A comparative study of the membrane proteins from Naegleria species: A 23-kDa protein participates in the virulence of Naegleria fowleri.

The plasma membrane is essential in the pathogenicity of several microorganisms. However, to date, there are few studies related to the plasma membrane proteins in Naegleria fowleri; this amoeba produces a fatal disease called primary amoebic meningoencephalitis. In the present study, we analyzed the electrophoretic pattern of the membrane proteins of N. fowleri and compared it with the nonpathogenic N. lovaniensis and N. gruberi. We detected a 23-kDa protein (Nf23) present at a higher level in N. fowleri than in the nonpathogenic amoebae. The mass spectrometry analysis showed that the Nf23 protein has a sequence of 229 amino acids that corresponds to a membrane protein. The mRNA level of nf23 was overexpressed 4-fold and 40,000-fold in N. fowleri compared with N. lovaniensis and N. gruberi, respectively. Moreover, we found a 5-fold overexpression of nf23 in N. fowleri trophozoites recovered from mouse brains compared with trophozoites axenically cultivated. In addition, the cytopathic effect on Madin-Darby Canine Kidney cells coincubated with N. fowleri diminished in the presence of antibodies against Nf23; nevertheless, the nonpathogenic amoebae did not produce damage to the monolayer cells. These results suggest that the plasma membrane protein Nf23 is probably involved in the virulence of N. fowleri.

Potentially pathogenic genera of free-living amoebae coexisting in a thermal spring.

Little is known about the prevalence of Balamuthia mandrillaris within the environment due to its difficult isolation, but once an axenic culture is established, it is relatively easy to maintain. As most of the time researchers are interested mainly in isolating B. mandrillaris from environmental samples, the flora that accompanies it becomes second in importance. Therefore, this study aimed to determine which potentially pathogenic free-living amoebae, in addition to B. mandrillaris, could be found co-inhabiting a source of natural thermal water called "Agua Caliente" (Mexico), where this amoeba has previously been detected twice by molecular methods. A third sampling from this same source was carried out to try to isolate B. mandrillaris and other free-living amoebae using 37 and 45 °C as isolation temperatures. For PCR techniques, specific primers were used for B. mandrillaris, Naegleria fowleri, and Acanthamoeba species, plus a universal primer set for the eukaryotic 18S SSU rRNA gene for other isolated amoebae. PCR products were sequenced for final identification. 42 strains of the primary isolate were obtained, but only 34 could be kept in culture. Of them, 23 strains were identified as Naegleria lovaniensis, eight strains as Acanthamoeba jacobsi, two strains as Stenamoeba sp. and only one was identified as Vermamoeba vermiformis. The isolation of B. mandrillaris was once again not successful, but the presence of potentially pathogenic and nonpathogenic free-living amoebae is reported for the first time in this type of water in Mexico thanks to molecular methodology.

The therapeutic strategies against Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate most prominently known for its role as the etiological agent of the Primary Amoebic Meningoencephalitis (PAM), a disease that afflicts the central nervous system and is fatal in more than 95% of the reported cases. Although being fatal and with potential risks for an increase in the occurrence of the pathogen in populated areas, the organism receives little public health attention. A great underestimation in the number of PAM cases reported is assumed, taking into account the difficulty in obtaining an accurate diagnosis. In this review, we summarize different techniques and methods used in the identification of the protozoan in clinical and environmental samples. Since it remains unclear whether the protozoan infection can be successfully treated with the currently available drugs, we proceed to discuss the current PAM therapeutic strategies and its effectiveness. Finally, novel compounds for potential treatments are discussed as well as research on vaccine development against PAM.

Amphotericin B induces apoptosis-like programmed cell death in Naegleria fowleri and Naegleria gruberi.

