Membrane fusion during phage lysis

Abstract

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.spanin | membrane fusion | spheroplast | holin | endolysin P hage lysis, the most common cytolytic event in the biosphere, has been extensively studied in phage λ, where four genes encoding five proteins (Fig. 1A) effect a three-step lytic process that releases the progeny virions (1, 2). The infection cycle suddenly terminates when the S105 holins, small membrane proteins encoded by gene S, are redistributed into large 2D foci, resulting in the formation of μm-scale holes in the cytoplasmic membrane (3). This event, called holin "triggering," occurs at a time specific to the allelic state of S and is temporally regulated by the proportion of a second S product, the antiholin S107 (4, 5). The R endolysin is then able to escape through the holes to attack the peptidoglycan (PG). Because the PG layer confers shape and mechanical integrity to the cell, holin and endolysin function was long thought to be necessary and sufficient for lysis (6, 7). However, recent genetic and physiological studies revealed that two other λ proteins, Rz and Rz1, are also required (Fig. 1B) (8). Rz and Rz1 are a type II integral membrane protein (N-in, C-out) and an outer membrane (OM) lipoprotein, respectively (9-11). Interacting by the C-termini of their periplasmic domains, Rz and Rz1 form a complex spanning the entire periplasm, designated as the spanin complex to denote its topology in the envelope. Accordingly, Rz and Rz1 are designated as the inner membrane (IM) (i-spanin) and OM (o-spanin) subunits of the spanin complex ( Fig. 1 A and B) (12). Experiments with GFP-Rz chimeras and biochemical analysis of envelope proteins indicate that the spanin complexes accumulate in the envelope throughout the morphogenesis period of the infection cycle (8, 13). The available data indicate that, after destruction of the PG by the endolysin, the spanin complex functions to disrupt the OM. In the absence of spanin function, the infection cycle termin...

Localization and Regulation of the T1 Unimolecular Spanin

“…The PG meshwork acts as a negative inhibitor of gp11 function. Previously, we showed that PG depletion in the presence of physiological levels of Rz-Rz1 resulted in lysis, independent of holin or endolysin function (8). This implied the intact PG network acts a negative regulator of two-component spanin function, preventing IM-OM fusion until the lytic pathway is initiated by holin triggering (8).…”

“…Proposed structural models of these complexes rely heavily on detailed genetic and biochemical analysis (4-7), focused on the predominantly alpha-helical, coiled-coil structure of the i-spanin. Moreover, there is compelling evidence that the spanin complexes disrupt the OM by causing IM-OM fusion and that this topological function is blocked as long as the PG is intact (8).…”

Diversity, specificity and molecular evolution of the lytic arsenal of Pseudomonas phages: in silico perspective

“…Phages infecting Gram-negative bacteria harbour an additional lytic tool; the spanins. Two-component spanins are integral membrane proteins (i-spanins and o-spanins for inner and outer membranes, respectively) that form a complex spanning the periplasm, and recently also unimolecular spanins (u-spanins) have been described (Summer et al, 2007;Rajaure et al, 2015). At the time of lysis, spanins change their conformation to fuse the outer and inner membranes.…”

Bursting out: linking changes in nano-topography and biomechanical properties of biofilm-forming Escherichia coli to T4 lytic cycle

“…Although the lysis event has been well-studied operationally through the use of video-microscopic as well as molecular and physiological methods 9–11 , there is little information on the impact of the lytic pathway on biomechanics and physical structure of the infected cell. During the last decade, several researchers have used atomic force microscopes (AFM) to characterize the infection cycle of E. coli by different phages.…”