RESUMO
Free-living amoebae (FLA) are protozoa dispersed in different environments and are responsible for different infections caused to humans and other animals. Microorganisms such as Acanthamoeba spp., Vermamoeba sp., and Naegleria sp. are associated with diseases that affect the central nervous system, in addition to skin infections and keratitis, as occurs in the genus Acanthamoeba and with Vermamoeba vermiformis. Due to the concerns of these FLA in anthropogenic aquatic environments, this work aimed to identify these microorganisms present in waters of Porto Alegre, Brazil. One litre sample was collected in two watercourses during the summer of 2022 and inoculated onto non-nutrient agar plates containing heat-inactivated Escherichia coli. Polymerase chain reaction results indicated the presence of FLA of the genera Acanthamoeba, Vermamoeba, and Naegleria in the study areas. Genetic sequencing indicated the presence of V. vermiformis and Naegleria gruberi. These aquatic and anthropogenic environments can serve as a means of spread and contamination by FLA, which gives valuable information on public health in the city.
Assuntos
Acanthamoeba , Amoeba , Naegleria , Humanos , Animais , Amoeba/genética , Acanthamoeba/genética , Naegleria/genética , Filogenia , BrasilRESUMO
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.
Assuntos
Anfotericina B/farmacologia , Antiprotozoários/farmacologia , Apoptose/efeitos dos fármacos , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Naegleria fowleri/efeitos dos fármacos , Naegleria/efeitos dos fármacos , Naegleria/citologia , Naegleria/genética , Naegleria/crescimento & desenvolvimento , Naegleria fowleri/citologia , Naegleria fowleri/genética , Naegleria fowleri/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trofozoítos/efeitos dos fármacos , Trofozoítos/crescimento & desenvolvimentoRESUMO
Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target.
Assuntos
Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases , Naegleria/genética , Proteínas de Protozoários , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/sangue , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Naegleria/enzimologia , Domínios Proteicos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Selenium (Se) is an essential trace element primarily found in selenoproteins as the 21st amino acid (selenocysteine, Sec, or U). Selenoproteins play an important role in growth and proliferation and are typically involved in cellular redox balance. Selenocysteine is encoded by an in-frame UGA codon specified by a stem-loop structure, the Sec insertion sequence element (SECIS), which, in eukaryotes, is located in the 3'-untranslated region (UTR). The availability of the Naegleria gruberi (ATCC 30224) genome sequence and the use of this organism as a model system for the pathogenic amoeba N. fowleri allowed us to investigate the Sec incorporation pathway in this primitive eukaryote. Using bioinformatics tools, we identified gene sequences encoding PSTK (O-phosphoseryl-tRNA(Sec) kinase), SepSecS (O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase), SelD/SPS2 (selenophosphate synthetase), EFSec (selenocysteine-specific elongation factor) and SBP (SECIS binding protein). These findings were confirmed by RT-PCR and by sequencing. A potential tRNA(Ser)Sec (SelC) gene and a putative selenoprotein with sequence similarity to a mitochondrial thioredoxin reductase (TR3) were also identified. Our results show that the selenocysteine incorporation machinery is indeed present in N. gruberi. Interestingly, the SelD/SPS2 gene is 2214 bp in length and contains two distinct domains. The N-terminal region shows sequence similarity to predicted methyltransferase proteins, and the C-terminal region is homologous to prokaryotic SelD/SPS2. Our results suggest the possibility of novel selenoproteins.
Assuntos
Vias Biossintéticas/genética , Naegleria/genética , Naegleria/metabolismo , Selenocisteína/biossíntese , Selenoproteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biologia Computacional , Perfilação da Expressão Gênica , Genes de Protozoários , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The amoeboflagellate genus Naegleria includes a few species that are virulent in experimental animals. One of these species, Naegleria italica, has been isolated from the environment only in Italy and Australia. I report here the isolation of a strain of N. italica from a water sample collected in Peru. This broadens the occurrence of this species to encompass three different continents. This new N. italica isolate from Peru has the same ITS1, 5.8S rDNA and ITS2 sequence as that of the type strain from Italy and the isolate from Australia. From the same water body in Peru a Naegleria strain was isolated that differs from N. italica by only one additional base pair in the ITS2 sequence. The maximum growth temperature tolerated by this particular isolate is 40 degrees C, which is different from that of N. italica, which is 42 degrees C.