Identification of aNaegleria fowleriMembrane Protein Reactive with Anti-Human CD59 Antibody

Abstract

Naegleria fowleri, the causative agent of primary amebic meningoencephalitis, is resistant to complement lysis. The presence of a complement regulatory protein on the surface of N. fowleri was investigated. Southern blot and Northern blot analyses demonstrated hybridization of a radiolabeled cDNA probe for CD59 to genomic DNA and RNA, respectively, from pathogenic N. fowleri. An 18-kDa immunoreactive protein was detected on the membrane of N. fowleri by Western immunoblot and immunofluorescence analyses with monoclonal antibodies for human CD59. Complement component C9 immunoprecipitated with the N. fowleri "CD59-like" protein from amebae incubated with normal human serum. In contrast, a gene or protein similar to CD59 was not detected in nonpathogenic, complement-sensitive N. gruberi amebae. Collectively, our studies suggest that a protein reactive with antibodies to human CD59 is present on the surface of N. fowleri amebae and may play a role in resistance to lysis by cytolytic proteins.Naegleria fowleri, an ameboflagellate found in freshwater lakes and ponds, is the causative agent of primary amebic meningoencephalitis, a rapidly fatal disease of the central nervous system (1, 7). The determinants of virulence for this ameba are unknown, but resistance to complement lysis appears to play a major role in its pathogenicity (13,30). Both pathogenic and nonpathogenic Naegleria species activate the alternative pathway of complement (16). However, pathogenic N. fowleri amebae are resistant to the lytic effect of complement (36). Immunoelectrophoretic studies have established that complement components C3 and C5 are converted to C3b and C5b, respectively, following incubation of complementresistant N. fowleri in normal human serum (NHS) (37). These results suggest that complement regulation by N. fowleri occurs at the stage of membrane attack complex (MAC) formation.CD59 is an 18-to 20-kDa glycosyl-phosphatidylinositol/inositol-anchored glycoprotein found on the surface of a variety of cell types which functions to inhibit complete formation of the MAC of complement. CD59 inhibition of complement lysis occurs by binding complement components C8 and C9, ultimately preventing C9 insertion into and polymerization in the cell membrane (9). The purpose of the present study was to determine whether complement-regulatory protein CD59, which is present on mammalian cells, could be detected on pathogenic N. fowleri amebae.Molecular and immunology-based assays were used to test the hypothesis. Southern blot analysis was performed to determine whether N. fowleri possesses the CD59 gene. Northern blot analysis was utilized to identify the presence of CD59 transcripts expressed by complement-resistant N. fowleri amebae. With monoclonal antibodies to human CD59, an immunoreactive protein was detected in the membrane fraction of N. fowleri by Western immunoblot analysis. Immunoprecipitation studies were used to establish whether the ameba "CD59-like" protein was able to associate with human complement component C9. In addition, an...

Trophozoites of Entamoeba histolytica express a CD59-like molecule in human colon

“…It has been described as a surface protein with antigenic and functional similarity to human CD59 in E. histolytica and Naegleria fowleri trophozoites in vitro (Braga et al 1992;Fritzinger et al 2006). The evidence suggests that the molecule CD59 like is the Gal/GalNAc binding lectin of E. histolytica that contains a region with antigenic crossreactivity with CD59 (Braga et al 1992;Petri et al 2002).…”

A high affinity inhibitor of human CD59 enhances complement-mediated virolysis of HIV-1: Implications for treatment of HIV-1/AIDS

“…CD59 is a GPI-linked complement regulatory protein, which specifically inhibits MAC formation and is universally expressed on the surface of mammalian cells (1,2). It is well established that many pathogenic enveloped viruses, including HIV-1, CMV, herpes virus, Ebola virus, and influenza virus, escape complement-mediated virolysis by incorporating host cell complement-regulatory proteins into their own viral envelope (3)(4)(5)(6)(7)(8)(9). The presence of complement regulators such as CD59 on the external surface of the viral envelope confers resistance to Ab-dependent, complement-mediated lysis.…”

Fulminant Amoebic Colitis: Role of the Innate and Adaptive Immune Responses

“…16c), suggesting that only the trophozoites positive to CD59 manage to evade the immune response and thus induce cellular damage in the intestine. On the other hand, on extract proteins of E. histolytica trophozoites, it was confirmed by Western Blot that the protein located on the surface of trophozoites corresponds to a molecular weight of approximately 21 kDa, similar to CD59 of mammalian cells (18-20 kDa) (Davies & Lachmann, 1993) as well as to the CD59 protein on N. fowleri (Fritzinger et al, 2006). In spite of this information, the source of these CD59 molecules in intestines with FAC is not clear.…”

“…Previous studies with pathogenic E. histolytica trophozoites have demonstrated the presence of proteins that share epitopes with human CD59, which probably protects amebas against lysis by the complement (Braga et al, 1992;Flores-Romo et al, 1994;Petri et al, 2002;Fritzinger et al, 2006). Nevertheless, the group of Ventura-Juárez (2009) found the antigen of CD59 by using immunohistochemical and immune-gold techniques.…”