EHEC

Suomennettavia asioita:
Ruotsinkielinen lähde:
http://www.lakartidningen.se/07engine.php?articleId=16614

 Englanninkielinen  lähde:
http://repositories.tdl.org/utswmed-ir/bitstream/handle/2152.5/298/waltersmatthew.pdf?sequence=3

Enterohemorrhagic E. coli O157:H7 (EHEC) causes outbreaks of
bloody diarrhea and
hemolytic-uremic syndrome (HUS) throughout the world.

The locus of enterocyte effacement (LEE)
consists of five major operons (LEE1 – LEE5) and is required for formation of
attaching and effacing (AE) lesions that disrupt intestinal epithelial microvilli.
(Auttaisiko toipilasajan gluteenivapaa ravinto!)
We have previously reported that expression of EHEC LEE genes is regulated
by the luxS quorum sensing system.

The luxS gene in EHEC affects the production of autoinducer-3 (AI-3),
which activates the LEE.

Epinephrine and norepinephrine also activate the LEE in a manner
similar to AI-3.

The luxS mutant had diminished transcription from the LEE promoters
during mid-exponential growth phase, decreased levels of the LEE-encoded proteins EscJ, Tir, and EspA, and reduced secretion of EspA and EspB, encoded by LEE4.

Epinephrine
enhanced LEE expression in both wildtype (WT) and the luxS mutant, but WT still exhibited greater LEE activation.
The results suggest a possible synergistic relationship between AI-3
and epinephrine. The combined effects of these two signaling molecules may lead to greater LEE expression and a more efficient infection.

Given the virulence defects resulting from the luxS mutation, we next examined
pathways which may be affected that lead to reduced AI-3 synthesis.
We show that several species of bacteria synthesize AI-3, suggesting a possible role for AI-3 in inter-species bacterial communication.
The LuxS enzyme produces the autoinducer-2 (AI-2) precursor
4,5-dihydroxy-2,3-pentanedione (DPD) and homocysteine. Homocysteine is required for the de novo synthesis of methionine in the cell.
The luxS mutation leaves the cell with only one pathway for the synthesis of homocysteine, involving the use of oxaloacetate and Lglutamate.
The exclusive use of this pathway appears to alter metabolism in the luxS mutant,
leading to decreased production of AI-3. Addition of aspartate and increasing the cellular concentration of aromatic amino acids, such as tyrosine, restored AI-3-dependent phenotypes in a luxS mutant. The defect in AI-3 production, but not in AI-2 production, was also restored by expressing the P. aeruginosa S-adenosylhomocysteine hydrolase, which produces homocysteine directly from S-adenosylhomocysteine, in the luxS mutant. Furthermore, Phenotype MicroArrays (Biolog) revealed that the luxS mutation caused numerous metabolic deficiencies, while AI-3 signaling had little effect on metabolism. These studies examine the
effects of the luxS mutation on LEE expression, how AI-3 production is affected by mutation of luxS, and explores the roles of the LuxS / AI-2 system in metabolism and QS.

CHAPTER 1. ESCHERICHIA COLI O157:H7 LITERATURE REVIEW.............................. 1
I. Classification of Enterohemorrhagic Escherichia coli O157:H7..................................... 1
II. Historical Perspective...................................................................................................... 1
III. Epidemiology................................................................................................................. 3
IV. Clinical Presentation, Diagnosis, and Treatment........................................................... 7
V. Pathology ........................................................................................................................ 9
VI. Genetic Content ........................................................................................................... 11
VII. Pathogenesis ............................................................................................................... 14
A. Colonization of the Gut............................................................................................. 14
B. LEE Encoded TTSS .................................................................................................. 14
C. EHEC Virulence Factors........................................................................................... 20
1. LEE-Encoded Effectors ......................................................................................... 20
2. Prophage-Encoded Effectors ................................................................................. 22
3. pO157 Effectors ..................................................................................................... 23
4. Shiga Toxin............................................................................................................ 24
VIII. Animal Models.......................................................................................................... 25
IX. Host Immune Response to EHEC Infection ................................................................ 27
X. Vaccinology .................................................................................................................. 29
XI. Quorum Sensing .......................................................................................................... 31
XII. Bacteria-Host Signaling ............................................................................................. 36
XIII. Quorum Sensing in EHEC ........................................................................................ 37
CHAPTER 2. OVERALL OBJECTIVE AND SYNOPSIS..................................................... 42
CHAPTER 3. MATERIALS AND METHODS ..................................................................... 46
I. Bacterial strains, plasmids, and cultivation .................................................................... 46
II. Transformation of E. coli .............................................................................................. 46
A. Preparation of chemically competent E. coli DH5α ................................................. 46
B. Electroporation of EHEC .......................................................................................... 49
III. DNA and RNA Isolation.............................................................................................. 50
IV. Recombinant DNA Techniques................................................................................... 52
V. Protein Expression and Purification.............................................................................. 54
VII. Preparation of Secreted Proteins ................................................................................ 55
VIII. Real-time RT-PCR.................................................................................................... 56
IX. β-galactosidase assays ................................................................................................. 57
X. In vitro synthesis of AI-2 ..............................................................................................58




PRIOR PUBLICATIONS
Sircili, M.P., Walters, M., Trabulsi, L.R., and Sperandio, V. 2003. Modulation of
Enteropathogenic E. coli (EPEC) virulence by quorum sensing. Infection and
Immunity 72:2329-2337.
Walters, M. and Sperandio, V. 2006. Quorum sensing in E. coli and Salmonella.
International Journal of Medical Microbiology. 296(2-3):125-31.
Almengor A.C., Walters M.S., McIver K.S. 2006. Mga Is Sufficient To Activate
Transcription In Vitro of sof-sfbX and Other Mga-Regulated Virulence Genes in the
Group A Streptococcus. Journal of Bacteriology 188(6):2038-47.
Walters, M. and Sperandio, V. (In Press) AI-3 / Epinephrine Signaling in the Kinetics of
LEE Gene Expression in EHEC. Infection and Immunity.
Walters, M., Sircili, M.P., and Sperandio, V. (In Press) AI-3 Synthesis is Not Dependent
on luxS in E. coli. Journal of Bacteriology.