Document Type

Dissertation

Date of Degree

Summer 2011

Degree Name

PhD (Doctor of Philosophy)

Degree In

Microbiology

First Advisor

Steven Clegg

Abstract

Klebsiella pneumoniae is an opportunistic, Gram-negative pathogen that is responsible for a variety of infections, including those of the respiratory and urinary tracts, following insertion of medical devices. It is believed that epithelial surfaces are mechanically disrupted through insertion of these devices thereby exposing the underlying extracellular matrix components. This disruption, as well as the in situ accumulation of matrix components on the indwelling device, has been proposed to provide a unique colonization niche for K. pneumoniae as the type 3 fimbriae of this organism have been shown to adhere to, and subsequently facilitate biofilm formation on, human extracellular matrix coated surfaces.

To identify regulators of type 3 fimbrial expression, a bank of transposon mutants was generated and screened using a colony immunoblotting assay, for isolates that were phenotypically non-fimbriate. One insertion identified was within a gene encoding a putative transcriptional regulator named MrkI. Mutants of mrkI were found to be non-fimbriate only under aerobic conditions, and as a result, were deficient for biofilm formation on both biotic and abiotic surfaces. The decrease in type 3 fimbrial production was at the level of fimbrial structural gene (mrk) transcription, though purified MrkI could not be shown to specifically bind the mrk promoter.

The two genes that flank mrkI, mrkH and mrkJ, encode proteins that contain domains involved in the binding or degradation, respectively, of the second messenger molecule cyclic diguanylate (c-di-GMP). Deletion of mrkJ from the chromosome resulted in an increase in type 3 fimbrial surface expression, which led to a concomitant increase in biofilm formation capability. This increase in fimbrial production was found to be due to intracellular accumulation of c-di-GMP as MrkJ was shown to be a functional phosphodiesterase.

Further study found that MrkH binds c-di-GMP and positively regulates type 3 fimbrial expression independently of aerobic growth conditions. The ability of MrkH to induce the production of type 3 fimbriae was also found to be dependent on binding c-di-GMP as evidenced by the inability of a site-directed mutant of MrkH to bind c-di-GMP and restore fimbrial expression in a mutant background. Using an mrk transcriptional reporter, it was found that production of MrkH and MrkI together was required for maximal expression of mrk. This observation, as well as others, alludes to an apparent synergistic effect whereby MrkH and MrkI may interact, forming a novel, c-di-GMP-dependent transcriptional activation complex.

Pages

x, 141 pages

Bibliography

Includes bibliographical references (pages 128-141).

Copyright

Copyright 2011 Jeremiah Johnson

Included in

Microbiology Commons

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