In vitro characterization of the phage lysis protein MS2-L

Abstract

Background: The peptide MS2-L represents toxins of the ssRNA Leviviridae phage family and consists of a predicted N-terminal soluble domain followed by a transmembrane domain. MS2-L mediates bacterial cell lysis through the formation of large lesions in the cell envelope, but further details of this mechanism as a prerequisite for applied bioengineering studies are lacking. The chaperone DnaJ is proposed to modulate MS2-L activity, whereas other cellular targets of MS2-L are unknown. Methods: Here, we provide a combined in vitro and in vivo overexpression approach to reveal molecular insights into MS2-L action and its interaction with DnaJ. Full-length MS2-L and truncated derivatives were synthesized cell-free and co-translationally inserted into nanodiscs or solubilized in detergent micelles. By native liquid bead ion desorption mass spectrometry, we demonstrate that MS2-L assembles into high oligomeric states after membrane insertion. Results: Oligomerization is directed by the transmembrane domain and is impaired in detergent environments. Studies with truncated MS2-L derivatives provide evidence that the soluble domain acts as a modulator of oligomer formation. DnaJ strongly interacts with MS2-L in membranes as well as in detergent environments. However, this interaction affects neither the MS2-L membrane insertion efficiency nor its oligomerization in nanodisc membranes. In accordance with the in vitro data, the assembly of MS2-L derivatives into large membrane located clusters was monitored by overexpression of corresponding fusions with fluorescent monitors in E. coli cells. Analysis by cryo-electron microscopy indicates that lesion formation is initiated in the outer membrane, followed by disruption of the peptidoglycan layer and disintegration of the inner membrane. Conclusion: MS2-L forms oligomeric complexes similar to the related phage toxin ΦX174-E. The oligomeric interface of both peptides is located within their transmembrane domains. We propose a potential function of the higher-order assembly of small phage toxins in membrane disintegration and cell lysis.

Lysis Physiology of Pseudomonas aeruginosa Infected with ssRNA Phage PRR1

“…K + leakage, indicating an increase in the permeability of the PM of the lysis protein-expressing cells, starts ~10 min later than the permeabilization of the OM. Low concentrations of the lysis protein in the cell envelope could cause stress and the dissociation of bonds between the OM and peptidoglycan layer, leading to lesion formation in the OM [ 12 ] and vesicle release [ 30 ]. It is possible that a high amount of lysis protein is necessary to form the permeabilizing structures in the PM, like holin protein does at the end of infection by dsDNA-containing phages [ 4 ].…”

“…The MS2-infected cells captured in the transmission and scanning electron micrographs showed various morphological aspects of the intermediates in the lysing process [ 12 , 37 ]. In contrast to endolysin–holin–spanin-mediated lysis, single-lysis protein-induced cell envelope degradation is limited and observed only locally [ 6 , 7 , 31 ].…”

“…In both cases, the lysis proteins were shown to inhibit a specific enzyme in peptidoglycan synthesis. The mechanism of the protein L-mediated lysis of phage MS2-infected cells remains unknown, but recent studies have shown that the interaction of this protein with the cell envelope induces lesion formation in the outer membrane (OM), followed by a disruption of the peptidoglycan layer and the disintegration of the PM [ 11 , 12 ].…”