Studies on the cyst stage of Naegleria fowleri in vivo and in vitro.

Naegleria fowleri is a pathogenic, free-living amoeba that causes primary amebic meningoencephalitis (PAM), a highly fatal disease of the central nervous system. N. fowleri demonstrates three forms: the trophozoite, flagellate, and cyst. Most studies have focused on the trophozoite limiting information on the cyst. The present study examined the ability of cysts to attach to, excyst into the trophozoite form, and destroy cell cultures. Additionally, the study assessed the ability of cysts to cause PAM in a murine model. The results demonstrated that exposure to cysts and transformation into trophozoites resulted in destruction of cell cultures. Specifically, the mixed glial cells exhibited an increase in lactate dehydrogenase (LDH) release compared with cells without cyst exposure. On day eight postexposure, there was a nearly fourfold increase in LDH. The cysts of N. fowleri were shown not to be infective in vivo in a murine model. The mediation of the encystment process by the intracellular concentration of cAMP was also investigated. Trophozoites were treated with dipyridamole, an inhibitor of cAMP-specific phosphodiesterases. Dipyridamole increased the rate of encystment by nearly twofold and increased the intracellular concentration of cAMP in cysts by nearly sixfold throughout this period suggesting that cAMP is a mediator of encystment for N. fowleri.

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri.

BACKGROUND: The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely. RESULTS: Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system. CONCLUSIONS: In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.

Identification and characterisation of a cryptic Golgi complex in Naegleria gruberi.

Although the Golgi complex has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organisation in several lineages, raising the question of what range of morphologies is possible for the Golgi. In order to understand this diversity, it is necessary to characterise the Golgi in many different lineages. Here, we identify the Golgi complex in Naegleria, one of the first descriptions of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two species of Naegleria and show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker (Ng)CopB by fluorescence in Naegleria gruberi, identifying membranous structures that are disrupted by Brefeldin A treatment, consistent with Golgi localisation. Confocal and immunoelectron microscopy reveals that NgCOPB localises to tubular membranous structures. Our data identify the Golgi organelle for the first time in this major eukaryotic lineage, and provide the rare example of a tubular morphology, representing an important sampling point for the comparative understanding of Golgi organellar diversity.This article has an associated First Person interview with the first author of the paper.

The Effect of Different Environmental Conditions on the Viability of Naegleria fowleri Amoebae.

Naegleria fowleri, a free-living amoeba found in soil and freshwater environments, is the causative agent of Primary Amoebic Meningoencephalitis. Infection occurs when amoebae enter the nasal cavity, attach to the nasal mucosa and travel along olfactory neurons towards the olfactory bulb. Upon reaching the central nervous system, the amoebae replicate very rapidly and can cause death in 3-10 days. Little is known about the conditions in which the amoeba can survive in the environment. We have tested conditions beyond the known boundaries on the viability of amoebae by introducing them into moderate and extreme salinity, pH, and temperatures. Our data shows that although viability expectedly decreases towards each of these extreme conditions, their tolerance was much greater than anticipated, including viability in moderate salinity, a wide pH range, and temperatures higher than the previously reported 45 °C.

Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds and is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). PAM occurs when amoebae attach to the nasal epithelium and invade the CNS, a process that involves binding to, and degradation of, extracellular matrix (ECM) components. This degradation is mediated by matrix metalloproteinases (MMPs), enzymes that have been described in other pathogenic protozoa, and that have been linked to their increased motility and invasive capability. These enzymes also are upregulated in tumorigenic cells and have been implicated in metastasis of certain tumours. In the present study, in vitro experiments linked MMPs functionally to the degradation of the ECM. Gelatin zymography demonstrated enzyme activity in N. fowleri whole cell lysates, conditioned media and media collected from invasion assays. Western immunoblotting indicated the presence of the metalloproteinases MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 [membrane type-1 matrix metalloproteinase (MT1-MMP)]. Highly virulent mouse-passaged amoebae expressed higher levels of MMPs than weakly virulent axenically grown amoebae. The functional relevance of MMPs in media was indicated through the use of the MMP inhibitor, 1,10-phenanthroline. The collective in vitro results suggest that MMPs play a critical role in vivo in invasion of the CNS and that these enzymes may be amenable targets for limiting PAM.

