Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2019

Membrane vesicles (MVs) as an outcome of bacteriophage mediated bacterial lysis (#83)

Pappu K Mandal 1 , Giulia Ballerin 1 , Nico K Petty 1 , Cynthia B Whitchurch 1
  1. The ithree institute, University of Technology, Ultimo, NSW, Australia

Membrane vesicles (MVs) prevail in all the three domains of life. Bacterial MVs are mostly spherical nanostructures (20- 400 nm), which involve in diverse biological processes such as pathogenicity, horizontal gene transfer, biofilm formation, and decoys to defend bacteria from antibiotics, antimicrobial peptides and bacteriophage predation. MVs biogenesis through explosive cell lysis in Gram-negative bacteria has been reported recently, which is due to expression of endolysin, a muralytic enzymes mostly used by bacteriophage to lyse its bacterial host. Bacteriophage mediated lysis in Gram-negative bacteria has been reported as a cell bursting phenomenon similar to that of explosive cell lysis and observations of vesicular structures within phage preparations have also been reported. However, there is a lack of knowledge on the source of MVs in phage lysates and also no clear evidence of bacteriophage mediated MVs formation. This work aimed to gain a better understanding on the processes of MVs biogenesis to determine if MVs formation occurs through phage-mediated lysis of Gram-negative bacteria, using the live-cell imaging techniques together with transmission electron microscopy (TEM). This study demonstrated explosive cell lysis of E. coli K-12 utilizing two distinct standard virulent phages T4 and T7. Analysis of multiple lysis events revealed that the bacteria lysed explosively either as direct blowout without any gross changes in cellular morphology or in progressive changes into spheroid morphology before rupturing. Live-cell super-resolution microscopy demonstrated that the exploding bacteria produced shattered membrane fragments that curled up inward and then rounded up forming circular MVs that persisted in the same topology thereafter. TEM revealed the presence of different forms of MVs within phage lysates, consistent with MVs formation through phage mediated bacterial lysis. To our knowledge, this is the first direct observation of MV biogenesis via phage mediated bacterial lysis. This study not only explored an unappreciated route for MVs formation but also suggested the phenomenon could be the major process responsible for the abundance of MVs in nature.

  1. Toyofuku M, Nomura N, Eberl L. Types and origins of bacterial membrane vesicles. Nat Rev Microbiol. 2019; 17(1): 13-24.
  2. Turnbull L, Toyofuku M, Hynen AL, Kurosawa M, Pessi G, Petty NK, Osvath SR, Carcamo-Oyarce G, Gloag ES, Shimoni R, Omasits U, Ito S, Yap X, Monahan LG, Cavaliere R, Ahrens CH, Charles IG, Nomura N, Eberl L, Whitchurch CB. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms. Nat Commun. 2016; 7:11220.