Abortive infection (Abi) – committing suicide upon phage infection

Primarily describe defense by Abi in the strict sense, in which cell suicide or growth impairment clearly
plays a major role in mechanism of defense. In some cases, Abi systems have bacteriostatic effects
(inflicting metabolic arrest) rather than bactericidal effects (killing the cell). Prolonged bacteriostasis can
eventually result in cell death so it is also described.

Core principle is that a bacterial cell, after sensing the infection, commits suicide before the phage can
complete its replication cycle. Whereas restriction enzymes target the phage DNA at very early stages of
the infection, Abi systems are expected to be activate only when the phage reaches middle or late
stages. Abi is activated as a defense of last resort.

Two functional modules: one senses the phage and one kills the cell or shuts down metabolism.

Diversity of Abortive Infection Systems in Bacteria

Rex System – Loss of membrane potential and drop in ATP levels

One of the first Abi systems to be deciphered in E. coli was Rex.

The Rex system is expressed from a repressed lambda prophage (lysogenic rather than lytic cycle) and
comprises of two genes, rexA and rexB.

System is shown to restrict plaque formation of lambdoid phages, as well as particular strains of T4, T7,
T5 phages.

The RexA protein is thought to sense a protein DNA complex that is produced as an intermediate of
phage replication or recombination.

Two copies of RexA activate one copy of RexB, a membrane-anchored protein containing four
transmembrane helices.

When activated, RexB forms an ion channel in the inner membrane. Results in a loss of membrane
potential and drop in cellular ATP levels; inhibiting bacterial growth and aborts phage infection.

Some studies suggest RexB activation results in cell death, others propose it induces a stationary phase-
like state from which some cells can recover after viral infection is mitigated.

Phages have evolved ways to overcome the Rex system. The wild-type T4 phage encodes two proteins
(RIIA and RIIB) that mitigate the activity of the Rex system via an unknown mechanism, hence only
mutant T4 strains in which RII locus is inactivated are blocked by the Rex system.

PifA system – Leakage of ATP

Another Abi system described in E. coli is PifA, which aborts infection of phage T7 midway through
infection. Late gene transcription is severely reduced and phage DNA replication is abolished. Activation
of PifA leads to leakage of ATP through loss of membrane integrity. Though cells do not lyse, they die.
The pifA gene is encoded on the F plasmid. pifA is coexpressed on the same operon with pifC, a
transcriptional repressor that inhibits its own expression as well as pifA, a concept later referred to as
type IV toxin-antitoxin (TA) and is also manifested by the Abi system AbiE.

The PifA protein was found to be associated with the membrane and contains an ATP/GTP binding
domain essential for defensive activity.

T7 capsid protein gp10 is trigger for activation of PifA toxicity, although mode which PifA sense this
protein is unknown. Another protein that induces toxicity if gp1.2, an inhibitor of dNTP hydrolysis in the
infected cell.

T7 phages containing mutations in both gp1.2 and gp10 genes escape PifA-mediated defense.

Lit and PrrC Systems – Host translation inactivation

(Lit)

Several Abi systems, including Lit and PrrC, have shown to induce cell death by inactivating host
translation machinery. Lit is a protease encoded by defective e14 prophage of E. coli K12. Acronym for
Late Inhibition of T4 – inhibits T4 late gene expression. Lit is activated when gp23, major head protein of
T4, is expressed in the cell.

Gp23 binds translation elongation factor EF-Tu, and complex is identified by Lit. Once activated, Lit
cleaves EF-Tu between Gly59 and Ile60 in the nucleotide-binding domain of EF-Tu, inhibiting translation
and arresting bacterial growth. Mutations in gp23 that resulted in escape from Lit localized the domain
identified by Lit to a short sequence (29 amino acids) denoted Gol that is highly conserved in head
proteins of T-even phages.

(PrrC)

prrC is a unique Abi gene that is activated as a secondary line of dense. prrC gene was found to encoded
by the clinical E. coli strain CT 196. Under normal conditions, PrrC binds the type I restriction
endonuclease EcoprrI and does not interfere. PrrC monitors normal activity and becomes activated
when the restriction enzyme is tampered with.

Phage T4 encodes a short peptide called Stp, which binds and inhibits EcoprrI. Inhibition of EcoprrI by
Stp activates a ribonuclease domain in PrrC and PrrC cleaves tRNA lys, causing protein synthesis to halt.
The T4 phage encodes a mechanism that enables relegation of tRNA lys (manifest through enzymes
polynucleotide kinase and RNA ligase), and it is thought that it has evolved to overcome PrrC defense.
Therefore, PrrC defends through Abi only against T4 phages in which either polynucleotide kinase or
RNA ligase have been inactivated.

