The objective of the proposed NMR studies is to determine the protein-protein interactions involved in the assembly of a bacterial nanoinjector that is essential in the pathogenesis of many bacterial pathogens. The nanoinjector is a needle-like macromolecular assembly and forms the structural part of bacterial protein export machinery, the type III secretion system (T3SS). The T3SS also consists of effector proteins, which are injected into the host cell cytoplasm, and chaperones, which form complexes with T3SS proteins while inside the bacterial cytoplasm. The T3SS needle apparatus is assembled from over 20 different proteins and consists of a basal structure anchored on the bacterial membranes, the needle itself on the bacterial surface, a tip complex, and a translocon. The translocon is the membrane spanning structure that punctures a hole on the host cell membrane to allow the passage of bacterial proteins into the host cell cytoplasm. On the first round of funding, we have determined how the needle protein interacts with the tip protein. Currently, how the tip protein interacts with the translocon is completely unknown. For this renewal, our Aim #1 is to determine how the tip protein interacts with the translocon proteins. Another unknown is how the tip proteins interact with their chaperones, thus our Aim #2 is to determine how the tip proteins interact with their chaperones. To advance our understanding of the biology of the T3SS, our NMR studies will be complemented with electron microscopy and functional assays. Many pathogens rely on the type III secretion system to infect millions of people worldwide. Because the needle apparatus is exposed on the bacterial surface, disrupting the needle assembly is an attractive target for the development of novel anti- infectives. This approach requires a detailed understanding of the protein-protein interactions involved in needle assembly.

Public Health Relevance

Before the age of antibiotics about 80 years ago, infectious diseases were the major cause of mortality in humans. Thus, the increased incidence of antibiotic resistance in bacterial pathogens poses a major public health concern. Many pathogens that cause millions of death worldwide and are major agents of food- borne outbreaks, bubonic plague, and secondary hospital infections require the assembly of a bacterial needle-like nanoinjector for pathogenesis. The proposed research seeks to determine how bacteria assemble the nanoinjector by determining the protein-protein interactions of its components. This knowledge is important in designing novel anti-infectives, which will prevent pathogens from invading human cells but will not contribute to the development of antibiotic resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI074856-06
Application #
8696949
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Mukhopadhyay, Suman
Project Start
2007-07-01
Project End
2019-01-31
Budget Start
2014-02-05
Budget End
2015-01-31
Support Year
6
Fiscal Year
2014
Total Cost
$329,301
Indirect Cost
$104,301
Name
University of Kansas Lawrence
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Rathinavelan, Thenmalarchelvi; Lara-Tejero, Maria; Lefebre, Matthew et al. (2014) NMR model of PrgI-SipD interaction and its implications in the needle-tip assembly of the Salmonella type III secretion system. J Mol Biol 426:2958-69
Chatterjee, Srirupa; Chaudhury, Sukanya; McShan, Andrew C et al. (2013) Structure and biophysics of type III secretion in bacteria. Biochemistry 52:2508-17
Chaudhury, Sukanya; Battaile, Kevin P; Lovell, Scott et al. (2013) Structure of the Yersinia pestis tip protein LcrV refined to 1.65?Å resolution. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:477-81
Chatterjee, Srirupa; Zhong, Dalian; Nordhues, Bryce A et al. (2011) The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate. Protein Sci 20:75-86
Rathinavelan, Thenmalarchelvi; Tang, Chun; De Guzman, Roberto N (2011) Characterization of the interaction between the Salmonella type III secretion system tip protein SipD and the needle protein PrgI by paramagnetic relaxation enhancement. J Biol Chem 286:4922-30
Rathinavelan, Thenmalarchelvi; Zhang, Lingling; Picking, Wendy L et al. (2010) A repulsive electrostatic mechanism for protein export through the type III secretion apparatus. Biophys J 98:452-61
Wang, Yu; Nordhues, Bryce A; Zhong, Dalian et al. (2010) NMR characterization of the interaction of the Salmonella type III secretion system protein SipD and bile salts. Biochemistry 49:4220-6
Wang, Yu; Zhang, Lingling; Picking, Wendy L et al. (2008) Structural dissection of the extracellular moieties of the type III secretion apparatus. Mol Biosyst 4:1176-80