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Myanmar aims to eliminate malaria by 2030. However, recent increase of malaria incidence is a great challenge to archive that goal. Increasing prevalence of Plasmodium vivax also hinders this endeavor. Monitoring genetic structure of the parasite is necessary to understand genetic nature and evolutionary aspect of P. vivax population in Myanmar. Partial fragment flanking blocks I and II of merozoite surface protein-3 alpha of P. vivax (pvmsp-3α) was amplified from P. vivax isolates collected in Pyin Oo Lwin, Mandalay Region, Myanmar in 2013-2015. Sequence analysis of pvmsp-3α was performed to determine genetic diversity and natural selection of this gene. Spatio-temporal genetic changes of pvmsp-3α in Myanmar P. vivax population were also investigated via comparative analysis of gene sequences obtained in this study and previously reported Myanmar pvmsp-3α sequences. Genetic diversity of Myanmar pvmsp-3α was detected in P. vivax isolates analyzed. Size polymorphisms in block I and amino acid changes and recombination events in block II were main factors contributing to the genetic diversity of pvmsp-3α. Comparative spatio-temporal analysis with previously reported Myanmar pvmsp-3α populations revealed the presence of genetic differences by population with moderate genetic differentiation between populations. Similar pattern of natural selection was also detected in Myanmar pvmsp-3α populations. These suggested that enough size of the P. vivax population sufficient to generate or maintain the genetic diversity remains in the population. Thus, continuous molecular surveillance of genetic structure of Myanmar P. vivax is necessary.
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Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 µg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.
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Naegleria fowleri , Pinus , Extratos Vegetais , Folhas de Planta , Naegleria fowleri/efeitos dos fármacos , Naegleria fowleri/genética , Extratos Vegetais/farmacologia , Pinus/química , Folhas de Planta/química , Animais , Ratos , Antiprotozoários/farmacologia , Linhagem Celular , Trofozoítos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/parasitologia , Encéfalo/metabolismo , Encéfalo/patologia , Perfilação da Expressão Gênica , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Concentração Inibidora 50 , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Acanthamoeba is an opportunistic pathogen that can cause human infections such as granulomatous amebic encephalitis and acanthamoeba keratitis. However, no specific drug to treat the diseases has been developed. Therefore, the discovery or development of novel drugs for treating Acanthamoeba infections is urgently needed. The anti-protozoan activity of (â)-epicatechin (EC) has been reported, suggesting it is an attractive anti-protozoal drug candidate. In this study, the amoebicidal activity of EC against A. castellanii was assessed and its mechanism of action was unveiled. METHODS: The amoebicidal activity of EC against A. castellanii trophozoites and the cytotoxicity of EC in HCE-2 and C6 cells were determined with cell viability assay. The underlying amoebicidal mechanism of EC against A. castellanii was analyzed by the apoptosis/necrosis assay, TUNEL assay, mitochondrial dysfunction assay, caspase-3 assay, and quantitative reverse transcription polymerase chain reaction. The cysticidal activity of EC was also investigated. RESULTS: EC revealed amoebicidal activity against A. castellanii trophozoites with an IC50 of 37.01 ± 3.96 µM, but was not cytotoxic to HCE-2 or C6 cells. EC induced apoptotic events such as increases in DNA fragmentation and intracellular reactive oxygen species production in A. castellanii. EC also caused mitochondrial dysfunction in the amoebae, as evidenced by the loss of mitochondrial membrane potential and reductions in ATP production. Caspase-3 activity, autophagosome formation, and the expression levels of autophagy-related genes were also increased in EC-treated amoebae. EC led to the partial death of cysts and the inhibition of excystation. CONCLUSION: EC revealed promising amoebicidal activity against A. castellanii trophozoites via programmed cell death events. EC could be a candidate drug or supplemental compound for treating Acanthamoeba infections.