Identification of Naegleria fowleri proteins linked to primary amoebic meningoencephalitis.

Naegleria fowleri (N. fowleri) causes primary amoebic meningoencephalitis, a rapidly fatal disease of the central nervous system. N. fowleri can exist in cyst, flagellate or amoebic forms, depending on environmental conditions. The amoebic form can invade the brain following introduction into the nasal passages. When applied intranasally to a mouse model, cultured N. fowleri amoebae exhibit low virulence. However, upon serial passage in mouse brain, the amoebae acquire a highly virulent state. In the present study, a proteomics approach was applied to the identification of N. fowleri amoeba proteins whose expression was associated with the highly virulent state in mice. Mice were inoculated intranasally with axenically cultured amoebae or with mouse-passaged amoebae. Examination by light and electron microscopy revealed no morphological differences. However, mouse-passaged amoebae were more virulent in mice as indicated by exhibiting a two log10 titre decrease in median infective dose 50 (ID50). Scatter plot analysis of amoebic lysates revealed a subset of proteins, the expression of which was associated with highly virulent amoebae. MS-MS indicated that this subset contained proteins that shared homology with those linked to cytoskeletal rearrangement and the invasion process. Invasion assays were performed in the presence of a select inhibitor to expand on the findings. The collective results suggest that N. fowleri gene products linked to cytoskeletal rearrangement and invasion may be candidate targets in the management of primary amoebic meningoencephalitis.

Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba.

Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporin's active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.

Identification of peptidases in highly pathogenic vs. weakly pathogenic Naegleria fowleri amebae.

Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse-passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba-CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.

The mitochondrial genome and a 60-kb nuclear DNA segment from Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis.

Naegleria fowleri is a unicellular eukaryote causing primary amoebic meningoencephalitis, a neuropathic disease killing 99% of those infected, usually within 7-14 days. Naegleria fowleri is found globally in regions including the US and Australia. The genome of the related nonpathogenic species Naegleria gruberi has been sequenced, but the genetic basis for N. fowleri pathogenicity is unclear. To generate such insight, we sequenced and assembled the mitochondrial genome and a 60-kb segment of nuclear genome from N. fowleri. The mitochondrial genome is highly similar to its counterpart in N. gruberi in gene complement and organization, while distinct lack of synteny is observed for the nuclear segments. Even in this short (60-kb) segment, we identified examples of potential factors for pathogenesis, including ten novel N. fowleri-specific genes. We also identified a homolog of cathepsin B; proteases proposed to be involved in the pathogenesis of diverse eukaryotic pathogens, including N. fowleri. Finally, we demonstrate a likely case of horizontal gene transfer between N. fowleri and two unrelated amoebae, one of which causes granulomatous amoebic encephalitis. This initial look into the N. fowleri nuclear genome has revealed several examples of potential pathogenesis factors, improving our understanding of a neglected pathogen of increasing global importance.

The 12th International Workshops on Opportunistic Protists (IWOP-12).

The 12th International Workshops on Opportunistic Protists (IWOP-12) was held in August 2012 in Tarrytown, New York. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency-associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and (2) foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists, e.g. the free-living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference that brings together research groups working on these opportunistic pathogens. Slow but steady progress is being achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune-deficient and immune-competent hosts, and is providing critical insights into these emerging and reemerging pathogens. This IWOP meeting demonstrated the importance of newly developed genomic level information for many of these pathogens and how analysis of such large data sets is providing key insights into the basic biology of these organisms. A great concern is the loss of scientific expertise and diversity in the research community due to the ongoing decline in research funding. This loss of researchers is due to the small size of many of these research communities and a lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms.

