Many gram negative bacterial pathogens, including Yersinia, Salmonella, Pseudomonas, Shigella, enteropathogenic E. coli, Chylamydia, and Burkholderia, use type III secretion systems (TTSS) to translocate effector proteins from the bacterial cytosol into mammalian cells. Translocated effector proteins, called Yops in Yersinia, subvert normal host processes to promote the survival of the pathogen, and thus TTSS play an essential role in the infectious process and virulence of these bacteria. TTSS are comprised of a base, which spans the inner and outer membranes of the bacteria, a needle, which extends from the base to the host cell, and a translocon, which is inserted into host cell membranes and through which Yops are thought to travel to reach host cell cytoplasm. In Yersinia, the needle is primarily composed of one 7kD protein, YscF, which polymerizes to form a long tube. The needle is thought to be a conduit for the passage of Yops from the bacteria to the translocon and is thought to conduct signals from the host cell membrane to the TTSS base when the pathogen comes in contact with a host cell. These signals trigger Yop translocation. A tip protein, LcrV, is found at the distal end of the needle. One function of LcrV is to position, assemble, and/or insert the translocon, into plasma membranes, which is essential for Yop delivery into cells. Two proteins, YopB and YopD comprise the translocon. The overall goals of this proposal are to understand the molecular interactions between TTSS components that are required for translocating Yops upon contact with mammalian cells. We have identified 3 dominant negative YscF alleles and 8 small molecules that inhibit Yop translocation. These unique mutants and compounds will be studied intensively as probes to understand the required molecular interactions for Yop translocation into host cells. Our working hypothesis is that our inhibitors interfere with critical interactions between YscF, LcrV, YopB and/or YopD. Understanding the mechanism of translocation and needle assembly will support the development of new strategies that interfere with these processes and thus neutralize the bacteria's pathogenecity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
3R56AI073759-01A2S1
Application #
7839596
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Mukhopadhyay, Suman
Project Start
2009-09-01
Project End
2010-08-31
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
1
Fiscal Year
2009
Total Cost
$412,500
Indirect Cost
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
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