Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Chris Minion

Abstract

To better understand the genetics and function(s) of biofilm formation in Escherichia coli

O157:H7, a series of studies were performed beginning with a genome wide mutational

analysis. A mini-Tn5 transposon library was constructed and screened for biofilm phenotype using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm-negative, and transposon insertions were located in 51 distinct genes/intergenic regions. Thirty-six genes were unique to this study. All of the fifty-one biofilm-negative phenotype (bnp) mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Notably, the involvement of virulence plasmid pO157 genes ehxD, an enterohemolysin auto transporter, and espP were identified. In addition, EhxD and EspP were also shown to be important for adherence to T84 intestinal epithelial cells suggesting a role for these genes in tissue interactions in vivo.

The involvement of Z2086, a phage-encoded putative regulator, in biofilm formation was

confirmed, the first such demomstration of a function for the cryptic phage-encoded genes in biofilm formation. Transcriptional analysis of a Z2086 deletion (ΔZ2086) compared to wild type showed that at p < 0.01, 363 genes were differentially expressed. Six (1.6%) were upregulated and 357 (98.3%) were down-regulated. The function of the differentially-regulated genes varied widely and included 11 biofilm-related genes, 20 virulence genes, genes of metabolic pathways, ABC transporters, ribosomal proteins and DNA binding proteins. Of the 357 down-regulated genes, 213 (59.6%) were phage-encoded, 16 (4.5%) were pO157-encoded.

During the establishment of E. coli O157:H7 infection, its capacity to adhere to host

intestinal epithelial cells is the critical first step in pathogenesis. To gain insight into a

potential linkage between biofilm formation and adherence to epithelial cells, the ability of the fifty-one bnp mutants to adhere to two human epithelial-like cell lines was examined. The analysis showed that all bnp mutants were incapable of adhering to T84 and HEp2 cells. In addition, adherence mutants Δeae and ΔespAB were fully competent in biofilm formation.Thus, these results show that while the biofilm forming activity is required for adherence to these cells, it is not sufficient. Additional adhesins or other supplemental structures are also required; the loss of either activity results in an inability to adhere.

Copyright Owner

Supraja Puttamreddy

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

150 pages

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