טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBarzilai Allon Sharon
SubjectThe Effect of Low Intensity Ultrasound on Mechanosensitive
Channel in Escherichia Coli
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisors Professor Eitan Kimmel
Assistant Professor Jonathan Kuhn
Full Thesis textFull thesis text - English Version


Abstract

Ultrasound creates mechanistic waves which at a special range of frequencies and intensities, known as of low intensity therapeutic ultrasound (LITUS), has been shown to induce delicate and reversible biological effects. A recent novel model, named the bilayer sonophore (BLS), was proposed in our laboratory and concerns the effects of LITUS on bilayer membranes. In the present research, the effect of LITUS on Escherichia coli was examined.

Mechanosensation is ubiquitous in all living organisms and encompasses a wide range of phenomena in response to mechanical forces. An example of a mechanical sensor is the mechanosensitive channel of large conductance (MscL) of the bacterium Escherichia coli (E. coli). This nano-valve channel has been proven to function as an overpressure regulator and responds to pressure changes that approach those that would cause cell death. As a mechano-sensor for membrane mechanical deformation and tension changes (the result of turgor pressure or asymmetric curvature), MscL opens to form a large pore that allows the passage of osmolites and cytoplasmic proteins such as Thioredoxin (Trx) under osmotic shock conditions.

In this study, the influence of LITUS on natural E. coli membranes was examined using the protein sensor MscL as a model. The major question asked was does LITUS activate this system. LITUS radiation was applied to three types of E. coli strains distinguished only in their MscL content. Initially the outer membrane of E. coli was perforated and then the cells were exposed to LITUS for a few minutes. Levels of cytoplasmic proteins released by the treatment were measured using immunoassay methods. This release is due to changes in the internal, cytoplasmic membrane of the cell. We propose two different mechanisms that might explain our findings: i) LITUS increases membrane tension and thus increases membrane permeability to water through pore formation and/or channel activation which can be confirmed via the escape of Trx molecules; and ii) LITUS creates sonophore inflation around the inner membrane which is bounded by the outer cell wall and this acts as a pump on the inner liquid of the cytoplasm, leading to cytoplasm overpressure.

Although the results from the current study cannot validate any mechanistic theory, we can conclude that LITUS does affect the transport of Trx protein through the inner membrane of E. coli. More than that, in the lower range of LITUS intensities there was a different response between bacteria with and without MscL channels and we recommend that further investigations be carried out in this range.