Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Biomedical Sciences
Non Technical Summary
In spite of persistent intestinal infection by Brachyspira pilosicoli spirochetes and their association with colitis in a broad range of hosts including human beings, non-human primates, pigs, dogs, domestic poultry, and certain wildlife species, the molecular basis of infection and disease is poorly understood. Consequently, the long term contribution of intestinal infection by B. pilosicoli remains unknown and current intervention strategies are empirical. This project will determine the contribution of B. pilosicoli genes in infection of host target cells. It is anticipated that greater understanding of the life cycle and host interactions of this pathogenic intestinal spirochete with a broad host range will provide a molecular framework for directed therapeutic approach for pathogenic spirochetes of humans and animals in general and intestinal Brachyspira species specifically.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goal of this project is to characterize the molecular mechanisms of host-parasite interactions and their contribution to susceptibility or resistance against colitis caused by the pathogenic intestinal spirochete Brachyspira pilosicoli. Preliminary in vitro studies examining the interaction of B. pilosicoli with eukaryotic cells have revealed that intimate attachment of B. pilosicoli on the cell membrane of cultured human intestinal epithelial cells elicits the secretion of chemokine interleukin 8, a key mediator of host mucosal innate and inflammatory responses to bacterial infection. Further studies examining the uptake of B. pilosicoli by human monocytic cells showed intracellular survival and replication of B. pilosicoli within vacuoles capable of evading fusion with lysosomal-associated membrane protein 1 (LAMP-1)-positive late endosomes post-infection. More recently, detailed analysis of the complete genome sequence of B. pilosicoli identified a large complement of genes encoding proteins with eukaryotic-like motifs, particularly ankyrin-repeats containing proteins (Ank). On the basis of these observations, we hypothesize that during interaction of B. pilosicoli with eukaryotic cells certain Ank have effector functions essential to intestinal infection. We will test this hypothesis by investigating the contribution of B. pilosicoli Ank proteins during spirochetal attachment onto cultured human intestinal epithelial cell lines and intracellular trafficking following phagocytic uptake by macrophage cell lines. Specifically, Aim 1 will determine the temporal expression and contribution of Ank proteins to pro-inflammatory cytokine/chemokine response of intestinal epithelial cells and monocytes post-infection. Aim 2 will explore the role of Ank proteins in directing the intracellular trafficking of B. pilosicoli-containing vacuole and inhibition of lysosomal fusion within monocytes post-infection.
Project Methods
Comparative analysis of B. pilosicoli ankyrin-repeat gene family, particularly the genetic organization and identification of homologous sequences among members of the genus Brachyspira, pathogenic spirochetes in the order Spirochaetales, and environmental and pathogenic bacteria will provide a more complete understanding and potential function of these proteins in infection and disease. Concurrently, rabbit polyclonal antibodies will be prepared against each recombinant His-tagged fusion Ank protein. Next, the level and kinetics of each ank gene expression and protein synthesis will be assessed respectively by real-time RT-PCR and immunoblotting over time post-infection of control and chloramphenicol-treated (protein) human intestinal epithelial and monocytic cell lines. Taken together the data will form the basis of selection of candidate Ank genes for inactivation by insertional mutagenesis with a kanamycin resistance cassette that can function in Brachyspira intestinal spirochete and homologous recombination. Gene inactivation in each mutant strain will be confirmed by immunoblotting and PCR assay. Subsequent experiments will correlate secretion of Ank proteins by B. pilosicoli during epithelial cell attachment and intracellular trafficking in monocytic cell lines with the pro-inflammatory cytokine responses and growth kinetics of wild type and isogenic B. pilosicoli mutant strains based on colony forming units over time post-infection. Potential growth defects of mutant strains will be confirmed by single-cell confocal laser scanning microscopy analysis of fluorescent labeled spirochetes over time post-infection. Each mutant strain also will be assessed for intracellular trafficking by co-localization of labeled spirochetes and specific markers of the endosomal-lysosomal pathway and confocal laser scanning microscopy analysis over time post-infection. Finally, rescue of mutant strains with an intracellular replication defect attributable to ank genes will be attempted by determining the intracellular trafficking of communal phagosomes also harboring a GFP-labeled wild-type strain in mixed infection of monocytic cell lines.