This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A.
Specific Aims :
The specific aims of this grant have not been modified although due to technical difficulties encountered the specific target cell line for the proteomic profile has been changed from PMNs to a liver cell line, HepG2 cells. The specifics of this change are described below. The cholesterol dependent cytolysins (CDCs) are a family of large pore forming toxins that are virulence factors produced by a variety of Gram-positive pathogens. Although the cytolytic mechanism of these multimeric pore-forming toxins has been largely conserved among the various CDC producing bacteria, it is becoming increasingly apparent that the CDCs may have evolved additional roles in pathogenesis. The cytolytic mechanism requires the formation of a large oligomeric structure that is comprised of approximately 30-40 monomers (reviewed in (Tweten et al., 2001). However, within the site of infection, as the toxins diffuse farther away from the site of the infection the concentration of toxin decreases and the likelihood of the formation of the complete oligomeric structures subsequently decreases. Yet a role for these """"""""non-lytic"""""""" structures in disease has not been fully examined. Streptococcus intermedius is unique among CDC producing pathogens because its CDC, intermedilysin (ILY), requires a specific cellular receptor for cytolytic function. ILY has been shown to be human cell specific and requires the presence of its cellular receptor huCD59. CD59 is an important surface expressed glycosylphosphatidylinositol (GPI)- anchored protein that is found on many cell types and is up regulated on activated polymorphonuclear leukocytes (PMNs) (Gordon et al., 1994). It has been associated with a number of important cellular functions including inhibiting the formation of the membrane attack complex (MAC) on host cells (Rollins and Sims, 1990), serving as an adaptor molecule for a candidate C1q receptor (C1qRO2-) (Otabor et al., 2004), and acting as a cell signaling molecule . It is unclear why ILY has evolved this cellular specificity for CD59 and what role this may play in pathogenesis. Interestingly, Streptococcus intermedius isolated from deep-seated abscesses has been shown to produce levels of ILY 6-10 times greater than strains isolated from peripheral site infections indicating its importance in pathogenesis (Nagamune et al., 2000). As PMNs are the major immune cell present in abscesses we propose to examine the PMN response to the receptor-mediated interaction of non-lytic mutants of ILY. We hypothesis that the ILY receptor mediated interaction with huCD59 can interfere with CD59 cellular functions such as MAC inhibition and C1q activated superoxide production (specific aim 1). We further hypothesis that ILY binding to CD59 can modulate PMN hysiology differently than other CDCs that do not bind to a specific receptor (specific aim 2).
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