2007 Bacteriology Meritorious Research Travel Award Recipients

Name:	Chris Shelburne, Ph.D.
Lab:	Abraham
Conference:	American Association of Immunologists Conference
Date:	May 18-22, 2007
Location:	Miami, Florida
Purpose:	To present poster

Abstract:

Mast cells enable heightened humoral immunity during infection
Dendritic cell (DC) accumulation in T-cell zones of secondary lymph organs is a critical event in the maximization of the primary immune response. Here, we demonstrate that mast cell (MC) control of elevated DC trafficking during infection enables the primary adaptive humoral immune response to proceed with heightened intensity. Elevated DC accumulation in draining lymph nodes (DLNs) was found to be the product of continual and incremental recruitment of DCs into the infected tissue site prior to their egress to DLNs. MCs contribute to this pattern of DC trafficking by the release of TNF, which was found to coordinately activate local blood vessel endothelium and DLNs to increase their expression of CD62E and CCL21, respectively. Blockade of either protein interfered with DC accumulation in DLNs, as well as the intensity of the humoral response to bacterial challenge. Thus, MCs enable the humoral immune response to proceed with enhanced intensity, which may be an important consideration in rationale vaccine design. Supported by grants DK50814 and AI50021 from the National Institutes of Health. 
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Abstract:

Dynamic association of the host cytoskeleton with the Chlamydia trachomatis inclusion
Chlamydiae sps are obligate intracellular pathogens that cause several human diseases including blinding trachoma, genital tract infections and atypical pneumonias. Within infected cells, Chlamydia resides in a large membrane-bound vacuole termed an “inclusion." The inclusion is largely segregated from classical endocytic and exocytic membrane transport pathways, and yet is able to efficiently acquire nutrients and lipids from the host. The vital role of cytoskeletal networks in maintenance of intracellular organelle-position as well as membrane protein and lipid traffic, prompted us to examine the arrangement of the host cytoskeleton in infected cells. Here, we report a dynamic interaction between inclusions and the host cytoskeletal elements. We have determined that the inclusion is encased by a network of actin and intermediate filaments (IF). IFs are one of the major cytoskeletal components of metazoans and mostly play a role in maintaining cell shape and motility. Surprisingly, cytoplasmic IF proteins encasing the inclusions (vimentin, cytokeratin-8 and cytokeratin-18) were proteolytically processed during infection. We determined that a chlamydial secreted protease (CPAF) was responsible for cleavage of these proteins and mapped the cleavage site to the N-terminal head region, a domain required for filament formation. Remarkably, despite removal of the Head domain, both vimentin and keratin filaments were retained as a filamentous cage enveloping the inclusion. Upon treatment with detergents, bacteria within the inclusions remained intact, suggesting that the IF cage provides structural integrity to the inclusion. Indeed, inclusions formed in MEFs from vimentin knockout mice were sensitive to detergent extraction and accumulated inclusion membrane-derived fibers, presumably the remnants of damaged inclusion membranes. Finally, maintenance of the vimentin cage around the inclusion required an intact actin filament network since disruption of actin, led to loss of the vimentin cage, hyper fibrillation of the inclusion membrane and disintegration of the inclusion. Our results provide evidence for a novel pathogenic strategy wherein Chlamydiae proteolytically processes and re-organizes the IF and the actin network around the inclusion to provide structural support for the growing inclusion.
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Abstract:

A conserved Toll-like receptor is required for Caenorhabditis elegans innate immunity
Pathogen recognition through Toll-like receptors (TLRs) is the first step required to mount an appropriate immune response against invading pathogens. The role of the only toll-like recepor, tol-1, in Caenorhabditis elegans innate immunity was shown to be limited to behavioral avoidance to a particular Gram-negative bacterium (1). Although no significant differences were observed in behavioral avoidance between wild type and the tol-1(nr2033) mutant to various other pathogens, the tol-1 mutant was hypersusceptible suggesting that TOL-1 may provide protection through other mechanisms. Based on survival assays and microscopy, we found that TOL-1 is essential for protecting the pharynx against the enteropathogenic bacterium, Salmonella enterica serovar Typhimuirum irrespective of behavioral avoidance. Loss of tol-1 not only led to rapid death of C. elegans but also resulted in rapid accumulation of S. enterica in the terminal bulb of the pharynx. We showed that TOL-1 is required for the proper expression of ABF-2 which is a defensin-like molecule expressed in the pharynx (2) and HSP-16.41 which is also expressed in the pharynx and is part of a family of heat shock proteins required for C. elegans immunity (3). These results indicate that TOL-1 plays a direct role in innate immunity.