BACKGROUNDNaegleria 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. METHODSAnti-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. RESULTSKPF 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. CONCLUSIONKPF 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.
Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is one of the most common X-linked recessive hereditary disorders in the world. Primaquine (PQ) has been used for radical cure of P. ...vivax to prevent relapse. Recently, it is also used to reduce P. falciparum gametocyte carriage to block transmission. However, PQ metabolites oxidize hemoglobin and generate excessive reactive oxygen species which can trigger acute hemolytic anemia in malaria patients with inherited G6PD deficiency.
A total of 252 blood samples collected from malaria patients in Myanmar were used in this study. G6PD variant was analysed by a multiplex allele specific PCR kit, DiaPlexC™ G6PD Genotyping Kit Asian type. The accuracy of the multiplex allele specific PCR was confirmed by sequencing analysis.
Prevalence and distribution of G6PD variants in 252 malaria patients in Myanmar were analysed. Six different types of G6PD allelic variants were identified in 50 (7 females and 43 males) malaria patients. The predominant variant was Mahidol (68%, 34/50), of which 91.2% (31/34) and 8.8% (3/34) were males and females, respectively. Other G6PD variants including Kaiping (18%, 9/50), Viangchan (6%, 3/50), Mediterranean (4%, 2/50), Union (2%, 1/50) and Canton (2%, 1/50) were also observed.
Results of this study suggest that more concern for proper and safe use of PQ as a radical cure of malaria in Myanmar is needed by combining G6PD deficiency test before PQ prescription. Establishment of a follow-up system to monitor potential PQ toxicity in malaria patients who are given PQ is also required.
Plasmodium falciparum merozoite surface protein-3 (PfMSP-3) is a target of naturally acquired immunity against P. falciparum infection and is a promising vaccine candidate because of its critical ...role in the erythrocyte invasion of the parasite. Understanding the genetic diversity of pfmsp-3 is important for recognizing genetic nature and evolutionary aspect of the gene in the natural P. falciparum population and for designing an effective vaccine based on the antigen.
Blood samples collected from P. falciparum-infected patients in Naung Cho and Pyin Oo Lwin, Myanmar, in 2015 were used in this study. The pfmsp-3 was amplified by polymerase chain reaction, cloned, and sequenced. Genetic polymorphism and natural selection of Myanmar pfmsp-3 were analysed using the programs DNASTAR, MEGA6, and DnaSP 5.10.00. Genetic diversity and natural selection of the global pfmsp-3 were also comparatively analysed.
Myanmar pfmsp-3 displayed 2 different alleles, 3D7 and K1. The 3D7 allelic type was predominant in the population, but genetic polymorphism was less diverse than for the K1 allelic type. Polymorphic characters in both allelic types were caused by amino acid substitutions, insertions, and deletions. Amino acid substitutions were mainly occurred at the alanine heptad repeat domains, whereas most insertions and deletions were found at the glutamate rich domain. Overall patterns of amino acid polymorphisms detected in Myanmar pfmsp-3 were similar in the global pfmsp-3 population, but novel amino acid changes were observed in Myanmar pfmsp-3 with low frequencies. Complicated patterns of natural selection and recombination events were predicted in the global pfmsp-3, which may act as major driving forces to maintain and generate genetic diversity of the global pfmsp-3 population.
Global pfmsp-3 revealed genetic polymorphisms, suggesting that the functional and structural consequences of the polymorphisms should be considered in designing a vaccine based on PfMSP-3. Further examination of genetic diversity of pfmsp-3 in the global P. falciparum population is necessary to gain in-depth insight for the population structure and evolutionary aspect of global pfmsp-3.
Plasmodium falciparum merozoite surface protein-1 (PfMSP-1) and -2 (PfMSP-2) are major blood-stage vaccine candidate antigens. Understanding the genetic diversity of the genes, pfmsp-1 and pfmsp-2, ...is important for recognizing the genetic structure of P. falciparum, and the development of an effective vaccine based on the antigens. In this study, the genetic diversities of pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum were analysed.
The pfmsp-1 block 2 and pfmsp-2 block 3 regions were amplified by polymerase chain reaction from blood samples collected from Myanmar patients who were infected with P. falciparum in 2013-2015. The amplified gene fragments were cloned into a T&A vector, and sequenced. Sequence analysis of Myanmar pfmsp-1 block 2 and pfmsp-2 block 3 was performed to identify the genetic diversity of the regions. The temporal genetic changes of both pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum population, as well as the polymorphic diversity in the publicly available global pfmsp-1 and pfmsp-2, were also comparatively analysed.
