This document summarizes various bacterial defense mechanisms against bacteriophages (phages) and how phages have adapted to evade these defenses. The major bacterial defenses discussed are adsorption inhibition, restriction-modification systems, CRISPR-Cas systems, and abortive infection mechanisms. The document then describes strategies phages use to adapt, such as evolving new receptor binding proteins to access host receptors, masking restriction sites in their genomes, mutating protospacers to evade CRISPR recognition, and inhibiting abortive infection toxin-antitoxin systems. The constant evolutionary arms race between bacterial defenses and phage counter-defenses is explored.

1. Revenge of the phages: defeating bacterial
defences
Raffia Siddique
Raffiasiddique_93@hotmail.com
National University Of Sciences And Technology
Julie E. Samson, Alfonso H. Magadán, Mourad Sabri and Sylvain Moineau
NATURE REVIEWS | MICROBIOLOGY

3. Abstract
• Constant fight b/w bacteria
and bacteriophage
• What bacteria has?
strong defense
• What bacteriophage has?
1. adsorption inhibition,
2. restriction–modification
3. CRISPR–Cas
4. abortive infection.

4. Bacterial anti-phage system
1. Inhibition of the phage
attachment to the host cell
surface receptors
2. Cleavage of the invading
phage genome
3. Abort phage infection by
making cell suicide

5. Access to host receptors
• Adsorption
• Interaction b/w RBP
present on the tail of
bacteriophage and the
bacterial cell surface
receptors.
• bacterial receptors are the
surface proteins,
polysaccharides and LPSs

6. Adapting to new receptors
Tailed phages are
able to modify
their RBPs to
acquire novel
receptor tropism
1- Evolving a new RBP
2- Gaining access to masked
receptors
3- Stochastic expression of RBPs

7. 1-Evolving a new RBP
Receptor of bacteria
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.

8. 2- Gaining access to masked receptors
surface molecules (for
example, capsule or
exopolysaccharide (EPS))
at the receptor site can
limit or prevent phage
access if the phage possesses a
depolymerase, it can
degrade these
substances to unmask
the receptor.

10. 3- stochastic expression of RBPs
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.

13. Adapting to new receptors

14. Battling restriction–modification
systems
• When a phage
genome manages to
enter the cell, it can
still face a myriad of
intracellular antiviral
barriers.
• Restriction–
modification (R–M)
systems are
widespread in
bacteria.
These systems typically
use a restriction
endonuclease (REase) to
cut invading foreign
DNA at specific
recognition sites.

16. These systems are classified into four types based upon;
1-structure,
2- recognition
site
3- mode of
action

17. phage anti-restriction strategies
Passive mechanisms of phage evasion.
The phage genome can be modified by the
host methyltransferase (Mtase)
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

18. Active mechanisms of phage evasion
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

19. Evading 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 and
2. 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.

20. 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.

22. Evading CRISPR-Cas System
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,

23. The phage-encoded anti-CRISPR protein blocks the interference
step by preventing the formation or blocking the action of the
CRISPR–Cas complexes.

24. The CRISPR–Cas system of Vibrio cholerae
phages. target an uncharacterized antiphage
system of the V. cholerae host;
this 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.

25. Escaping abortive-infection
mechanisms
Abi systems inhibit various steps of the phage replication cycle
Abi systems typically consist of a
single protein or protein
complex and are often found on
mobile genetic elements, such as
prophages and plasmids,
Toxin–antitoxin (TA)
systems represent a
subgroup of Abi systems
that lead to bacterial death
following activation by
phage infection.
TA systems are composed of
a toxin and a neutralizing
antitoxin that renders the
toxin ineffective during
normal bacterial growth
The antitoxin is more labile than the
toxin, so when a stress is encountered,
the antitoxin is degraded and the toxin
is free to induce either dormancy or
cell death

26. 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.

27. Thanks
Questions?