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Acanthamoeba castellanii , Amebíase , Amebicidas , Catequina , Dieldrin/análogos & derivados , Doenças Mitocondriais , Animais , Humanos , Amebicidas/farmacologia , Amebicidas/uso terapêutico , Caspase 3 , Catequina/farmacologia , Amebíase/tratamento farmacológico , Trofozoítos , Apoptose , Doenças Mitocondriais/tratamento farmacológicoRESUMO
Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is one of the most common X-linked hereditary disorders worldwide. G6PD deficiency provides resistance against severe malaria, but paradoxically, G6PD deficiency is also a stumbling block in fighting against malaria. Primaquine (PQ), a drug for the radical cure of Plasmodium vivax, can cause lethal acute hemolytic anemia in malaria patients with inherited G6PD deficiency. In this study, we analyzed the phenotypic and genotypic G6PD deficiency status in 1721 individuals (963 males and 758 females) residing in three malaria-endemic areas within the Gia Lai province, Vietnam. The G6PD activity in individuals ranged from 3.04 to 47.82 U/g Hb, with the adjusted male median (AMM) of 7.89 U/g Hb. Based on the G6PD activity assay results, no phenotypic G6PD deficiency was detected. However, the multiplex polymerase chain reaction to detect G6PD variations in the gene level revealed that 26 individuals (7 males, 19 females) had Viangchan mutations (871 G > A). Sequencing analyses suggested that all the males were hemizygous Viangchan, whereas one was homozygous, and 18 were heterozygous Viangchan in females. These results suggested a relatively low prevalence of G6PD deficiency mutation rate (1.51%) in the minor ethnic populations residing in the Gia Lai province, Vietnam. However, considering these areas are high-risk malaria endemic, concern for proper and safe use of PQ as a radical cure of malaria is needed by combining a G6PD deficiency test before PQ prescription.
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Antimaláricos , Deficiência de Glucosefosfato Desidrogenase , Malária Vivax , Malária , Feminino , Humanos , Masculino , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/uso terapêutico , Prevalência , Vietnã/epidemiologia , Primaquina/uso terapêutico , Malária/tratamento farmacológico , Malária Vivax/epidemiologia , Malária Vivax/tratamento farmacológico , Antimaláricos/efeitos adversosRESUMO
Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established for FLA infections of the CNS, emphasizing the urgent need to discover or develop safe and effective drugs. Flavonoids, natural compounds from plants and plant-derived products, are known to have antiprotozoan activities against several pathogenic protozoa parasites. The anti-FLA activity of flavonoids has also been proposed, while their antiamoebic activity for FLA needs to be emperically determined. We herein evaluated the antiamoebic activities of 18 flavonoids against Naegleria fowleri and Acanthamoeba species which included A. castellanii and A. polyphaga. These flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. Demethoxycurcumin, kaempferol, resveratrol, and silybin (A+B) showed in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species. Apigenin, costunolide, (â)-epicatechin, (â)-epigallocatechin, rosmarinic acid, and (â)-trans-caryophyllene showed selective antiamoebic activity for Acanthamoeba species. Luteolin was more effective for N. fowleri. However, afzelin, berberine, (±)-catechin, chelerythrine, genistein, (+)-pinostrobin, and quercetin did not exhibit antiamoebic activity against the amoeba species. They neither showed selective antiamoebic activity with significant cytotoxicity to C6 glial cells. Our results provide a basis for the anti-FLA activity of flavonoids, which can be applied to develope alternative or supplemental therapeutic agents for FLA infections of the CNS.
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Acanthamoeba , Amebíase , Amoeba , Naegleria fowleri , Humanos , Flavonoides/farmacologia , Amebíase/tratamento farmacológicoRESUMO
Plasmodium falciparum erythrocyte binding antigen 175 (PfEBA-175) plays essential role in erythrocyte invasion by the parasite and is a leading vaccine candidate. However, its genetic diversity in global isolates is a concern in developing an universal vaccine incorporating this protein. This study aimed to investigate genetic polymorphisms and natural selection of pfeba-175 region II (RII) in Myanmar and Vietnam P. falciparum isolates. Vietnam pfeba-175 RII displayed a low genetic polymorphism, while Myanmar pfeba-175 RII showed high levels of genetic diversity across the region. Point mutations, deletion, and recombinations were main factors contributing to genetic diversities in P. falciparum populations. Global pfeba-175 RII revealed similar, but not identical, genetic polymorphisms and natural selection profiles. Despite profiles of amino acid substitutions differed among populations, five major amino acid changes (K279E, E403K, K481I, Q584K, and R664) were commonly detected in global pfeba-175 RII populations. Haplotype network and genetic differentiation analyses of global pfeba-175 RII populations demonstrated no geographical relationships. Non-neglectable level of genetic diversity was observed in global pfeba-175 RII populations, emphasizing the need to consider this when designing an effective vaccine based on this protein. This study underscores the importance of the continuous monitoring of genetic diversity of pfeba-175 RII in the global P. falciparum populations.
