Douglas K. Struck

Breaking free: "protein antibiotics" and phage lysis.

Bacteriophages must destroy the bacterial cell wall to lyse their host and release their progeny into the environment. There are at least two distinct mechanisms by which phages destroy the cell wall. Bacteriophages with large genomes use a holin-endolysin system, while bacteriophages with small genomes encode a single lysis protein. Three unrelated single protein lysis systems are known and these proteins will be the focus of the review. Recent results indicate that at least two of these proteins inhibit cell wall synthesis and are thus the phage analogs of antibiotics like penicillin.

Topological dynamics of holins in programmed bacterial lysis.

The fate of phage-infected bacteria is determined by the holin, a small membrane protein that triggers to disrupt the membrane at a programmed time, allowing a lysozyme to attack the cell wall. S2168, the holin of phage 21, has two transmembrane domains (TMDs) with a predicted N-in, C-in topology. Surprisingly, TMD1 of S2168 was found to be dispensable for function, to behave as a SAR (“signal-anchor-release”) domain in exiting the membrane to the periplasm, and to engage in homotypic interactions in the soluble phase. The departure of TMD1 from the bilayer coincides with the lethal triggering of the holin and is accelerated by membrane depolarization. Basic residues added at the N terminus of S2168 prevent the escape of TMD1 to the periplasm and block hole formation by TMD2. Lysis thus depends on dynamic topology, in that removal of the inhibitory TMD1 from the bilayer frees TMD2 for programmed formation of lethal membrane lesions.

The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes.

Bacteriophage λ has four adjacent genes –S, R, Rz and Rz1– dedicated to host cell lysis. While S, encoding the holin and antiholin, and R, encoding the endolysin, have been intensively studied, the products of Rz and Rz1 have not been characterized at either the structural or functional levels. Rz1 is an outer membrane lipoprotein and our results indicate that Rz is a type II signal anchor protein. Here we present evidence that an Rz–Rz1 complex that spans the periplasm carries out the final step in the process of host lysis. These results are discussed in terms of a model where endolysin‐mediated degradation of the cell wall is a prerequisite for conformational changes in the Rz–Rz1 complex leading to the juxtaposition and fusion of the IM and OM. Fusion of the two membranes removes the last physical barrier to efficient release of progeny virions.

The Pinholin of Lambdoid Phage 21: Control of Lysis by Membrane Depolarization

The phage 21 holin, S(21), forms small membrane holes that depolarize the membrane and is designated as a pinholin, as opposed to large-hole-forming holins, like S(lambda). Pinholins require secreted SAR endolysins, a pairing that may represent an intermediate in the evolution of canonical holin-endolysin systems.

Deletion analysis resolves cell-binding and lytic domains of the Pasteurella leukotoxin

A series of internal deletions in the IktA gene of Pasteurella haemofytica has been constructed. All of the deletions eliminated the lytic activity of the leukotoxin towards the bovine lymphoma cell line, BL‐3. Deletions removing segments of the amino‐proximal hydrophobic region, which is thought to constitute an essential membrane‐spanning domain, were found to agglutinate BL‐3 cells. Agglutination was similar to lysis by the wild‐type toxin in that it was dependent upon the presence of calcium and required expression of the lktC gene. The agglutinating deletion proteins protected BL‐3 cells from lysis by the wild‐type toxin in a competitive fashion. This suggests that these mutants bind to a surface feature of the leukocyte which interacts with the native leukotoxin. These findings demonstrate that the cell‐binding and lytic domains of the leukotoxin are separable.

Structure of the lethal phage pinhole

Perhaps the simplest of biological timing systems, bacteriophage holins accumulate during the phage morphogenesis period and then trigger to permeabilize the cytoplasmic membrane with lethal holes; thus, terminating the infection cycle. Canonical holins form very large holes that allow nonspecific release of fully-folded proteins, but a recently discovered class of holins, the pinholins, make much smaller holes, or pinholes, that serve only to depolarize the membrane. Here, we interrogate the structure of the prototype pinholin by negative-stain transmission electron-microscopy, cysteine-accessibility, and chemical cross-linking, as well as by computational approaches. Together, the results suggest that the pinholin forms symmetric heptameric structures with the hydrophilic surface of one transmembrane domain lining the surface of a central channel ≈15 Å in diameter. The structural model also suggests a rationale for the prehole state of the pinholin, the persistence of which defines the duration of the viral latent period, and for the sensitivity of the holin timing system to the energized state of the membrane.