High levels of genetic diversity of pfmsp-1 and pfmsp-2 were observed in the Myanmar P. falciparum isolates. Twenty-eight different alleles of pfmsp-1 (8 for K1 type, 14 for MAD20 type, and 6 for RO33 type) and 59 distinct alleles of pfmsp-2 (18 for FC27, and 41 for 3D7 type) were identified in the Myanmar P. falciparum population in amino acid level. Comparative analyses of the genetic diversity of the Myanmar pfmsp-1 and pfmsp-2 alleles in the recent (2013-2015) and past (2004-2006) Myanmar P. falciparum populations indicated the dynamic genetic expansion of the pfmsp-1 and pfmsp-2 in recent years, suggesting that a high level of genetic differentiation and recombination of the two genes may be maintained. Population genetic structure analysis of the global pfmsp-1 and pfmsp-2 also suggested that a high level of genetic diversity of the two genes was found in the global P. falciparum population.
Despite the recent remarkable decline of malaria cases, the Myanmar P. falciparum population still remains of sufficient size to allow the generation and maintenance of genetic diversity. The high level of genetic diversity of pfmsp-1 and pfmsp-2 in the global P. falciparum population emphasizes the necessity for continuous monitoring of the genetic diversity of the genes for better understanding of the genetic make-up and evolutionary aspect of the genes in the global P. falciparum population.
Plasmodium lactate dehydrogenase (pLDH) is a major target in diagnosing the erythrocytic stage of malaria parasites because it is highly expressed during blood-stage parasites and is distinguished ...from human LDH. Rapid diagnostic tests (RDTs) for malaria use pLDH as a target antigen; however, genetic variations in pLDH within the natural population threaten the efficacy of pLDH-based RDTs.
Genetic polymorphisms of Plasmodium vivax LDH (PvLDH) and Plasmodium falciparum LDH (PfLDH) in Myanmar isolates were analysed by nucleotide sequencing analysis. Genetic polymorphisms and the natural selection of PvLDH and PfLDH were analysed using DNASTAR, MEGA6, and DnaSP ver. 5.10.00 programs. The genetic diversity and natural selection of global PvLDH and PfLDH were also analysed. The haplotype network of global PvLDH and PfLDH was constructed using NETWORK ver. 5.0.0.3. Three-dimensional structures of PvLDH and PfLDH were built with YASARA Structure ver. 18.4.24 and the impact of mutations on structural change and stability was evaluated with SDM ver. 2, CUPSAT and MAESTROweb.
Forty-nine PvLDH and 52 PfLDH sequences were obtained from Myanmar P. vivax and P. falciparum isolates. Non-synonymous nucleotide substitutions resulting in amino acid changes were identified in both Myanmar PvLDH and PfLDH. Amino acid changes were also identified in the global PvLDH and PfLDH populations, but they did not produce structural alterations in either protein. Low genetic diversity was observed in global PvLDH and PfLDH, which may be maintained by a strong purifying selection.
This study extends knowledge for genetic diversity and natural selection of global PvLDH and PfLDH. Although amino acid changes were observed in global PvLDH and PfLDH, they did not alter the conformational structures of the proteins. These suggest that PvLDH and PfLDH are genetically well-conserved in global populations, which indicates that they are suitable antigens for diagnostic purpose and attractive targets for drug development.
Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are ...major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed.
Blood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed.
Thirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions.
Myanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the strong selective pressure of host immunity on the PfAMA-1 gene. These results have significant implications in understanding the nature of Myanmar PfAMA-1 along with global PfAMA-1. They also provide useful information for the development of effective malaria vaccine based on this antigen.
Circumsporozoite surface protein (CSP) of malaria parasites has been recognized as one of the leading vaccine candidates. Clinical trials of vaccines for vivax malaria incorporating Plasmodium vivax ...CSP (PvCSP) have demonstrated their effectiveness in preventing malaria, at least in part. However, genetic diversity of pvcsp in the natural population remains a major concern.
A total of 171 blood samples collected from patients infected with Plasmodium vivax in Myanmar were analysed in this study. The pvcsp was amplified by polymerase chain reaction, followed by cloning and sequencing. Polymorphic characteristics and natural selection of pvcsp population in Myanmar were analysed using DNASTAR, MEGA6 and DnaSP programs. The polymorphic pattern and natural selection of publicly accessible global pvcsp sequences were also comparatively analysed.