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

Highlights and summaries of the 11th International Workshops on Opportunistic Protists.

The 11th in the series of International Workshops on Opportunistic Protists (IWOP-11) was held in August 2010 on the Big Island of Hawaii. These meetings are devoted to agents of infections that cause serious problems in AIDS patients and other individuals with defective immune systems. International Workshops on Opportunistic Protists serves as a forum for exchange of current research information on Pneumocystis, Cryptosporidium and the Microsporidia, Toxoplasma, free-living amoebae, kinetoplastid flagellates and other pathogens that are particularly pathogenic in immunodeficient hosts. Studies on interactions between host and pathogen, especially host responses, were highlighted in this year's symposium. The lack of in vitro cultivation methods for luxuriant growth of Pneumocystis, Cryptosporidium and the Enterocytozoon bieneusi remains a major hindrance to understanding the basic biology of these organisms and precludes genetic manipulations. However, slow but steady progress is being achieved by hard work including data mining of some completed or partially completed genome sequencing of several IWOP organisms. Of great concern is evidence for dramatic decline in research funding for these pathogens and the lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms that can provide critical insight into emerging and reemerging pathogens.

Acanthamoeba culbertsoni elicits soluble factors that exert anti-microglial cell activity.

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.

Free-living amoebae, Legionella and Mycobacterium in tap water supplied by a municipal drinking water utility in the USA.

Legionella and Mycobacterium can proliferate within free-living amoebae (FLA) where they are protected from disinfectants at concentrations that can kill bacteria but not protozoa. Despite effective treatment of drinking water, microbes can enter water utility distribution systems (DS) and hence the plumbing within building premises. Additionally, biofilm formation may account for the persistence of microbes in the DS. In the present study a domestic water tap in north-central United States (USA) was sampled in March and September 2007 and analysed for FLA, Legionella and Mycobacterium. Identification of organisms was determined by growth on specific culture media, light and electron microscopy, and amplification of DNA probes specific for each organism. In both the spring and fall samples, amoebae, Legionella and Mycobacterium were detected. However, Acanthamoeba was prominent in the spring sample whereas Vahlkampfia and Naegleria were the amoebae detected in the autumn. Bacterial proliferation in laboratory cultures was noticeably enhanced in the presence of amoebae and biofilms rapidly formed in mixed amoebae and bacteria cultures. It is hypothesized that temperature affected the dynamics of FLA species population structure within the DS and that pathogenic bacteria that proliferate within FLA, which are themselves opportunistic pathogens, pose dual public health risks.

Acanthamoeba culbertsoni: analysis of amoebic adhesion and invasion on extracellular matrix components collagen I and laminin-1.

Acanthamoeba are free-living amoebae found in most environments that can cause brain and corneal infections. To infect humans, these pathogens must interact with host cells and the extracellular matrix (ECM). In order to define the mode by which amoebae recognize ECM components and process this recognition, we analyzed Acanthamoeba culbertsoni attachment and invasion, respectively, on collagen I and laminin-1 and on tridimensional collagen I and matrigel matrices. We determined that amoebae surface proteins are involved in adhesion, that exogenous sugars can decrease adhesion and invasion, and that adhesion and invasion are dependent on microfilament reorganization. In addition, we determined the role of serine- and metallo-proteases on invasion and found that adhesion was blocked when amoebae were treated with a metallo-protease inhibitor. Collectively, these results suggest that adhesion and invasion are protease- and microfilament-dependent events in which amoebic surface proteins play a pivotal role.

Identification of Naegleria fowleri in warm ground water aquifers.

