POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
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<strong>BLAST</strong> X Poster #21<br />
CHARACTERIZATION OF EtgA, A MURAMIDASE ASSOCIATED WITH THE TYPE III<br />
SECRETION SYSTEM OF ENTEROPATHOGENIC ESCHERICHIA COLI<br />
Elizabeth García-Gómez, Norma Espinosa, Bertha González-Pedrajo<br />
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional<br />
Autónoma de México. Ap. Postal 70-243. Ciudad Universitaria, México D. F., 04510.<br />
Tel. (5255) 56-22-59-65 egarcia@ifc.unam.mx, bpedrajo@ifc.unam.mx<br />
Enteropathogenic E. coli (EPEC) is a human pathogen that produces severe diarrhea<br />
and death among children from developing countries. EPEC infection is characterized by the<br />
formation <strong>of</strong> an attaching and effacing histopathology (A/E lesion) that consists <strong>of</strong> gut epithelial<br />
microvilli destruction, intimate adherence <strong>of</strong> the bacterium to the enterocyte and the<br />
development <strong>of</strong> an actin-rich pedestal-like structure beneath the adherent non-invasive bacteria.<br />
EPEC utilizes a type three secretion system (T3SS) to translocate effector proteins<br />
directly from the bacterial cytoplasm into the host cell cytosol, subverting diverse enterocytic cell<br />
signaling pathways and producing drastic cytoskeletal reorganization. EPEC genes required for<br />
the assembly <strong>of</strong> the T3SS and A/E lesion development are contained on a 35.6 kb pathogenicity<br />
island known as the locus <strong>of</strong> enterocyte effacement (LEE).<br />
The T3SS or injectisome is a macromolecular protein complex that needs to span the<br />
periplasmic space for its assembly; however, the peptidoglycan cell wall constitutes a barrier<br />
that allows the free passage <strong>of</strong> only small proteins. To overcome this obstacle, lytic<br />
transglycosylases (LTs) have been identified as specialized enzymes associated with different<br />
transport systems. It has been proposed that during T3SS biogenesis a LT facilitates a<br />
temporally and spatially controlled opening <strong>of</strong> the peptidoglycan layer.<br />
In this study, we have identified and characterized a LEE open reading frame with<br />
unknown function, rorf3 (renamed etgA), which encodes a protein with a lytic transglycosylase<br />
domain and a signal sequence. A truncated version <strong>of</strong> EtgA lacking its signal sequence<br />
(EtgAns) was purified by nickel chromatography as an N-terminal His-tagged recombinant<br />
protein. Lytic activity was determined by zymograms. An invariantly conserved glutamate<br />
residue at position 42 was replaced by alanine through site-directed mutagenesis, and its<br />
enzymatic activity was evaluated. The effect <strong>of</strong> over-producing EtgA, EtgAns and EtgAE42A<br />
over EPEC growth and secretion was evaluated. Furthermore, the subcellular localization <strong>of</strong> the<br />
protein was determined. Additionally, an etgA null mutant strain was generated to evaluate the<br />
role <strong>of</strong> EtgA in T3SS assembly.<br />
Our results show that EtgA is a T3SS associated muralytic enzyme. The highly<br />
conserved glutamate residue (catalytic residue) is essential for EtgA function. EPEC growth rate<br />
was affected when EtgA was overproduced, but not with EtgAns or EtgAE42A. Our data<br />
demonstrate that the protein is secreted by the Sec pathway and that it disrupts the<br />
peptidoglycan layer, causing bacterial lysis. Periplasmic localization <strong>of</strong> EtgAns was determined.<br />
Finally, we show that EtgA is essential for efficient protein secretion.<br />
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