Antimicrobial and Pore-forming Peptides of Free-living and Potentially Highly Pathogenic Naegleria fowleri Are Released from the Same Precursor Molecule

Abstract

The pore-forming polypeptides of Naegleria fowleri, naegleriapores A and B, are processed from separate multipeptide precursor structures. According to their transcripts, each precursor molecule appears to contain additional naegleriapore-like polypeptides, all of which share a structural motif of six invariant cysteine residues within their amino acid sequence. To identify the putative pronaegleriapore-derived peptides at the protein level, amoebic extracts were screened for small cysteine-rich polypeptides by fluorescently labeling their cysteine residues. Three novel naegleriapore isoforms derived from the precursor molecule of naegleriapore B were identified. Two of the isoforms were purified to homogeneity and tested for their biological activity. The pore-forming activity of the novel peptides was remarkably lower than that of the originally isolated naegleriapores, but both peptides killed bacteria by permeabilizing their cytoplasmic membranes. Collectively, these results indicate that naegleriapore isoforms with antibacterial and pore-forming activity are proteolytically released from the same precursor protein, presumably to generate a phylogenetically ancient complementary antimicrobial arsenal.Naegleria fowleri is a free-living amoeboflagellate of soil and freshwater habitats throughout the world. Although the trophozoites are able to fulfil their life cycle without the intervention of a parasitic stage, they can invade the human brain via the nasal mucosa and olfactory nerve and cause primary amoebic meningoencephalitis (1, 2). This disease is characterized by massive host tissue destruction indicating that the cytolytic activity of the amoebae is a major factor for their pathogenicity.In vitro, intact amoebae (1), as well as cell-free extracts thereof (3), lyse efficiently a variety of target cells. The amoebic cytolytic capacity has been attributed mainly to pore-forming molecules that have been identified more than a decade ago (4). Recently, we characterized at the molecular level naegleriapores A and B, proteins that potently display pore-forming activity that kills prokaryotic as well as eukaryotic target cells (5). According to the respective transcripts, both naegleriapores were processed from larger precursor structures, each of which contains several naegleriapore isoforms that share the structural motif of six invariant cysteine residues within their highly diverse amino acid sequences. The precursor molecule of naegleriapore A contains three isoforms, and that of naegleriapore B contains five isoforms (5). For convenience, the putative isoforms within the precursor molecules were designated according to their location on the precursor of naegleriapore A as A1-A3 and on that of naegleriapore B as B1-B5. Isoforms A2 and B1 represent proteins that have been isolated recently by us according to their pore-forming activity (5).As the existence, fate, and function of additional naegleriapores contained in the precursor molecules are unclear, we initiated a survey pursuant to their identificatio...

Mucins in the host defence against Naegleria fowleri and mucinolytic activity as a possible means of evasion

“…On the other hand, it has been shown that N. fowleri has a variety of virulence factors that may help the parasite to evade the host immune response and finally invade the tissue. These factors are mainly cysteine proteases (Aldape et al, 1994;Mat Amin, 2004;Serrano-Luna et al, 2007), phospholipases (Fulford & Marciano-Cabral, 1986 2001), pore-forming proteins (Young & Lowrey, 1989;Herbst et al, 2002Herbst et al, , 2004, and membrane capping and shedding formation (Shibayama et al, 2003). In the current study, we evaluated the role of mucins as a natural immune response, and the role of mucinolytic activity in trophozoites of the pathogen N. fowleri and the non-pathogen N. gruberi as an evasive mechanism.…”

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri

“…Several proteins, such as Nf-actin (Jung et al, 2009;Lee et al, 2007;Sohn et al, 2010), pore-forming naegleriapores A and B (Herbst et al, 2002(Herbst et al, , 2004, cysteine protease (Aldape et al, 1994) and virulence-related protein (Hu et al, 1991(Hu et al, , 1992, have been implicated in Naegleria pathogenesis, but the exact roles of these proteins are not yet well understood. The present study enables modulation of the in vivo pathogenicity of N. fowleri by using different compositions of axenic growth media.…”

“…In order to assess transcriptional differences between N. fowleri trophozoites grown in the three different media, a quantitative real-time RT-PCR using primers against potential pathogenicity factors (Aldape et al, 1994;Herbst et al, 2002Herbst et al, , 2004Hu et al, 1991Hu et al, , 1992Jung et al, 2009;Lee et al, 2007;Sohn et al, 2010) was performed. The primers used for the PCR are listed in Table 1.…”

“…Furthermore, a diverse array of proteins has been associated with Naegleria-mediated cell lysis (Marciano-Cabral & Cabral, 2007). Two pore-forming proteins, termed naegleriapores A and B, were isolated and the gene coding for these polypeptides was identified by Herbst et al (2002Herbst et al ( , 2004. After biochemical and functional characterization, naegleriapores have been found to be cytotoxic to human cells (Herbst et al, 2004(Herbst et al, , 2002.…”

“…Two pore-forming proteins, termed naegleriapores A and B, were isolated and the gene coding for these polypeptides was identified by Herbst et al (2002Herbst et al ( , 2004. After biochemical and functional characterization, naegleriapores have been found to be cytotoxic to human cells (Herbst et al, 2004(Herbst et al, , 2002. Another protein involved in N. fowleri-induced cell destruction is Nfactin, which is specifically located around phagocytic foodcups and therefore plays an important role in phagocytic activity (Sohn et al, 2010).…”