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Isolation and characterization of a family of porin proteins from Helicobacter pylori

University of British Columbia

Porins are pore forming proteins found in the outer membranes of gram negative bacteria. They are involved in the transport of small, hydrophilic molecules across the membrane, and are thus important in the uptake of a variety of substances from the external environment. An attempt was made to identify porin(s) in the outer membrane of the gastric pathogen Helicobacter pylori in order to gain insight into the physiology of this organism. Growth studies were done in order to optimize large scale growth conditions for H. pylori, and a novel growth media was developed such that large masses of membrane proteins could be obtained. Five porin proteins, (HopA, B, C, D and E) were identified and purified. N-terminal sequence analysis revealed that all five shared a strong degree of homology with each other which indicated that this was a group of related proteins. Model membrane analyses showed that the HopA, B, C, and D proteins functioned as channels. They all were functional as monomers, and they possessed similar channel forming properties in that they formed channels with conductances between 0.24 and 0.36 nS in 1.0 M KC1 pH 7.0. HopE also formed channels, but it showed different pore forming characteristics, as even though it still appeared to function as a monomer, it formed large channels with conductances of 1.5 nS in 1.0 M KC1. Since most porins previously isolated function as trimers, a series of major outer membrane proteins which were isolated as monomers from a variety of organisms were examined for pore forming ability in order to further show precedence for the existence of functional monomeric porins. Six of these proteins functioned as porins, indicating that porins which are functional as monomers may be more common than previously thought. Growth studies utilizing a defined medium demonstrated that the expression of HopA, B, and C was regulated by the amount of iron in the medium, and the different expression levels of the porins appeared to affect the uptake of a number of different antibiotics. Degenerate PCR primers created from N-terminal and internal protein sequences allowed amplification of DNA sequences encoding the N-terminal portions of two of the H. pylori porin proteins, and these DNA sequences were used to identify hopA and hopB clones from a H. pylori chromosomal DNA library that was created. Hybridization with H. pylori chromosomal DNA indicated that the hopA and hopB genes are located at different loci on the chromosome.

Thesis/Dissertation

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2009-03-27

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http://hdl.handle.net/2429/6624

Microbiology and Immunology

University of British Columbia

UBCV

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