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Malária Falciparum , Vacinas , Humanos , Plasmodium falciparum/metabolismo , Mianmar , Vietnã , Proteínas de Protozoários/metabolismo , Antígenos de Protozoários , Polimorfismo Genético , Malária Falciparum/parasitologia , Seleção Genética , Eritrócitos/metabolismo , Variação GenéticaRESUMO
Extracellular vesicles (EVs) of protozoan parasites have diverse biological functions that are essential for parasite survival and host-parasite interactions. In this study, we characterized the functional properties of EVs from Naegleria fowleri, a pathogenic amoeba that causes a fatal brain infection called primary amoebic meningoencephalitis (PAM). N. fowleri EVs (NfEVs) have been shown to be internalized by host cells such as C6 glial cells and BV-2 microglial cells without causing direct cell death, indicating their potential roles in modulating host cell functions. NfEVs induced increased expression of proinflammatory cytokines and chemokines such as TNF-α, IL-1α, IL-1ß, IL-6, IL-17, IFN-γ, MIP-1α, and MIP-2 in BV-2 microglial cells; these increases were initiated via MyD88-dependent TLR-2/TLR-4. The production levels of proinflammatory cytokines and chemokines in NfEVs-stimulated BV-2 microglial cells were effectively downregulated by inhibitors of MAPK, NF-κB, or JAK-STAT. Phosphorylation levels of JNK, p38, ERK, p65, JAK-1, and STAT3 were increased in NfEVs-stimulated BV-2 microglial cells but were effectively suppressed by each corresponding inhibitor. These results suggest that NfEVs could induce proinflammatory immune responses in BV-2 microglial cells via the NF-κB-dependent MAPK and JAK-STAT signaling pathways. Taken together, these findings suggest that NfEVs are pathogenic factors involved in the contact-independent pathogenic mechanisms of N. fowleri by inducing proinflammatory immune responses in BV-2 microglial cells, further contributing to deleterious inflammation in infected foci by activating subsequent inflammation cascades in other brain cells.
Assuntos
Antígenos de Grupos Sanguíneos , Vesículas Extracelulares , Naegleria fowleri , Microglia , NF-kappa B , Citocinas , ImunidadeRESUMO
BACKGROUND: Naegleria fowleri is a brain-eating amoeba causing a fatal brain infection called primary amoebic meningoencephalitis (PAM). Despite its high mortality over 95%, effective therapeutic drug for PAM has not been developed yet. Therefore, development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated anti-amoebic effect of kaempferol (KPF) against N. fowleri and its underlying anti-amoebic molecular mechanisms. METHODS: Anti-amoebic activity of KPF against N. fowleri trophozoites, as well as cytotoxicity of KPF in C6 glial cells and CHO-K1 cells were investigated. The programmed cell death mechanisms in KPF-treated N. fowleri were also analyzed by apoptosis-necrosis assay, mitochondrial dysfunction assay, TUNEL assay, RT-qPCR, and CYTO-ID assay. RESULTS: KPF showed anti-amoebic activity against N. fowleri trophozoites with an IC50 of 29.28 ± 0.63 µM. However, it showed no significant cytotoxicity to mammalian cells. KPF induced significant morphological alterations of the amoebae, resulting in death. Signals associated with apoptosis were detected in the amoebae upon treatment with KPF. KPF induced an increase of intracellular reactive oxygen species level, loss of mitochondrial membrane potential, increases of expression levels of genes associated with mitochondria dysfunction, and reduction of ATP levels in the amoebae. Autophagic vacuole accumulations with increased expression levels of autophagy-related genes were also detected in KPF-treated amoebae. CONCLUSION: KPF induces programmed cell death in N. fowleri trophozoites via apoptosis-like pathway and autophagy pathway. KPF could be used as a candidate of anti-amoebic drug or supplement compound in the process of developing or optimizing therapeutic drug for PAM.
