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Title: Responses of the Opportunistic Pathogen Moraxella (Branhamella) catarrhalis to Clinically Relevant Changes in Environmental Conditions
Author: Cumbes, Bevan Christopher
ISNI:       0000 0004 2695 7791
Awarding Body: University of the West of England, Bristol
Current Institution: University of the West of England, Bristol
Date of Award: 2010
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The Gram-negative bacterium, Moraxella ca tarrhalis, is an opportunistic human pathogen that colonises the nasopharynx of healthy individuals. The disease most often associated with this organism is otitis media in children, though it is increasingly implicated in lower respiratory tract infections and even some cases of meningitis, endocarditis and bacteraemia. The most commonly quoted sites of colonisation and infection are the nasopharynx, tympanic cavity and lower respiratory tract. The different microenvironments found at these infection sites suggest a range of conditions in which the bacterium is an effective pathogen. An investigation was undertaken to determine what effect changing between these environments had on the proteome of M. catarrhalis. The effect of temperature was examined by comparing protein expression between cultures grown at 26, 30, 35, 37 and 40°C and also following an up-shift in temperature from 30 to 37°C. The effect of pH was examined by shifting the growth media from pH 7.2 to either pH 6.5 or pH 8.0. The effect of oxygen was examined by growing M. catarrhalis in continuous culture with 5-6 % excess O2 and comparing this to M. catarrhalis grown with no excess O2 . Two dimensional electrophoresis and difference in-gel electrophoresis were used to separate out the proteins from whole cell Iysates and to measure the relative abundance of proteins that were affected by the test conditions. The number of proteins affected by temperature was: 51 at 26°C, 10 at 30°C, 9 at 35°C and 23 at 40°C. Up-shifting pH affected 12 proteins and down-shifting pH affected 12 separate proteins. 52 proteins were affected by restricting oxygen. In addition, the identities and positions of 60 proteins were found and used to produce the most complete annotated M. catarrhalis 20 proteomic map to date. Among those proteins found to be affected by changes in environmental conditions, isoforms of the adhesin Omp CO were found to increase when oxygen availability was reduced. Attempts were made to produce a knockout ompCD mutant but these proved unsuccessful, indicating that Omp CD may have a fundamentally important role in viability of M. catarrhalis strain NCTC 11020. The general porin M35 increased in reduced oxygen and during stationary phase, suggesting a role in modifying membrane permeability similar to that of its homolog Omp C from E. coli. An isogenic m35 knockout mutant showed reduced growth in nutrient poor media, was more tolerant of osmotic pressure, was killed more quickly by exposure to an anaerobic environment and was less able to autoagglutinate. The mutation affected little difference in ability to grow at different pH levels and there was no difference in susceptibility to gentamicin, or in cell morphology or membrane thickness. Also, there was no significant difference in adherence or invasion of human pharyngeal or alveolar epithelial cells compared to the wild type, indicating that M35 is not a virulence factor
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available