AbiZ System – Damaging cell membrane (premature lysis)

The Abi gene abiZ, which protects L. lactis against infection of phage phi31, also induces cell killing by
damaging the cell membrane of the infected cell. The abiZ protein is membrane bound and acts
cooperatively with lysin and holin proteins of the phage that are expressed late in the infection cycle and
accelerates cell lysis. Premature cell lysis causes infected cells to release unassembled phage particles.

Kinase gene, Stk2 – Phosphorylation leading to cell death

Shown to protect species of Staphylococcus against Siphoviridae phages via phosphorylation of proteins

Kinase gene, Stk2, is activated by phage protein PacK, which is hypothesized to play a role in phage DNA
packing. Once activated, Stk2 phosphorylates multiple proteins in the cell, leading to cell death.

Toxin-Antitoxin Systems and Abortive Infection

A TA system comprises a pair of genes, usually transcribed from the same operon. The first is toxic, and
the second confers immunity to this toxicity. TA systems have been divided into six types. The most
common type is type II, where toxic and antitoxin are proteins. Antitoxin physically binds to the toxin.
Antitoxin is less stable than toxin and more prone to be degraded by proteases. When cell enters
conditions of stress and protein production halts, the antitoxin is rapidly degraded and stable toxin is no
longer suppressed.
Type II TA systems are extremely abundant in microbial genomes. Some genomes encode more than 80
such systems. They play roles in bacterial responses to stress conditions, confer resistance to antibiotics,
take part in biofilm formation regulation and plasmid maintenance. TA systems have been proposed to
play a role in phage defense via Abi, but evidence is limited to few well-established examples. The
general role for TA systems in phage defense is sill under dispute.

RnIAB is a type II TA system encoded by E. coli K12 that protects against certain strains of phage T4 via
Abi. RnIA is a endoribonuclease toxin that is inhibited by its cognate antitoxin, RnIB. The half life of RnIA
is more than 25 min, while the RnIB half life is estimated to be 2 min. T4 infection rapidly shuts off host
gene expression and RnIB gets degraded soon, releasing RnIA to become an active endoribonuclease
that indiscriminately degrades host and phage mRNA, leading to cell death.

A homologous system called LsoAB encoded on a plasmid of E. coli strain O157:II7 shows similar
antiphage functionality

Wild-type T4 phage encodes a gene called dmd that can overcome defense by RnIAB. This gene is
expressed early in infection and encodes an antitoxin mimic that binds and neutralizes RnIA toxin. RnIAB
provides resistance against only T4 phages strains mutated in dmd.

ToxIN system, was shown to provide phage resistance in various enteric babcteria, including Erwinia
carotovora, Serratia marcescens, and E. coli. ToxIN is a type III TA system where the antitoxin (oxI) is a
ncRNA that physically binds the ToxN toxin protein and inhibits its activity. IN uninfected cells ToxI and
ToxN are found as an inactive RNA-protein complex comprising 3 ToxI RNAs and three ToxN proteins.
ToxN is an endoribonuclease and it becomes activated and presumably cleaves cellular and phage RNA,
stopping phage production and causing bacteriostasis or cell death. Interestingly, some phages escape
ToxIN by expressing a ncRNA that mimics the ToxI antitoxin and inhibits ToxN toxicity.
Cyclic Oligonucleotide-Based Antiphage Signaling Systems (CBASS)

In these systems, the phage-sensing module is disconnected from the cell-killing module and the
proteins that encode these two functions do not physically interact. Rather, when the phage sensing
protein identifies phage infection, it produces a small secondary messenger molecule comprising 2 or 3
nucleotides covalently linked to form a cyclic molecule and this secondary messenger activates the cell-
killing effect protein that carries out Abi.

The most studied CBASSs to date are two homologous systems encoded by Vibriocholerae El Tor and E.
coli TW11681. When cloned into a lab strain of E. coli that lacks CBASS, these systems protected against
a variety of phages. The key components are a protein called DncV that produces cyclic GMP-AMP
(cGAMP) molecules and CapV, which is a phospholipase activated by cGAMP. DncV protein is inactive in
uninfected cells, but infection triggers production of cGAMP that accumulates to micromolar level
concentrations. cGAMP binds and activates CapV, which degrades inner membrane, leading to cell lysis
and death.

CBASS is so temporally coordinated that, on one hand, cell death occurs before phage generates mature
particles, and on the other hand, the cell commits to death only after the phage has advanced to late
stages.

When infected by phage P1, cells encoding E. coli TW11681 CBASS generated secondary messenger
cGAMP 30-40 min after initial infection, and culture lysis due to CapV phospholipase a few minutes
after.

Considering that P1-infected CBASS-lacking cultures lyse after 60-70 min, the CBASS systems seems
active only when the phage has temporally completed about half of its replicative cycle.

Cell lysis does not release mature particles: CBASS encoding culture in which 20% of bacteria were
initially infected does not undergo collapse.

