טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentJammal Joanna
SubjectMolecular Basis for Chemo-Sensitization of Gram-Negative
Bacteria
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Amram Mor
Full Thesis textFull thesis text - English Version


Abstract

The rise of multidrug resistant bacteria is a global health concern, due to the rapid spread of resistance coupled with a sharp decline in the number of new antibiotics under development. This problem is particularly imperative for pathogenic Gram-negative bacteria (GNB), representing one of the main challenges for the healthcare system and public health in general.

Membrane active compounds, exerting antibacterial activities through non-specific modes of action are considered attractive candidates for de-novo design of new antimicrobial agents. Namely, the peptidomimetic family of oligomers of acylated cations (OACs) that were designed and synthesized in our lab to investigate their ability to mimic host defense peptides. The OACs represent a potentially useful approach for developing new antibacterial agents owing to their structural simplicity, pharmacokinetics stability and relative low production cost.

In this work we focused on short, membrane-active, lipopeptide-like (SMALL) compounds. They are linear peptidomimetic sequences consisting of three cationic amino acids and two acyl chains, which showed a significant activity in vivo in combination with ineffective antibiotics. According to these studies, the in vivo activity was due to a combination of two main properties: i) the capacity to permeabilize GNB outer membrane (OM); ii) improved bioavailability. To extend this work, the present study aims to improve one or both properties towards identifying systemically efficient antibacterial lead analogs with higher safety profile.

Our findings suggest that less hydrophobic analogs such as C10OOc12O and C6OOc12O, which, on their own, are inefficient in affecting GNB growth (i.e., MICs were >50µM) in broth medium, can induce OM permeabilization and partial inner membrane depolarization at low sub-minimal inhibitory concentration (MIC) levels. In plasma, however, they induce potent bactericidal activity. This activity was enhanced in presence of exogenous lysozyme or conventional antibiotics while reduced in presence of anti-complement antibodies or upon heat treatment. These membrane damages induced by the SMALL OAC can allow the passage of various antibacterial molecules through the outer and/or inner membranes that serve as their barriers. This activity was also maintained in vivo in affecting disease course using two different mouse infection models for both mono- and combination therapies. Moreover, by comparing their concentrations in the urine to polymyxin B (well-known for its nephrotoxicity), we collected evidence for significantly higher (>10 folds) urine excretion levels of C6OOc12O, thereby providing indication of potentially lower nephrotoxicity of this SMALL analog.

Collectively, the results support the notion that our observations are linked to the capacity of mildly hydrophobic compounds to induce mild permeabilization and/or depolarization of bacterial membranes, in vitro and in vivo. In addition, these findings suggest a potentially useful combination therapy approach for expanding the sensitivity spectrum of GNB to include impermeable antibiotics or plasma components.