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Naegleria fowleri , Animais , Quempferóis/farmacologia , Apoptose , Necrose , Autofagossomos , MamíferosRESUMO
Emergence and spreading of antimalarial drug resistant malaria parasites are great hurdles to combating malaria. Although approaches to investigate antimalarial drug resistance status in Myanmar malaria parasites have been made, more expanded studies are necessary to understand the nationwide aspect of antimalarial drug resistance. In the present study, molecular epidemiological analysis for antimalarial drug resistance genes in Plasmodium falciparum and P. vivax from the Mandalay region of Myanmar was performed. Blood samples were collected from patients infected with P. falciparum and P. vivax in four townships around the Mandalay region, Myanmar in 2015. Partial regions flanking major mutations in 11 antimalarial drug resistance genes, including seven genes (pfdhfr, pfdhps, pfmdr-1, pfcrt, pfk13, pfubp-1, and pfcytb) of P. falciparum and four genes (pvdhfr, pvdhps, pvmdr-1, and pvk12) of P. vivax were amplified, sequenced, and overall mutation patterns in these genes were analyzed. Substantial levels of mutations conferring antimalarial drug resistance were detected in both P. falciparum and P. vivax isolated in Mandalay region of Myanmar. Mutations associated with sulfadoxine-pyrimethamine resistance were found in pfdhfr, pfdhps, pvdhfr, and pvdhps of Myanmar P. falciparum and P. vivax with very high frequencies up to 90%. High or moderate levels of mutations were detected in genes such as pfmdr-1, pfcrt, and pvmdr-1 associated with chloroquine resistance. Meanwhile, low frequency mutations or none were found in pfk13, pfubp-1, pfcytb, and pvk12 of the parasites. Overall molecular profiles for antimalarial drug resistance genes in malaria parasites in the Mandalay region suggest that parasite populations in the region have substantial levels of mutations conferring antimalarial drug resistance. Continuous monitoring of mutations linked with antimalarial drug resistance is necessary to provide useful information for policymakers to plan for proper antimalarial drug regimens to control and eliminate malaria in the country.
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The circumsporozoite surface protein of Plasmodium vivax (PvCSP) plays a critical role in parasite biology. It has been extensively studied as a leading vivax-malaria-vaccine candidate. In this study, the genetic polymorphism and natural selection of pvcsp in P. vivax isolates collected from the Central Highlands, Vietnam were analyzed to understand the genetic structure of the parasite circulating in the endemic area and to provide baseline information for effective vaccine development based on the protein. Only two major alleles, VK210 and VK247, were detected in Vietnamese pvcsp, with VK247 being the predominant one. The N-terminal and C-terminal regions of Vietnamese VK210 and VK247 variants showed a low genetic diversity. Amino acid substitutions, insertions of a single amino acid or octapeptide (ANKKAEDA in VK210 and ANKKAGDA in VK247), and tetrapeptide repeat motifs (GGNA) were the main factors generating genetic diversity in the two regions of the Vietnamese VK210 and VK247 variants. Interestingly, these two regions of Vietnamese pvcsp displayed a unique natural selection pressure distinct from global pvcsp, particularly with the neighboring Southeast Asian pvcsp population. Meanwhile, the central repeat region (CRR) in both the VK210 and VK247 variants showed a high degree of polymorphic characters, caused by varying numbers, types, and combinations of peptide repeat motifs (PRMs) in Vietnamese pvcsp. Highly complicated polymorphic patterns of the CRR were also detected in global pvcsp. These results expand our understanding of the genetic structure of Vietnamese pvcsp and the population dynamics of P. vivax in the Central Highlands, Vietnam.