The phage component sensed by E. coli TW11681 CBASS is currently unknown.

Bacterial CBASS shows structural and functional homology with cGAS-STING antiviral pathway of animal
cells. CBASS were identified in more than 10% of sequenced bacterial and archaeal genomes and span a
remarkable diversity of oligonucleotide cyclase and effector protein activities.

Oligonucleotide cyclase proteins can produce a variety of cyclic oligonucleotide signals in addition to
cGAMP, including AMP-UMP cyclic UMP-UMP… The cell killing domain the effector gene also varies,
with effectors including, phospholipases, endonucleases, transmembrane domains that form
membrane-spanning ion channels, and other domains whose mechanisms of cell killing are unknown.

In many cases CBASS operons contain, in addition to two core genes, ancillary genes of unknown
function that were shown to be necessary for protection against some but not all phages.

CBASS in microbes suggest transferring via secondary messenger mediator is successful. The enzymatic
activity of the oligonucleotide cyclase rapidly amplifies the signal when the phage is sensed, so a single
sensor can activate multiple cell-killing effectors. This accelerates the process and shortens the time
elapsed from phage sensing to execution of cell killing.
Abortive Infection in CRIPSR-Associated Defense

CRISPR-Cas is the adaptive immune system of bacteria and archaea, capable of acquiring short pieces of
phage DNA and storing them as spacers to form immune memory.

Type I, which are the most abundant in nature, and type II systems, which include Cas9, both degrade
phage DNA and are capable of abolishing the infection without necessitating cell death. However,
findings show that type II systems can sometimes lead to Abi.

In these systems, the crRNA-Cas complex binds to phage RNA rather than DNA. The phage RNA is
recognized during active transcription from the phage genome and cleaved by Csm3 (IIIA) or Cmr4 (IIIB)
subunit of the crRNA/protein complex.

Another component of the complex (Cas10) cleaves the DNA from which the RNA was transcribed. It
was recently shown recognition and cleave of phage RNA induces a third enzymatic by the Palm domain
of Cas10. Once phage RNA is identified by base pairing with crRNA, Cas10 synthesizes a cyclic
oligoadenylate (cOA) secondary messenger comprising of 4 or 6 AMP molecules in a cyclic configuration.
The cOA bind and activate n RNase called Csm6 that indiscriminately degrades phage and host RNAs,
leading to dormancy and sometimes death.

These systems become activated only if first lines have failed to inactivate the phage by degrading its
DNA because transcription is a signature for active propagation. When phage transcription is recognized,
type III CRISPR-Cas system attempts to inactivate it by cleaving both transcribed RNA and phage DNA.

Presumable, a limited amount of cOA will not be sufficient to induce full toxicity by Csm6, but if multiple
loci are identified, it would mean that phage genome has already managed to replicate and phage gene
expression is abundant.

Interestingly, recent findings show that bacteria encode enzymes that can hydrolyze cOA molecules;
these enzymes can clear the cell from residual cOAs and survive the phage infection and resuscitate it
from Csm6-induced dormancy.

Abi Was also suggested to be the outcome of type VI CRISPR-Cas systems. The effector protein in these
systems is Cas13. Base pairing with phage RNA results in cleavage of the RNA but also induces a
nonspecific RNase activity of Cas13 that cleaves both cell and phage mRNAs, resulting in dormancy.

Summary and Outlook

This article reviewed Abi systems that have been discovered and studied over a period of more than
four decades. Researchers expanded the discussion on newer discoveries from the past few years. They
did not enumerate of all Abi systems, rather those in which cell death or dormancy was clearly
attributed to the activity of the defense system. They also focused on Abi systems in which at least part
of the mechanism has been elucidated.

While mechanistic diversity of Abi systems have been recognized for several decades, their abundance
in nature was not fully appreciated. Recent discovery of CBASS and type III CRISPR-Cas systems as
widespread and abundant suggests cell suicide in an immunological strategy that may be more
abundant in bacteria and archaea than previously relized.

Historically, Abi was studied mainly in E. coli and Lactococci. With expansion of genomic information,
studies focusing on defense systems in nonmodel organisms likely will reveal additional systems that
rely on cell suicide for colony survival.

New antiphage systems were found to be encoded by prophages in Mycobacaterium and Gordonia were
suggested to function via Abi.

Future studies on these systems, as well as other defense systems may result in an additional wave of
discoveries that would expand the already fascinating diversity of known Abi mechanisms.
Useful Information:

The lysogenic cycle: The phage infects a bacterium and inserts its DNA into the bacterial chromosome
allowing the phage DNA (prophage) to be copied and passed on along with the cell’s own DNA. The cycle
allows a phage to reproduce without killing its host. Lambda phage can switch between lytic and
lysogenic cycle.

Protease: enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single
amino acids