Naegleria fowleri and Naegleria gruberi belong to the free-living amoebae group. It is widely known that the non-pathogenic species N. gruberi is usually employed as a model to describe molecular pathways in this genus, mainly because its genome has been recently described. However, N. fowleri is an aetiological agent of primary amoebic meningoencephalitis, an acute and fatal disease. Currently, the most widely used drug for its treatment is amphotericin B (AmB). It was previously reported that AmB has an amoebicidal effect in both N. fowleri and N. gruberi trophozoites by inducing morphological changes that resemble programmed cell death (PCD). PCD is a mechanism that activates morphological, biochemical and genetic changes. However, PCD has not yet been characterized in the genus Naegleria. The aim of the present work was to evaluate the typical markers to describe PCD in both amoebae. These results showed that treated trophozoites displayed several parameters of apoptosis-like PCD in both species. We observed ultrastructural changes, an increase in reactive oxygen species, phosphatidylserine externalization and a decrease in intracellular potassium, while DNA degradation was evaluated using the TUNEL assay and agarose gels, and all of these parameters are related to PCD. Finally, we analysed the expression of apoptosis-related genes, such as sir2 and atg8, in N. gruberi. Taken together, our results showed that AmB induces the morphological, biochemical and genetic changes of apoptosis-like PCD in the genus Naegleria.

Naegleria fowleri: enolase is expressed during cyst differentiation.

Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.

[Molecular diagnosis of a fatal primary amoebic meningoencephalitis in Guadeloupe (French West Indies)]. / Diagnostic moléculaire d'une méningoencéphalite amibienne primitive à l'occasion d'un cas fatal en Guadeloupe.

We report the first case of primary amoebic meningoencephalitis in a 9-year-old boy in Guadeloupe. The outcome was rapidly fatal in 7 days. The patient presumably acquired the infection by swimming and diving in a basin supplied by natural thermal water 1 week before onset of the disease. The possibility of a free-living amoeba infection was suspected both on the negativity of all bacterial and viral initial tests and on the observation of peculiar cells in stained cerebrospinal fluid samples. Although the amoeba was not isolated, Naegleria fowleri could be identified by polymerase chain reaction with specific primers on DNA extracted from frozen cerebrospinal fluid samples. Furthermore, as the internal transcribed spacer (ITS1) region of DNA is variable in length between the different strains of N. fowleri, sequencing of the amplified ITS1 demonstrated that the responsible N. fowleri strain belongs to a common genotype present in the American and European continent.

Successful treatment of Naegleria fowleri meningoencephalitis by using intravenous amphotericin B, fluconazole and rifampicin.

BACKGROUND: Primary amebic meningoencephalitis (PAM) is an emerging disease with a rapidly fatal outcome. Only eight reports of cured cases have appeared in the medical literature to date. METHODS: A 10-year-old boy developed PAM caused by Naegleria fowleri 1 week after swimming in an irrigation canal. He was admitted to our hospital after 9 h of severe headache and vomiting, fever, ataxic gait, mild confusion, and seizures were evident. Trophozoites were identified in the cerebrospinal fluid (CSF). Treatment with intravenous (i.v.) dexamethasone, amphotericin B, fluconaloze, and oral rifampicin was started. After several hours of conflicting clinical signs, recovery began, and on the third day he was conscious again. Hospital discharge occurred on day 23, after a normal brain CT scan. There was no sequel to the disease during the following 12 months. RESULTS: The amebas present in the CSF were identified and confirmed as N. fowleri after observation of wet mounts and of cultures seeded on 1.5% non-nutrient agar plates covered with Escherichia coli, vegetative and cystic forms, enflagellation experiments in distilled water at 98 degrees F, temperature tolerance testing and by indirect immunofluorescence using N. fowleri LEE antibody. The genotype was determined by PCR amplification and sequencing of the internal transcribed spacers (ITS) including the 5.8S rDNA. CONCLUSIONS: Early treatment of PAM by i.v. administration of amphotericin B and fluconazole, and oral administration of rifampicin can offer some hope of cure for this devastating disease.