Degree Type

Dissertation

Date of Award

2009

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Chris Minion

Abstract

Most Escherichia coli reside harmlessly in the animal intestine and share a mutualistic interaction with its human host. This relationship endures until the scales are shifted toward the pathogen such as a break in the intestinal mucosal barrier, host immunosuppression, the introduction of the species into a non-native environment such as the urinary tract, acquisition of a new phenotype, appearance of a strain with the ability to cause disease in a healthy host, etc. The focus of this dissertation is the interaction of the pathogen E. coli O157:H7 with the ruminant host. Four separate microarray studies were conducted to ascertain the transcriptome of E. coli O157:H7 under environmental conditions found within the ruminant host. These conditions were heat shock, growth under anaerobic conditions, exposure to rumen fluid, and interaction with a protozoan.

The response to elevated temperature (heat shock) is highly conserved. The transcriptome of Escherichia coli K-12 has been studied over a variety of conditions while such studies involving E. coli O157:H7 are just now being conducted. To better understand the impact of heat shock on E. coli O157:H7, global transcript levels of strain EDL933 cells shifted from 37yC to 50yC for 15 min were compared to cells left at 37yC by microarray. Using a mixed model analysis, 193 genes were found to be differentially transcribed at p < 0.0042 with a q-value of < 0.1. The 111 down-regulated genes include the curli pili associated genes csgABCDEFG, maltose transport associated proteins malEFK, and NADH dehydrogenase subunit encoding nuoCEHIJN. The 82 genes up-regulated include the heat shock induced genes rpoH, dnaK, dnaJ, groEL, groES, and grpE along with two LEE encoded genes; hypothetical gene Z5121 and sepZ. Twenty-three additional genes located in O-islands were found to be differentially expressed. qRT-PCR was performed to validate microarray results. Also, samples subjected to a 30˚C to 42˚C shift were examined by qRT-PCR to confirm differential transcription of selected genes. These results indicate that this pathogen may regulate its virulence factors in response to temperature changes.

Escherichia coli O157:H7 encounters two environmental conditions within the ruminant gastrointestinal tract, limiting oxygen and rumen fluid. Both of these conditions may have implications in the general physiology of E. coli and its survival during passage through the rumen. The impact of these environmental conditions on the transcriptome of E. coli O157:H7 is unclear. To study this further, the transcriptome of E. coli O157:H7 strain EDL933 grown under anaerobic conditions was compared to cells exposed to rumen fluid and to cells grown aerobically. Four hundred nineteen genes were differentially expressed during anaerobic growth at p < 0.014 with a false discovery rate < 10%. Among the 280 up-regulated genes were the curli pili associated genes csgBA and csgDEFG, adherence associated genes ompA, tdcA, and cadA, heat shock genes dnaK, dnaJ, groEL, and groES and the σ38 encoding gene rpoS. Ten genes, 8 up- and 2 down-regulated, located in E. coli O157:H7 virulence-related O-islands were differentially transcribed with a fold change >1.3 suggesting that these genes may play a role in host adaptation. Translational β-galactosidase fusions with csgBA confirmed its regulation during anaerobic conditions. As a result of rumen fluid exposure, 83 genes were differentially transcribed at p < 0.002 with a false discovery rate < 10%, 38 up- and 45 down-regulated. There were 11 up-regulated, 11 down-regulated, and 3 opposite-regulated genes common to both conditions. Our analysis indicated that protein synthesis was retarded during anaerobiasis and may be further slowed by exposure to rumen fluid.

Free-living protozoa, such as Acanthamoeba castellanii, are environmental hosts for several bacterial species pathogenic to humans and animals. Intracellular residence within protozoa has been implicated in enhancing virulence by selecting for virulence factors and antibiotic resistance outside of the animal host. To better understand this relationship with E. coli O157:H7, we characterized E. coli O157:H7 transcriptomes from extracellular bacteria in grown in broth to E. coli O157:H7 residing within A. castellanii using two-color microarrays. Statistical analysis indicated that 969 genes were differentially expressed at a p value < 0.018, with a false discovery rate of 1.9% and a fold change cutoff of 1.3. There were 655 up-regulated transcripts that include 40 genes involved in virulence of which 32 are encoded on O-islands. These up-regulated virulence-associated genes included shiga toxin genes (stx1A, stx1B stx2A), 14 genes involved in LEE and non-LEE encoded type three secretion system components and secreted factors. Additional induced genes include SOS response genes such as lexA and recA, genes involved in or predicted to be involved in antibiotic resistance (rarD, macAB, marABR, mdtK, yojI, yhgN), the quorum sensing operon lsrACDB, the efe and feo iron acquisition systems There were 314 down-regulated transcripts that include 19 transcripts associated with virulence, 7 of which are encoded on O-islands. Our results demonstrate a significant portion of the E. coli O157:H7 genome, including many virulence related genes, was differentially expressed as a result of the protozoan intracellular environment.

In summary, these studies attempt to shed light on the biology of E. coli O157:H7 within the ruminant host. These results show that E. coli O157:H7 demonstrates gene regulation similar to that of non-pathogenic E. coli and other bacteria in response to the aforementioned environments. In addition, E. coli O157:H7 demonstrated differential expression of virulence factors and genes found within other pathogenic E. coli along with genes that have not been characterized as being differentially transcribed or associated with the previously described environments. The characterization of E. coli O157:H7 within these environments has provided insights into unanswered questions and on unknown mechanisms of survival and persistence of this important pathogen.

Copyright Owner

Michael Dwight Carruthers

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

197 pages

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