Ry Young

Phages will out: strategies of host cell lysis

Most phages accomplish host lysis using a muralytic enzyme, or endolysin, and a holin, which permeabilizes the membrane at a programmed time and thus controls the length of the vegetative cycle. By contrast, lytic single-stranded RNA and DNA phages accomplish lysis by producing a single lysis protein without muralytic activity.

Holins kill without warning

Holins comprise the most diverse functional group of proteins known. They are small bacteriophage-encoded proteins that accumulate during the period of late-protein synthesis after infection and cause lysis of the host cell at a precise genetically programmed time. It is unknown how holins achieve temporal precision, but a conserved feature of their function is that energy poisons subvert the normal scheduling mechanism and instantly trigger membrane disruption. On this basis, timing has been proposed to involve a progressive decrease in the energized state of the membrane until a critical triggering level is reached. Here, we report that membrane integrity is not compromised after the induction of holin synthesis until seconds before lysis. The proton motive force was monitored by the rotation of individual cells tethered by a single flagellum. The results suggest an alternative explanation for the lysis “clock,” in which holin concentrations build to a critical level that leads to formation of an oligomeric complex that disrupts the membrane.

Two beginnings for a single purpose: the dual-start holins in the regulation of phage lysis.

For most large phages of both Gram‐positive and Gram‐negative bacteria, there appears to be a single pathway for achieving disruption of the host envelope, requiring at least two phage‐encoded lysis functions (a holin and an endolysin). The holin is a small membrane protein which causes a non‐specific lesion in the cytoplasmic membrane, which allows the endolysin to gain access to its substrate, the peptidoglycan. The scheduling of host lysis is effected by regulatory mechanisms which govern the synthesis and activity of the holin protein accumulating in the membrane. Accordingly, aspects of expression and function of holin genes are considered here, focusing mainly on the lambdoid S genes. This group of genes, of which lambda S is the prototype, are characterized by a dual‐start motif consisting of two Met start codons separated by one or two codons, at least one of which specifies Arg or Lys. Two protein products are elaborated, differing only by two or three N‐terminal residues but apparently possessing opposing functions: the shorter polypeptide is the active holin, or lysis‐effector, whereas the longer polypeptide apparently acts as an inhibitor of holin function. Models will be considered which may account for the ability of the holin to form a ‘hole’ in the cytoplasmic membrane at a programmed time, as well as for the inhibitory properties of the longer product. Finally, we discuss recent results suggesting that the dual‐start motif can be viewed as a level of regulation superimposed on a timing function intrinsic to the canonical holin structure.

Sizing the Holin Lesion with an Endolysin-β-Galactosidase Fusion

Double-stranded DNA phages require two proteins for efficient host lysis: the endolysin, a muralytic enzyme, and the holin, a small membrane protein. In an event that defines the end of the vegetative cycle, the λ holin S acts suddenly to permeabilize the membrane. This permeabilization enables the R endolysin to attack the cell wall, after which cell lysis occurs within seconds. A C-terminal fusion of the R endolysin with full-length β-galactosidase (β-Gal) was tested for lytic competence in the context of the late-gene expression system of an induced λ lysogen. Under these conditions, the hybrid R-β-Gal product, an active tetrameric β-Gal greater than 480 kDa in mass, was fully functional in lysis mediated by the S holin. Western blot analysis demonstrated that the lytic competence was not due to the proteolytic release of the endolysin domain of the R-β-Gal fusion protein. The ability of this massive complex to be released by the S holin suggests that S causes a generalized membrane disruption rather than a regular oligomeric membrane pore. Similar results were obtained with an early lysis variant of the S holin and also in parallel experiments with the T4 holin, T, in an identical lambda context. However, premature holin lesions triggered by depolarization of the membrane were nonpermissive for the hybrid endolysin, indicating that these premature lesions constituted less-profound damage to the membrane. Finally, a truncated T holin functional in lysis with the endolysin is completely incompetent for lysis with the hybrid endolysin. A model for the formation of the membrane lesion within homo-oligomeric rafts of holin proteins is discussed.

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.

Biochemical and Genetic Evidence for Three Transmembrane Domains in the Class I Holin, λ S

λ S, the prototype class I holin gene, encodes three potential transmembrane domains in its 107 codons, whereas 21 S, the class II prototype spans only 71 codons and encodes two transmembrane domains. Many holin genes, including λS and 21 S, have the “dual-start” regulatory motif at the N terminus, suggesting that class I and II holins have the same topology. The primary structure of 21 S strongly suggests a bitopic “helical-hairpin” topology, with N and C termini on the cytoplasmic side of the membrane. However, λ S chimeras with an N-terminal signal sequence show Lep-dependent function, indicating that the N-terminal domain of S requires export. Here the signal sequence chimera is shown to be sensitive to the missense change A52V, which blocks normal S function. Moreover, cysteine-modification studies in isolated membranes using a collection of S variants with single-cysteine substitutions show that the positions in the core of the 3 putative transmembrane domains of λ S are protected. Also, S proteins with single-cysteine substitutions in the predicted cytoplasmic and periplasmic loops are more efficiently labeled in inverted membrane vesicles and whole cells, respectively. These data constitute direct evidence that the holin Sλ has three transmembrane domains and indicate that class I and class II holins have different topologies, despite regulatory and functional homology.

The lethal lambda S gene encodes its own inhibitor.

The 107 codon reading frame of the lambda lysis gene S begins with the codon sequence Met1‐Lys2‐Met3..., and it has been demonstrated in vitro that both Met codons are used for translational starts. Furthermore, the partition of initiation events at the two start codons strongly affects the scheduling of lysis. We have presented a model in which the longer product, S107, acts as an inhibitor of the shorter product, S105, the lethal lysis effector, despite the fact that the two molecules differ only in the Met‐Lys residues at the amino terminus of S107. Using immunological and biochemical methods, we show in this report that the two predicted protein products, S105 and S107, are detectable in vivo as stable, membrane‐bound molecules. We show that S107 acts as an inhibitor in trans, and that its inhibitory function is entirely defined by the positively charged Lys2 residue. Moreover, our data show that energy poisons abolish the inhibitory function of S107 and simultaneously convert S107 into a lysis effector. We propose a two step model for the lethal action of gene S: first, induction of the S gene results in the accumulation of S105 and S107 molecules in mixed oligomeric patches in the cytoplasmic membrane; second, S monomers rearrange by lateral diffusion within the patch to form an aqueous pore. The R gene product, a transglycosylase, is released through the pore to the periplasm, resulting in destruction of the peptidoglycan and bursting of the cell. According to this model, the lateral diffusion step is inhibited by the energized state of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Transposition mutagenesis of bacteriophage lambda: a new gene affecting cell lysis.

Abstract Insertions of Tn903, a transposable kanamycin-resistance element, in bacteriophage lambda at 0.95 on the lambda physical map adversely affect growth of the phage. These insertion mutants are able to assemble particles, but are unable to lyse the infected cell properly. The mutants define a new genetic complementation group that we have designated as gene Rz . Cells infected with the λRz:: Tn903 isolates will, at the normal time of lysis, change their shape from a rod to a sphere. These spheres are stable in dilute buffers with Mg 2+ but are lysed with EDTA. In addition, these results demonstrate the utility of transposition mutagenesis in refining the genetic map of even so intensely studied a genome as lambda.

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

ABSTRACT Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii. We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patients downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.