae host; the latter system is contained within a locus resembling a phage-inducible chromosomal island (PICI), referred to as a PICI-like element (PLE). The spacers in the phage CRISPR locus are complementary to PLE sequences, and the CRISPR machinery is then able to specifically target this genetic element and inactivate it.

Phage strategies to by-pass toxin–antitoxin systems

During normal bacterial growth, the antitoxin neutralizes the toxin, thereby preventing bacterial cell death.

During phage infection, an imbalance in the toxin–antitoxin ratio or inactivation of the antitoxin results in liberation of the toxin, which is free to act on its target and inhibit bacterial growth. This growth inhibition also leads to the abortion of phage infection.

When certain genes in Lactococcus spp. phages are mutated, the phages can by-pass abortive-infection (Abi) systems. For example, related Lactococcus spp. phages can by-pass the AbiQ mechanism through mutations in genes involved in nucleotide metabolism.

Some phages encode a molecule (for example, Dmd of coliphage T4) that functionally replaces the bacterial antitoxin, thereby counteracting toxin activity and avoiding host death. Phage ϕTE of Pectobacterium atrosepticum produces a pseudo-antitoxin RNA or hijacks the native antitoxin ToxI to neutralize the toxin ToxN during phage infection.