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Naegleria fowleri is a ubiquitous protozoa parasite that can cause primary amoebic meningoencephalitis (PAM), a fatal brain infection in humans. Cathepsin Bs of N. fowleri (NfCBs) are multifamily enzymes. Although their pathogenic mechanism in PAM is not clearly understood yet, NfCBs have been proposed as pathogenic factors involved in the pathogenicity of amoeba. In this study, the immune response of BV-2 microglial cells induced by NfCB was analyzed. Recombinant NfCB (rNfCB) evoked enhanced expressions of TLR-2, TLR-4, and MyD88 in BV-2 microglial cells. This enzyme also induced an elevated production of several pro-inflammatory cytokines such as TNF-α, IL-1α, IL-1ß, and IL-6 and iNOS in cells. The inhibition of mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK, effectively reduced the production of these pro-inflammatory cytokines. The rNfCB-induced production of pro-inflammatory cytokines in BV-2 microglial cells was suppressed by inhibiting NF-kB and AP-1. Phosphorylation and nuclear translocation of p65 in cells were also enhanced by rNfCB. These results suggest that NfCB can induce a pro-inflammatory immune response in BV-2 microglial cells via the NF-κB- and AP-1-dependent MAPK signaling pathways. Such a NfCB-induced pro-inflammatory immune response in BV-2 microglial cells might contribute to the pathogenesis of PAM caused by amoeba, by exacerbating deleterious immune responses and tissue damages in N. fowleri-infected foci of the brain.
Assuntos
Naegleria fowleri , Catepsina B/metabolismo , Citocinas/metabolismo , Humanos , Imunidade , Lipopolissacarídeos/farmacologia , Microglia/metabolismo , NF-kappa B/metabolismo , Naegleria fowleri/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismoRESUMO
Heliminthic paramyosin is a multifunctional protein that not only acts as a structural protein in muscle layers but as an immune-modulatory molecule interacting with the host immune system. Previously, we found that paramyosin from Clonorchis sinensis (CsPmy) is bound to human complement C9 protein (C9). To analyze the C9 binding region on CsPmy, overlapping recombinant fragments of CsPmy were produced and their binding activity to human C9 was investigated. The fragmental expression of CsPmy and C9 binding assays revealed that the C9 binding region was located at the C-terminus of CsPmy. Further analysis of the C-terminus of CsPmy to narrow the C9 binding region on CsPmy indicated that the region flanking731Leu-780 Leu was a potent C9 binding region. The CsPmy fragments corresponding to the region effectively inhibited human C9 polymerization. These results provide a precise molecular basis for CsPmy as a potent immunomodulator to evade host immune defenses by inhibiting complement attack.
Assuntos
Clonorchis sinensis , Animais , Complemento C9/metabolismo , Humanos , Fatores Imunológicos , Tropomiosina/química , Tropomiosina/metabolismoRESUMO
BACKGROUND: Coccidiosis is a poultry disease that occurs worldwide and is caused by Eimeria species. The infection is associated with reduced feed efficiency, body weight gain, and egg production. This study aimed to investigate the current status of coccidiosis and anticoccidial resistance to anticoccidial drugs used as part of control strategies for this disease in Korean chicken farms. RESULTS: An overall prevalence of 75% (291/388) was found. Positive farms contained several Eimeria species (mean = 4.2). Of the positive samples, E. acervulina (98.6%), E. maxima (84.8%), and E. tenella (82.8%) were the most prevalent species. Compared with cage-fed chickens, broilers and native chickens reared in free-range management were more at risk of acquiring an Eimeria infection. Sensitivities to six anticoccidial drugs (clopidol, diclazuril, maduramycin, monensin, salinomycin, and toltrazuril) were tested using nine field samples. Compared with untreated healthy control chickens, the body weight gains of infected chickens and treated/infected chickens were significantly reduced in all groups. Fecal oocyst shedding was significantly reduced in four clopidol-treated/infected groups, three diclazuril-treated/infected groups, two toltrazuril-treated/infected groups, one monensin-treated/infected group, and one salinomycin-treated/infected group, compared with the respective untreated/infected control groups. Intestinal lesion scores were also reduced in three clopidol-treated/infected groups, one monensin-treated/infected group, and one toltrazuril-treated/infected group. However, an overall assessment using the anticoccidial index, percent optimum anticoccidial activity, relative oocyst production, and reduced lesion score index found that all field samples had strong resistance to all tested anticoccidial drugs. CONCLUSION: The results of this large-scale epidemiological investigation and anticoccidial sensitivity testing showed a high prevalence of coccidiosis and the presence of severe drug resistant Eimeria species in the field. These findings will be useful for optimizing the control of coccidiosis in the poultry industry.
