Haemophilus influenzae (Hi) is a Gram-negative bacteria that is the causative agent of bacterial meningitis. The outer membrane (OM) of Gram-negative bacteria functions as a selectively permeable barrier. The exchange of small hydrophilic solutes between the external environment and the periplasm is mediated by large water-filled channels, porins. Charged residues of the pore determine the functional properties of the protein, which include: ion conductance, ionic selectivity, and voltage gating. / To study the properties of the porin (341 amino acids; Mr 37,782) of Haemophilus influenzae type b (Hib), purified porin was subjected to chemical modification. The covalent modification of lysine residues with succinic anhydride (SA; Mr 100.08) results in charge reversal. The addition of up to 12 succinate groups per porin molecule was identified using electrospray ionization mass spectrometry (MS). Tryptic digestion of the modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight MS identified the sites of succinylation. The majority of modified lysines were positioned in surface-located loops, numbers 1 and 4 to 7. When the electrophysiological properties of SA-modified porin were analyzed in planar lipid bilayers (PLBs) and compared to Hib porin it was found that the single channel conductance was increased, while the threshold for voltage gating was decreased. The addition of extra negative charges increase the single channel conductance of Hib porin and function as voltage sensors. / Selected lysine residues that were found to be modified with SA were substituted with glutamic acid using site-directed mutagenesis. Single point mutations were made in a residue assigned to the barrel lumen and to three residues in each of loops 4 and 6. The mutant Hib porins had increased single channel conductances relative to wild-type Hib porin. Voltage gating of mutant Hib porins was altered by the introduction of negative charges into loops 4 and 6 and in the barrel lumen. Previous experiments had implicated surface-exposed loop 4 in voltage gating. This study ascribes a role for residues in loop 6 and a residue within the barrel lumen in the changes that accompany pore closure. / Hi strains causing infection in cystic fibrosis patients are capable of persistent infection despite prolonged antibiotic treatment with beta-lactam antibiotics. During the course of infection porin properties may be altered due to the changes in porin sequences that are attributed to antigenic drift. The electrophysiological properties of four porins from CF patient-derived Hi strains were characterized to examine changes in porin properties arising from persistent infection of the CF lung. The clinical Hi porins displayed altered channel properties that included increased voltage sensitivity and single channel conductances that were either greater or smaller than that of Hib porin. The decreased single channel conductance of one of the porins was associated with an increase in the minimal inhibitory concentration of the antibiotics novobiocin and streptomycin. These results demonstrate a porin-mediated decrease in OM permeability as an antibiotic resistance mechanism for Hi.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.38146 |
| Date | January 2002 |
| Creators | Arbing, Mark A. |
| Contributors | Coulton, James W. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001864235, proquestno: NQ78640, Theses scanned by UMI/ProQuest. |
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