Bacteriophage-encoded lethal membrane disruptors: Advances in understanding and potential applications

Abstract

Holins and spanins are bacteriophage-encoded membrane proteins that control bacterial cell lysis in the final stage of the bacteriophage reproductive cycle. Due to their efficient mechanisms for lethal membrane disruption, these proteins are gaining interest in many fields, including the medical, food, biotechnological, and pharmaceutical fields. However, investigating these lethal proteins is challenging due to their toxicity in bacterial expression systems and the resultant low protein yields have hindered their analysis compared to other cell lytic proteins. Therefore, the structural and dynamic properties of holins and spanins in their native environment are not well-understood. In this article we describe recent advances in the classification, purification, and analysis of holin and spanin proteins, which are beginning to overcome the technical barriers to understanding these lethal membrane disrupting proteins, and through this, unlock many potential biotechnological applications.

Identifying the components of theShewanellaphage LambdaSo lysis system

“…While two of the LambdaSo main lysis components were readily identified, no spanin components turned up by homology searches. Known spanins consist of one or two protein components (reviewed in (2, 3)), but have in common that interaction with the OM is mediated via a lipid anchor. In Gram-negatives such proteins are characterized by a signal sequence followed by one or more cysteine residues to which the lipid anchor is coupled (29).…”

“…Bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC 4 (3); Molecular Probes TM ) was used for staining of depolarized cells. A 10 mg/ml (w/v) stock solution was prepared in DMSO.…”

“…All steps were performed at room temperature. Pellets were resuspended in 200 ml PBS and stained with DiBAC 4 (3) at a final concentration of 1mg/ml (w/v) for 20 min in the dark. After three steps of washing with 200 ml PBS, cells were resuspended in 200 ml PBS and evaluated using the TECAN plate reader.…”

“…Lysis is initiated by holins (4), which are transmembrane proteins that, upon production, accumulate in the CM where they arrange to form non-specific lesions sufficiently large for the passage of phage-produced endolysins once phage assembly is complete (5). These endolysins are peptidoglycan-degrading enzymes, which, once released into the periplasm, perform the second step, the breakdown of the cell wall (2, 3). Alternatively, some phages possess non-canonical holins, so-called pinholins, in concert with signal-anchor-release (SAR) endolysins (6, 7).…”

“…Accumulation of the pinholins leads to formation of small transmembrane channels (7, 8), which results in breakdown of the proton motive force and the release of the SAR endolysins from the CM, upon which degradation of the cell wall proceeds. The third and final step of phage lysis is performed by spanins, which can occur in at least two versions as a unimolecular or a two-component spanin system (2, 3). In the more common two-component spanin systems, one component (the i-spanin) is integrated into the CM by a transmembrane region, while the second component (the o-spanin) is a lipoprotein, which is tightly anchored in the OM.…”