Structural and functional studies of porins from pathogenic bacteria

Abstract

Multi-drug resistant bacteria have become a real threat to public health worldwide. Gram-negative bacteria, in particular, have shown high level of antibiotic resistance due to the presence of an additional membrane, known as outer-membrane (OM), that acts as an extra barrier. Most antibiotics enter the cells via a particular class of outer-membrane proteins (OMPs) known as porins. Porins are β-barrel channels that allow the passive diffusion of hydrophobic compounds. The porins are known to select against molecules on the basis of size and charge. When exposed to antibiotics, bacteria can modify the OM permeability by altering their porins profile. Mutations affecting the size and conductivity of the pore channel, and modification of the level of porins expression are just a few examples of how the bacteria can decrease the influx of antibiotics.

In order to better understand their interaction with antibiotics, this thesis presents structural and functional studies on porins from pathogenic bacteria. The structure of the natively expressed major outer-membrane protein (MOMP) from Campylobacter jejuni was determines, revelling the presence of a calcium-binding site inside the channel. Electro-physiology and in silico modelling analysis have shown to be important for the stability and the function of the protein. Omp50 from C. jejuni was expressed in E. Coli and its tyrosine kinase activity was analysed in vitro. Finally the structures of the two major porins from Enterobacter aerogens were determined and compared to their orthologs within the Enterobacteriaceae family. Further, a liposome-swelling assay (LSA) was used to deter-mine the rate of permeation of clinically relevant antibiotics through a series of porins. Combining these data allow a more detailed molecular understanding of translocation.