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Coccidiose , Coccidiostáticos , Eimeria , Doenças das Aves Domésticas , Animais , Galinhas , Clopidol , Coccidiose/tratamento farmacológico , Coccidiose/epidemiologia , Coccidiose/veterinária , Coccidiostáticos/farmacologia , Coccidiostáticos/uso terapêutico , Resistência a Medicamentos , Fazendas , Monensin , Oocistos , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/epidemiologia , República da Coreia/epidemiologia , Aumento de PesoRESUMO
Cysteine proteases of malaria parasites have been recognized as potential targets in antimalarial drug development as they play pivotal roles in the biology of these parasites. However, strict regulation of their activities is also necessary to minimize or prevent deleterious damage to the parasite and the host. Previously, we have characterized falcipain family cysteine proteases of Plasmodium malariae, named as malapains (MPs). MPs are active hemoglobinases. They also may participate in the release of merozoites from mature schizonts by facilitating remodeling of erythrocyte skeleton proteins. In this study, we identified and characterized an endogenous inhibitor of cysteine protease of P. malariae (PmICP). PmICP shared similar structural and biochemical properties with ICPs from other Plasmodium species. Recombinant PmICP showed a broad range of inhibitory activities against diverse cysteine proteases such as falcipain family enzymes (MP-2, MP-4, VX-3, VX-4, and FP-3), papain, and human cathepsins B and L, with stronger inhibitory activities against falcipain family enzymes. The inhibitory activity of PmICP was not affected by pH. PmICP was thermo-labile, resulting in rapid loss of its inhibitory activity at a high temperature. PmICP effectively inhibited hemoglobin hydrolysis by MPs and regulated maturation of MPs, suggesting its role as a functional regulator of MPs.
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Aedes aegypti is an important mosquito vector transmitting diverse arboviral diseases in Myanmar. Pyrethroid insecticides have been widely used in Myanmar as the key mosquito control measure, but the efforts are constrained by increasing resistance. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) are related to pyrethroid resistance in Ae. aegypti. We analyzed the patterns and distributions of the kdr mutations in Ae. aegypti in the Mandalay area of Myanmar. The segment 6 regions of domains II and III of vgsc were separately amplified from individual Ae. aegypti genomic DNA via polymerase chain reaction. The amplified gene fragments were sequenced. High proportions of three major kdr mutations, including S989P (54.8%), V1016G (73.6%), and F1534C (69.5%), were detected in the vgsc of Ae. aegypti from all studied areas. Other kdr mutations, T1520I and F1534L, were also found. These kdr mutations represent 11 distinct haplotypes of the vgsc population. The S989P/V1016G/F1534C was the most prevalent, followed by S989P/V1016V and V1016G/F1534C. A quadruple mutation, S989P/V1016G/T1520I/F1534C, was also identified. High frequencies of concurrent kdr mutations were observed in vgsc of Myanmar Ae. aegypti, suggesting a high level of pyrethroid resistance in the population. These findings underscore the need for an effective vector control program in Myanmar.
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Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2-4 (MP2-4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.
