RESUMEN

Balamuthia mandrillaris is a pathogenic protozoan that causes a rare but almost always fatal infection of the central nervous system and, in some cases, cutaneous lesions. Currently, the genomic data for this free-living amoeba include the description of several complete mitochondrial genomes. In contrast, two complete genomes with draft quality are available in GenBank, but none of these have a functional annotation. In the present study, the complete genome of B. mandrillaris isolated from a freshwater artificial lagoon was sequenced and assembled, obtaining an assembled genome with better assembly quality parameter values than the currently available genomes. Afterward, the genome mentioned earlier, along with strains V039 and 2046, were subjected to functional annotation. Finally, comparative genomics analysis was performed, and it was found that homologous genes in the core genome potentially involved in the virulence of Acanthamoeba spp. and Trypanosoma cruzi. Moreover, eleven of fifteen genes were identified in the three strains described as potential target genes to develop new treatment approaches for B. mandrillaris infections. These results describe proteins in this protozoan's complete genome and help prioritize which target genes could be used to develop new treatments.

Asunto(s)

Acanthamoeba , Balamuthia mandrillaris , Balamuthia mandrillaris/genética , Virulencia/genética , Hibridación Genómica Comparativa , Acanthamoeba/genética , Genómica

RESUMEN

Free-living amoebae (FLA) are protozoa widely distributed in the environment, found in a great diversity of terrestrial biomes. Some genera of FLA are linked to human infections. The genus Acanthamoeba is currently classified into 23 genotypes (T1-T23), and of these some (T1, T2, T4, T5, T10, T12, and T18) are known to be capable of causing granulomatous amoebic encephalitis (GAE) mainly in immunocompromised patients while other genotypes (T2, T3, T4, T5, T6, T10, T11, T12, and T15) cause Acanthamoeba keratitis mainly in otherwise healthy patients. Meanwhile, Naegleria fowleri is the causative agent of an acute infection called primary amoebic meningoencephalitis (PAM), while Balamuthia mandrillaris, like some Acanthamoeba genotypes, causes GAE, differing from the latter in the description of numerous cases in patients immunocompetent. Finally, other FLA related to the pathologies mentioned above have been reported; Sappinia sp. is responsible for one case of amoebic encephalitis; Vermamoeba vermiformis has been found in cases of ocular damage, and its extraordinary capacity as endocytobiont for microorganisms of public health importance such as Legionella pneumophila, Bacillus anthracis, and Pseudomonas aeruginosa, among others. This review addressed issues related to epidemiology, updating their geographic distribution and cases reported in recent years for pathogenic FLA.

RESUMEN

Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.

Asunto(s)

Acanthamoeba castellanii/ultraestructura , Balamuthia mandrillaris/ultraestructura , Peroxinas/genética , Peroxisomas/ultraestructura , Acanthamoeba castellanii/enzimología , Acanthamoeba castellanii/genética , Balamuthia mandrillaris/enzimología , Balamuthia mandrillaris/genética , Catalasa/metabolismo , Microscopía Electrónica de Transmisión , Peroxinas/metabolismo , Peroxisomas/enzimología , Peroxisomas/genética , Filogenia

RESUMEN

Two amoeboid organisms were obtained from water samples taken from a thermal spring, "Agua Caliente", in Northwestern Mexico. The isolates were obtained when samples were cultivated at 37 °C on non-nutrient agar coated with Escherichia coli. The initial identification of the isolates was performed morphologically using light microscopy. The samples were found to have trophozoite morphology consistent with members of the genus Stenamoeba, a genus derived in 2007 from within the abolished polyphyletic genus Platyamoeba. Further analysis was performed by sequencing PCR products obtained using universal eukaryotic primers for the small subunit ribosomal ribonucleic acid (SSU rRNA) gene. Sequencing primers were designed to allow the comparison of the 18S rRNA gene sequences of the new isolates with previous sequences reported for Stenamoeba. Phylogenetic relationships among sequences from Stenamoeba were determined using Maximum Likelihood analysis. The results showed the two "Agua Caliente" sequences to be closely related, while clearly separating them from those of other Stenamoeba taxa. The degrees of sequence differentiation from other taxa were considered sufficient to allow us to propose that the Mexican isolates represent a new species.

RESUMEN

Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5 T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.

