Section: Rotations

Doyle Ward, Ph.D.

Other Affiliation(s):
   Interdisciplinary Graduate Program
   Molecular Genetics and Microbiology

Rotation Projects

1. Biogenesis of T4SS. It is becoming evident that large multi-component secretion systems are frequently associated with lytic transglycosylases that may locally remodel the peptidoglycan in order to permit assembly of the T4SS or allow passage of large substrates. The protein VirB1 has homology to lytic transglycosylases. We believe that VirB8 functions to spatially restrict VirB1 activity by direct interaction with VirB1. Both proteins are found in high molecular weight complexes in the presence of cross-linking agents but the composition of these complexes is not known. Gene replacement, protein tagging, affinity chromatography, and mass spectrometry are methods that will be used to determine the nature of the interacting partners and the mechanisms for spatial localization of VirB1 activity.

2. T4SS contribution to B. cepacia virulence. B. cepacia encodes a T4SS which may contribute to intracellular survival and virulence of this opportunistic human pathogen. We will determine the involvement of the T4SS in pathogenesis and ultimately the nature of the substrates secreted.  T4SS gene knockouts will determine the genetic requirements for pathogenesis. Expression studies will determine the transcriptional regulation and protein expression profile of the T4SS components. Ultimately, assays will be developed to screen for secreted substrates.  The long term goal is to determine how the secreted substrates effect disease in the host. Methods include PCR, construction of reporter cassettes, generation of in-frame deletion mutations, and in vitro and in vivo invasion assays.

3. Structural studies. We would like to identify subassemblies of the T4SS and determine the structure of its components. Initially, we will clone, express, and purify T4SS proteins and characterize them biochemically. We will attempt to purify and identify sub-assemblies. Suitable proteins or complexes will be subjected to structural determination.

Phone: 508-856-6861
Fax: 508-856-6863
E-mail: Doyle.Ward@umassmed.edu
Keywords: Microbial Pathogenesis, Biochemistry, Infectious Disease

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