Clostridium difficile is a spore-forming anaerobic bacillus that causes severe colitis with high mortality. The incidence and severity of C. difficile infections (CDIs) are both increasing at an alarming rate. Treatment is associated with unacceptable failure and relapse rates and no vaccine has been licensed. While disease is strongly dependent on the elaboration of cytotoxins, the mechanisms required for colonization are essentially unknown. This knowledge gap inhibits the development of novel strategies to prevent acquisition of infection. Type IV pili (T4Ps) represent the most widespread class of pili, broadly distributed among diverse phylogenetic divisions including the Clostridiales. C. difficile genomes have the complete set of T4P biogenesis genes including several pilin or pilin-like proteins. As T4Ps play essential roles in the colonization and adherence to tissues of numerous animals during the pathogenesis of multiple infectious diseases, we aim to test the hypothesis that T4Ps play a role in colonization and disease in CDI. To do so, we will generate a prepilin peptidase mutant of an epidemic C. difficile strain, complement the mutant, confirm its phenotype and compare the ability of the wild type, mutant and complemented strain in a murine model of CDI. The structures of several T4P pilin proteins have been solved and revealed significant insights into the biogenesis and function of these important proteins. These insights have been enhanced by solving the structures of distantly related pilin and pilin-like proteins. We have purified to homogeneity two C. difficile pilin proteins. We have obtained high-quality 2D NMR data from one of these proteins and have crystallized the other.
We aim to determine the structures of both proteins providing critical information about the most divergent members of this class thus far investigated. The proposed studies will establish whether T4Ps are important C. difficile colonization and virulence factors and deepen our appreciation of the structure and biogenesis of these ubiquitous pili.

Public Health Relevance

Clostridium difficile is a bacterium that causes severe and sometimes deadly intestinal infections, particularly in the most vulnerable hospitalized patients. The number of such infections has been increasing at an alarming rate, treatment failures and relapses are common and there is no vaccine. A thorough understanding of the molecular details of this infection, particularly how the bacteria attach to the intestine and become established, is lacking. We aim to determine whether newly discovered surface filaments play a role in colonization by this microbe and to determine the atomic structure of proteins that make up these filaments. These studies could lead to new strategies to reduce the burden of disease caused by this serious infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI105881-02
Application #
8626357
Study Section
Special Emphasis Panel (ZRG1-IDM-A (80))
Program Officer
Ranallo, Ryan
Project Start
2013-03-01
Project End
2015-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
2
Fiscal Year
2014
Total Cost
$189,303
Indirect Cost
$63,370
Name
University of Maryland Baltimore
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Piepenbrink, Kurt H; Maldarelli, Grace A; de la Peña, Claudia F Martinez et al. (2014) Structure of Clostridium difficile PilJ exhibits unprecedented divergence from known type IV pilins. J Biol Chem 289:4334-45
Maldarelli, Grace A; De Masi, Leon; von Rosenvinge, Erik C et al. (2014) Identification, immunogenicity, and cross-reactivity of type IV pilin and pilin-like proteins from Clostridium difficile. Pathog Dis 71:302-14