Naegleria fowleri is the causative agent of Primary Amebic Meningoencephalitis a rapidly fatal disease of the central nervous system. Pathogenic and nonpathogenic species have been described, but the determinants of pathogenicity and virulence are unknown. Dr. Marciano have demonstrated that an important determinant of virulence is the ability of the ameba to resist complement-mediated lysis. Therefore, the goal of this study is to define the mechanisms by which highly-pathogenic strains of N. fowleri develop resistance to complement.In conducting these studies, N. fowleri LEEmp, a highly-pathogenic strain and N. gruberi, a non-pathogenic strain will be employed. The rationale for using theses strains is that both pathogenic and nonpathogenic ameba activate the complement cascade, but pathogenic strains are complement-resistant while nonpathogenic strains are complement-sensitive.The applicant has determined that complement-resistant Naegleria have developed at least two mechanisms for rendering the membrane attack complex nonfunctional. Under conditions of low serum concentrations, complement-resistant Naegleria remove the assembled membrane attack complex from their surface by vesiculation. Alternatively, in the presence of high concentrations of serum, complement regulatory proteins may play a more prominent role in resistance to complement-mediated lysis. The involvement of surface proteins in resistance to complement has been demonstrated in experiments in which treatment of N. fowleri trophozoites with trypsin, papain, endoglycosidase H or tunicamycin, converted complement-resistant amebae to complement-sensitive organisms. Also, antiserum to CD59, a complement regulatory protein on erythrocytes and platelets, cross- reacts with a Naegleria protein of approximate molecular mass of 42-44 kDa. Their data suggest that surface membrane proteins, especially glycoproteins, of highly pathogenic amebae play a role in resistance to complement-mediated lysis by inhibiting the lytic membrane attack complex. They propose to test the hypothesis that cell surface membrane modifications of N. fowleri account for their resistance to complement lysis. In order to accomplish these objectives the following specific aims will serve as guidelines to the research: 1. To determine the cellular events that regulate ameba membrane vesiculation in response to complement which lysis to protection from complement-mediated lysis. The role of calcium ions, the activity of protein kinases, and cytoskeletal changes within Naegleria will be determined since these may play an important role in vesiculation. 2. To purify the Naegleria proteins GP28, GP42-44, GP49/50, GP62, GP65, and P95 which have been shown to be upregulated in Naegleria during the transformation from a state of complement-sensitive to that of complement-resistant. The basic biochemical properties of these proteins will be defined. 3. To establish the relationship of CD59, found on human erythrocytes, leukocytes, and platelets to the Naegleria membrane glycoprotein, GP42-44. GP42-44 cross-reacts with antibodies directed against human erythrocyte CD59, a complement regulatory protein and may share functional homology. 4. To determine the role of BP28, GP42-44, GP49/50, GP62, GP65, and P95 in inhibiting the membrane attack complex. Studies will include incorporation of purified proteins into the membranes of complement-sensitive amebae in order to determine whether they inhibit lysis by the membrane attack complex.
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