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Apical membrane antigen-1 of Plasmodium falciparum (PfAMA-1) is a leading malaria vaccine candidate antigen. However, the genetic diversity of pfama-1 and associated antigenic variation in global P. falciparum field isolates are major hurdles to the design of an efficacious vaccine formulated with this antigen. Here, we analyzed the genetic structure and the natural selection of pfama-1 in the P. falciparum population of Vietnam. A total of 37 distinct haplotypes were found in 131 P. falciparum Vietnamese isolates. Most amino acid changes detected in Vietnamese pfama-1 were localized in the ectodomain, domains I, II, and III. Overall patterns of major amino acid changes in Vietnamese pfama-1 were similar to those of global pfama-1, but the frequencies of the amino acid changes slightly differed by country. Novel amino acid changes were also identified in Vietnamese pfama-1. Vietnamese pfama-1 revealed relatively lower genetic diversity than currently analyzed pfama-1 in other geographical regions, and suggested a distinct genetic differentiation pattern. Evidence for natural selection was detected in Vietnamese pfama-1, but it showed purifying selection unlike the global pfama-1 analyzed so far. Recombination events were also found in Vietnamese pfama-1. Major amino acid changes that were commonly identified in global pfama-1 were mainly localized to predicted B-cell epitopes, RBC-binding sites, and IUR regions. These results provide important information for understanding the genetic nature of the Vietnamese pfama-1 population, and have significant implications for the design of a vaccine based on PfAMA-1.
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Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Haplótipos , Malária Falciparum/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Seleção Genética , Antígenos de Protozoários/química , Humanos , Malária Falciparum/genética , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Proteínas de Membrana/química , Plasmodium falciparum/imunologia , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/química , Homologia de Sequência de Aminoácidos , VietnãRESUMO
Avian coccidiosis is a disease caused by members of the genus Eimeria. Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.
Assuntos
Antígenos de Protozoários/genética , Eimeria tenella/genética , Proteínas de Protozoários/genética , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Galinhas/parasitologia , Coccidiose/parasitologia , Feminino , Variação Genética , Micronema/genética , Micronema/metabolismo , Doenças das Aves Domésticas/parasitologia , Seleção Genética/genéticaRESUMO
Despite a significant decline in the incidence of malaria in Myanmar recently, malaria is still an important public health concern in the country. Although Plasmodium falciparum is associated with the highest incidence of malaria in Myanmar, the proportion of P. vivax cases has shown a gradual increase in recent years. The genetic diversity of P. vivax merozoite surface protein-1 block 5-6 (pvmsp-1 ICB 5-6) in the P. vivax population of Myanmar was analyzed to obtain a comprehensive insight into its genetic heterogeneity and evolutionary history. High levels of genetic diversity of pvmsp-1 ICB 5-6 were identified in the P. vivax isolates collected from Myanmar between 2013 and 2015. Thirty-nine distinct haplotypes of pvmsp-1 ICB 5-6 (13 for Sal I type, 20 for recombinant type, and 6 for Belem type) were found at the amino acid level. Comparative analyses of the genetic diversity of pvmsp-1 ICB 5-6 sequences in the recent (2013-2015) and the past (2004) P. vivax populations in Myanmar revealed genetic expansion of the pvmsp-1 ICB 5-6 in recent years, albeit with a declined incidence. The recent increase in the genetic heterogeneity of Myanmar pvmsp-1 ICB 5-6 is attributed to a combination of factors, including accumulated mutations and recombination. These results suggest that the size of the P. vivax population in Myanmar is sufficient to enable the generation and maintenance of genetic diversity, warranting continuous molecular surveillance of genetic variation in Myanmar P. vivax.
RESUMO
Vietnam achieved outstanding success against malaria in the last few decades. The mortality and morbidity of malaria in Vietnam have decreased remarkably in recent years, but malaria is still a major public health concern in the country, particularly in the Central Highlands region. In this study, molecular analyses of malaria parasites in the Central Highlands were performed to understand the population structure and genetic diversity of the parasites circulating in the region. Plasmodium falciparum (68.7%) and P. vivax (27.4%) along with mixed infections with P. falciparum/P. vivax (3.9%) were detected in 230 blood samples from patients with malaria. Allele-specific nested-polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of pfmsp-1, pfama-1, pvcsp, and pvmsp-1 revealed complex genetic makeup in P. falciparum and P. vivax populations of Vietnam. Substantial multiplicity of infection (MOI) was also identified, suggesting significant genetic diversity and polymorphism of P. falciparum and P. vivax populations in the Central Highlands of Vietnam. These results provide fundamental insight into the current patterns of dispersion and genetic nature of malaria parasites as well as for the development of malaria elimination strategies in the endemic region.