The holin of bacteriophage lambda forms rings with large diameter

Holins control the length of the infection cycle of tailed phages (the Caudovirales) by oligomerizing to form lethal holes in the cytoplasmic membrane at a time dictated by their primary structure. Nothing is currently known about the physical basis of their oligomerization or the structure of the oligomers formed by any known holin. Here we use electron microscopy and single‐particle analysis to characterize structures formed by the bacteriophage λ holin (S105) in vitro. In non‐ionic or mild zwitterionic detergents, purified S105, but not the lysis‐defective variant S105A52V, forms rings of at least two size classes, the most common having inner and outer diameters of 8.5 and 23 nm respectively, and containing approximately 72 S105 monomers. The height of these rings, 4 nm, closely matches the thickness of the lipid bilayer. The central channel is of unprecedented size for channels formed by integral membrane proteins, consistent with the non‐specific nature of holin‐mediated membrane permeabilization. S105 present in detergent‐solubilized rings and in inverted membrane vesicles showed similar sensitivities to proteolysis and cysteine‐specific modification, suggesting that the rings are representative of the lethal holes formed by S105 to terminate the infection cycle and initiate lysis.

Regulation of a Phage Endolysin by Disulfide Caging

In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, Lyz(P1), of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz(103), the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz(103) does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz(103) is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, Lyz(P1) and Lyz(103) define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz(103) is the first enzyme found to be regulated by disulfide bond caging of its active site.

The T4 RI Antiholin Has an N-Terminal Signal Anchor Release Domain That Targets It for Degradation by DegP

Lysis inhibition (LIN) of T4-infected cells was one of the foundational experimental systems for modern molecular genetics. In LIN, secondary infection of T4-infected cells results in a dramatically protracted infection cycle in which intracellular phage and endolysin accumulation can continue for hours. At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. Here we show that the N-terminal sequence comprises a signal anchor release (SAR) domain, which causes the secretion of RI in a membrane-tethered form and then its subsequent release into the periplasm, without proteolytic processing. Moreover, the SAR domain confers both functional lability and DegP-mediated proteolytic instability on the released form of RI, although LIN is not affected in a degP host. These results are discussed in terms of a model for the activation of RI in the establishment of the LIN state.

Evolutionary dominance of holin lysis systems derives from superior genetic malleability

For the microviruses and the leviviruses, bacteriophages with small single-stranded genomes, host lysis is accomplished by expression of a single gene that encodes an inhibitor of cell wall synthesis. In contrast, phages with double-stranded DNA genomes use a more complex system involving, at minimum, an endolysin, which degrades peptidoglycan, and a holin, which permeabilizes the membrane in a temporally programmed manner. To explore the basis of this difference, a chimera was created in which lysis gene E of the microvirus phiX174 replaced the entire lysis cassette of phage lambda, which includes the holin gene S and the endolysin gene R. The chimeric phage was viable but more variability was observed both in the distribution of plaque sizes and in the burst sizes of single cells, compared to the isogenic S(+) parent. Using different alleles of E, it was found the average burst size increased with the duration of the latent period, just as observed with S alleles with different lysis times. Moreover, within a set of missense E alleles, it was found that variability in lysis timing was limited and almost exclusively derived from changes in the level of E accumulation. By contrast, missense mutations in S resulted in a wide variation in lysis times that was not correlated with levels of accumulation. We suggest that the properties of greater phenotypic plasticity and lesser phenotypic variation make the function of holin proteins more genetically malleable, facilitating rapid adaptation towards a lysis time that would be optimal for changed host and environmental conditions. The inferior malleability of single-gene systems like E would restrict their occurrence to phages in which coding capacity is the overriding evolutionary constraint.