The free-living amoeba Naegleria fowleri was identified as the etiological agent of primary amoebic meningoencephalitis that caused the deaths of two children in Peoria, Arizona, in autumn of 2002. It was suspected that the source of N. fowleri was the domestic water supply, which originates from ground water sources. In this study, ground water from the greater Phoenix Metropolitan area was tested for the presence of N. fowleri using a nested polymerase chain reaction approach. Phylogenetic analyses of 16S rRNA sequences of bacterial populations in the ground water were performed to examine the potential link between the presence of N. fowleri and bacterial groups inhabiting water wells. The results showed the presence of N. fowleri in five out of six wells sampled and in 26.6% of all ground water samples tested. Phylogenetic analyses showed that beta- and gamma-proteobacteria were the dominant bacterial populations present in the ground water. Bacterial community analyses revealed a very diverse community structure in ground water samples testing positive for N. fowleri.

Acanthamoeba interaction with extracellular matrix glycoproteins: biological and biochemical characterization and role in cytotoxicity and invasiveness.

Acanthamoeba are free-living amoebae that are dispersed in most environments. Occasionally, Acanthamoeba cause serious human infections, such as keratitis and encephalitis. During the infection process, amoebic adhesion to, and degradation of, host cells and their extracellular matrix (ECM) appear to be important requirements. We examined the interaction of Acanthamoeba with the ECM, and related this event to host cell destruction and tissue invasion. Pathogenic Acanthamoeba culbertsoni differentially attached on the ECM glycoproteins laminin-1, collagen-I, and fibronectin, as compared with non-pathogenic Acanthamoeba astronyxis. Binding to collagen-I and laminin-1 induced A. culbertsoni to become flattened and elongated. Because attachment on laminin-1 was higher in A. culbertsoni, laminin-1 was chosen for further analysis. A 55-kDa laminin-binding protein was identified in pathogenic amoebae, but it was not found in non-pathogenic amoebae. No differential cytotoxicity against distinct cell types was observed between A. culbertsoni incubated with or without ECM. On the other hand, binding on collagen-I or matrigel scaffolds induced a differential effect where A. culbertsoni invaded collagen-I matrices more rapidly. These results indicate that ECM recognition, as an antecedent to tissue invasion, may be a trait characteristic of pathogenic Acanthamoeba.

Survey for the presence of Naegleria fowleri amebae in lake water used to cool reactors at a nuclear power generating plant.

Water from Lake Anna in Virginia, a lake that is used to cool reactors at a nuclear power plant and for recreational activities, was assessed for the presence of Naegleria fowleri, an ameba that causes primary amebic meningoencephalitis (PAM). This survey was undertaken because it has been reported that thermally enriched water fosters the propagation of N. fowleri and, hence, increases the risk of infection to humans. Of 16 sites sampled during the summer of 2007, nine were found to be positive for N. fowleri by a nested polymerase chain reaction assay. However, total ameba counts, inclusive of N. fowleri, never exceeded 12/50 mL of lake water at any site. No correlation was obtained between the conductivity, dissolved oxygen, temperature, and pH of water and presence of N. fowleri. To date, cases of PAM have not been reported from this thermally enriched lake. It is postulated that predation by other protozoa and invertebrates, disturbance of the water surface from recreational boating activities, or the presence of bacterial or fungal toxins, maintain the number N. fowleri at a low level in Lake Anna.

Identification of Acanthamoeba sp. in paraffin-embedded CNS tissue from an HIV+ individual by PCR.

The opportunistic pathogens, Acanthamoeba and Balamuthia, are the causative agents of the fatal central nervous system (CNS) infection granulomatous amoebic encephalitis. We report an infection of Acanthamoeba in an HIV+ individual. In the present case, multiple lesions were observed in the skin, brain, lung, liver, and bone. A polymerase chain reaction (PCR) assay specific for Acanthamoeba was positive on tissue from a brain biopsy that had been embedded in paraffin. This report demonstrates the need for the consideration of Acanthamoeba infections in HIV+ individuals with skin lesions and multiple lesions throughout the body with CNS involvement. The results of the present study demonstrate that opportunistic amoebic infections can be diagnosed by PCR from paraffin-embedded biopsy material.