This thesis reports the characterisation of a novel outer membrane protein (OMP)
from M. catarrhalis, designated M35, with a molecular mass of 36.1 kDa. This protein is
structurally homologous to classic Gram-negative porins, such as OMP C from E. coli and
OMP K36 from K. pneumoniae, with a predicted structure of 8 surface loops connecting 16
antiparallel -sheets. Comparison of the DNA sequences of the M35 genes from 18 diverse
clinical isolates showed that the gene was highly conserved (99.6-100 % of nucleotides)
with only one isolate (ID78LN266) having base variations that resulted in amino acid
substitutions.
A single amino acid mutation in the 3rd external loop of M35 in isolate ID78LN266
significantly affected antibody recognition, indicating that loop 3 contains an immunodominant
B-cell epitope. The reduction in antibody-binding to M35 from ID78LN266
was similar to that caused by complete removal of loop 3. Since loop 3 folds into the porin
channel in the classic structure, the antibody specificity to loop 3 was hypothesised to be
a potential mechanism for evasion of host immune responses targeted to M35, potentially
explaining the high degree of conservation across isolates.
A series of recombinant proteins were constructed to analyse the binding to M35 of
antibodies specificity for loop 3 or the remainder of the protein. It was found that loop 3-
specific antibodies were not able to bind to M35 on the surface of M. catarrhalis and
that this corresponds both with a lack of ability to enhance opsonophagocytosis in vitro
and bacterial clearance in vivo. Additionally, antibodies raised against a version of M35
lacking loop 3 and M35 from the variant isolate ID78LN266 were both no less effective
than the full consensus M35 by both these measures. It therefore appears that while the
majority of antibodies raised against M35 are specific for loop 3 these antibodies do not
mediate anti-M. catarrhalis actions.
Two deletion mutant strains of M. catarrhalis that do not contain the outer membrane
protein M35 were created by insertional inactivation of the M35 gene. Growth comparisons
between these mutant strains and their wildtype parent strains initially led to the
hypothesis that M35 is necessary for efficient glutamic acid uptake by M. catarrhalis,
however this hypothesis was later shown to be incorrect. Efficient uptake of glutamic acid
seemed to be mediated by a novel 40 kDa protein that was up-regulated in the deletion
mutant strains, presumably to compensate for the lack of M35. M35 was also found to
be essential for in vivo survival of M. catarrhalis in the nasal cavities of mice, indicating
that it is an essential functional protein for colonisation of the mucosal surface.
| Identifer | oai:union.ndltd.org:ADTP/203488 |
| Date | January 2008 |
| Creators | Easton, Donna Meredith, n/a |
| Publisher | University of Canberra. n/a |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | ), Copyright Donna Meredith Easton |
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