Asunto(s)

Acanthamoeba/genética , Acanthamoeba/patogenicidad , Genotipo , Acanthamoeba/enzimología , Animales , Perros , Células Epiteliales/parasitología , Células Epiteliales/patología , Células de Riñón Canino Madin Darby , Serina Proteasas/metabolismo , Proteínas de Uniones Estrechas/metabolismo

RESUMEN

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.

Asunto(s)

Amoeba/patogenicidad , Manantiales de Aguas Termales/parasitología , Acanthamoeba/clasificación , Acanthamoeba/genética , Acanthamoeba/aislamiento & purificación , Acanthamoeba/patogenicidad , Amoeba/clasificación , Amoeba/genética , Amoeba/aislamiento & purificación , Balamuthia mandrillaris/clasificación , Balamuthia mandrillaris/genética , Balamuthia mandrillaris/aislamiento & purificación , Balamuthia mandrillaris/patogenicidad , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Genotipo , Manantiales de Aguas Termales/química , Concentración de Iones de Hidrógeno , México , Naegleria fowleri/clasificación , Naegleria fowleri/genética , Naegleria fowleri/aislamiento & purificación , Naegleria fowleri/patogenicidad , Filogenia , Reacción en Cadena de la Polimerasa , Espectrofotometría , Temperatura

RESUMEN

The presence of free-living amoebae of the genera Naegleria, Acanthamoeba and Balamuthia, which contain pathogenic species for humans and animals, has been demonstrated several times and in different natural aquatic environments in the northwest of Mexico. With the aim of continuing the addition of knowledge about immunology of pathogenic free-living amoebae, 118 sera from children and adolescents, living in three villages, were studied. Humoral IgG response against B. mandrillaris, N. fowleri and Acanthamoeba sp. genotype T4, was analyzed in duplicate to titers 1: 100 and 1: 500 by enzyme-linked immunosorbent assay (ELISA). Children and adolescents ages ranged between 5 and 16 years old, with a mean of 9 years old, 55% males. All tested sera were positive for the 1: 100 dilution, and in the results obtained with the 1: 500 dilution, 116 of 118 (98.3%) were seropositive for N. fowleri, 101 of 118 (85.6%) were seropositive for Acanthamoeba sp. genotype T4, and 43 of 118 (36.4%) were seropositive for B. mandrillaris. The statistical analysis showed different distributions among the three communities and for the three species of pathogenic free-living amoebae, including age. Lysed and complete cells used as Balamuthia antigens gave differences in seropositivity.

Asunto(s)

Acanthamoeba/inmunología , Anticuerpos Antiprotozoarios/sangre , Balamuthia mandrillaris/inmunología , Infecciones Protozoarias del Sistema Nervioso Central/epidemiología , Naegleria fowleri/inmunología , Adolescente , Amebiasis/epidemiología , Amebiasis/inmunología , Infecciones Protozoarias del Sistema Nervioso Central/inmunología , Infecciones Protozoarias del Sistema Nervioso Central/parasitología , Niño , Preescolar , Ecosistema , Encefalitis/epidemiología , Encefalitis/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/sangre , Masculino , México/epidemiología , Población Rural

RESUMEN

Since the first report of Balamuthia mandrillaris as a causative agent of granulomatous amoebic encephalitis in humans, the environmental niche of this amoeba was assumed to be restricted to soil and dust. A single isolation from water was recently made independently by us from Northern Mexico. Now we report the isolation of 8 new strains of B. mandrillaris from Mexico. This continues the pattern of an excess of isolates from North America, compared to other parts of the world. All of the new isolates are environmental isolates, 7 from water samples and one from soil. The identity of each isolate was confirmed by PCR and by examining the sequences of the mitochondrial 16S-like rRNA gene. Success in amplification was determined using comparisons of amplifications of DNA from the strain CDC: V039 and the water strain (ITSON-BM1) as positive controls. The DNA sequences of the new isolates were compared to older strains from clinical cases using phylogenetic analysis, showing very high sequence similarity. The similarity among the new isolates and with previous clinical and environmental isolates of B. mandrillaris was also examined using biochemical and immunological studies. High homogeneity of total protein products, and similarity in antigenic moiety among the eight new isolates and two controls was found. Taken together, the molecular and biochemical studies indicate very low levels of genetic variation within B. mandrillaris.

Asunto(s)

Balamuthia mandrillaris/genética , Agua Dulce/parasitología , Proteínas Protozoarias/química , Suelo/parasitología , Balamuthia mandrillaris/clasificación , Balamuthia mandrillaris/aislamiento & purificación , Western Blotting , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , México , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN