Holin/endolysin/spanin cell lysis

For most phages, active and timed lysis involves three steps corresponding to disruption of each of the three layers of the cell envelope: inner membrane, peptidoglycan and outer membrane .

Two proteins, an endolysin and a holin are usually required for programmed host cell lysis by dsDNA prokaryotic viruses . Additional proteins called spanins are involved in the lysis process in Gram-negative hosts. A multistep process allows a better control of lysis timing .

1. Cell membrane disruption (Gram-negative host, Gram-positive host)

Holins (from making “holes”) are small membrane proteins that control the lysis timing. They accumulate in the host cell membrane until, at a programmed time (probably when reaching a critical concentration), they aggregate into oligomers and permeabilize the host cytoplasmic membrane inducing loss of polarization.

Canonical holins form large pores at one side of the bacteria, exposing locally the peptidoglycans to cytoplasmic canonical endolysin molecules . The outcome of this kind of lysis is a localized envelope catastrophe: “local blow-out”.

Pinholins form small pores that result in membrane depolarization, triggering SAR endolysin activation and degradation of peptidoglycans in the whole cellular periplasmic space. The outcome of this kind of lysis is a general lysis of the outer membrane of the host cell.

Antiholins associate specifically with the holins or pinholins giving rise to inactive heteromultimers. This allows a larger accumulation of holin molecules without any risk of premature aggregation before the triggering time.

ssDNA and ssRNA prokaryotic viruses usually have a “single gene lysis” which is thought to inhibit the peptidoglycan layer polymerization.
The advantage of the two-component system is that holins allow a abrupt and time-determined lysis thus preventing slow host cell deterioration.

2. Peptidoglycan disruption (Gram-negative host, Gram-positive host)

Endolysins are muralytic enzymes that degrade the host cell wall. Depending on the absence or presence of a signal sequence, endolysins are classified as canonical endolysins (no signal sequence) and signal-anchor-release endolysins (SAR endolysins, N-terminal type II signal anchor). Canonical endolysins accumulate in the cytoplasm and are released at a precise timing through the large holes generated by the aggregation of holin molecules.

SAR endolysins are already bound on the periplasmic side of the cell membrane and thus do not need to transit through large holes in the cell membrane. Instead, their release occurs through membrane depolarization performed by pinholins, which make small holes in the host cell membrane (about 2 nm in diameter) .

3. Outer membrane disruption (Gram-negative host)

Spanins are essential for the lysis of bacteria with both internal (IM) and outer (OM) membranes (mostly Gram-negative). Lysis of these host bacteria additionally requires the disruption of the outer membrane. Spanins span the inner and outer membrane either as a unique (u-spanin) or as a heterodimer molecule (IM-spanin or i-spanin + OM-spanin or o-spanin) .
After cell wall degradation by the endolysin, the spanin complexes are free to diffuse and aggregate. the lytic blowout occurs when sufficient spanin complexes are liberated within the degraded area of the PG. They mediate the fusion of the IM and OM, thereby disrupting the cell membranes .

Examples of lysis cassettes:

Virus Family Genus Phage lysis protein Reference
Phage T4 Myoviridae T4likevirus Endolysin
Holin
Spanin, outer membrane subunit
Spanin, inner membrane subunit
Antiholin
Phage P2 Myoviridae P2likevirus Endolysin
Holin
Antiholin
Spanin, inner membrane subunit
Spanin, outer membrane subunit
Phage P22 Podoviridae P22likevirus Endolysin
Holin
Spanin, inner membrane subunit
Spanin, outer membrane subunit -
Phage φKMV Podoviridae Phikmvlikevirus SAR endolysin
Pinholin
Spanin, inner membrane subunit
Spanin, outer membrane subunit
Phage T7 Podoviridae T7likevirus Holin
Spanin, inner membrane subunit
Spanin, outer membrane subunit
Phage lambda Siphoviridae Lambdalikevirus Endolysin
Holin
Spanin, inner membrane subunit
Spanin, outer membrane subunit
Phage T1 Siphoviridae Tunalikevirus Endolysin
Holin
u-Spanin