Myanmar pvcsp sequences were divided into two subtypes VK210 and VK247 comprising 143 and 28 sequences, respectively. The VK210 subtypes showed higher levels of genetic diversity and polymorphism than the VK247 subtypes. The N-terminal non-repeat region of pvcsp displayed limited genetic variations in the global population. Different patterns of octapeptide insertion (ANKKAEDA in VK210 and ANKKAGDA in VK247) and tetrapeptide repeat motif (GGNA) were identified in the C-terminal region of global pvcsp population. Meanwhile, the central repeat region (CRR) of Myanmar and global pvcsp, both in VK210 and VK247 variants, was highly polymorphic. The high level of genetic diversity in the CRR has been attributed to the different numbers, types and combinations of peptide repeat motifs (PRMs). Interestingly, 27 and 5 novel PRMs were found in Myanmar VK210 and VK247 variants, respectively.
Comparative analysis of the global pvcsp population suggests a complex genetic profile of pvcsp in the global population. These results widen understanding of the genetic make-up of pvcsp in the global P. vivax population and provide valuable information for the development of a vaccine based on PvCSP.
Naegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. Cysteine proteases produced by the amoeba may play ...critical roles in the pathogenesis of infection. In this study, a novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was characterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells.
Recombinant fowlerstefin was expressed in Escherichia coli. The inhibitory activity of fowlerstefin against several cysteine proteases, including human cathepsins B and L, papain and NfCPB-L, was analyzed. Fowlerstefin-induced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay.
Fowlerstefin is a cysteine protease inhibitor with a monomeric structure, and belongs to the stefin family. Recombinant fowlerstefin effectively inhibited diverse cysteine proteases including cathepsin B-like cysteine proteases of N. fowleri (NfCPB-L), human cathepsins B and L, and papain. Expression of fowlerstefin in the amoeba was optimal during the trophozoite stage and gradually decreased in cysts. Fowlerstefin induced an inflammatory response in BV-2 microglial cells. Fowlerstefin induced the expression of several pro-inflammatory cytokines and chemokines including IL-6 and TNF in BV-2 microglial cells. Fowlerstefin-induced expression of IL-6 and TNF in BV-2 microglial cells was regulated by mitogen-activated protein kinase (MAPKs). The inflammatory response induced by fowlerstefin in BV-2 microglial cells was downregulated via inhibition of NF-κB and AP-1.
Fowlerstefin is a pathogenic molecule that stimulates BV-2 microglial cells to produce pro-inflammatory cytokines through NF-κB- and AP-1-dependent MAPK signaling pathways. Fowlerstefin-induced inflammatory cytokines exacerbate the inflammatory response in N. fowleri-infected areas and contribute to the pathogenesis of PAM.
Plasmodium falciparum circumsporozoite protein (PfCSP) is one of the most extensively studied malaria vaccine candidates, but the genetic polymorphism of PfCSP within and among the global P. ...falciparum population raises concerns regarding the efficacy of a PfCSP-based vaccine efficacy. In this study, genetic diversity and natural selection of PfCSP in Myanmar as well as global P. falciparum were comprehensively analysed.
Blood samples were collected from 51 P. falciparum infected Myanmar patients. Fifty-one full-length PfCSP genes were amplified from the blood samples through a nested polymerase chain reaction, cloned into a TA cloning vector, and then sequenced. Polymorphic characteristics and natural selection of Myanmar PfCSP were analysed using the DNASTAR, MEGA6, and DnaSP programs. Polymorphic diversity and natural selection in publicly available global PfCSP were also analysed.
The N-terminal and C-terminal non-repeat regions of Myanmar PfCSP showed limited genetic variations. A comparative analysis of the two regions in global PfCSP displayed similar patterns of low genetic diversity in global population, but substantial geographic differentiation was also observed. The most notable polymorphisms identified in the N-terminal region of global PfCSP were A98G and 19-amino acid length insertion in global population with different frequencies. Major polymorphic characters in the C-terminal region of Myanmar and global PfCSP were found in the Th2R and Th3R regions, where natural selection and recombination occurred. The central repeat region of Myanmar PfCSP was highly polymorphic, with differing numbers of repetitive repeat sequences NANP and NVDP. The numbers of the NANP repeats varied among global PfCSP, with the highest number of repeats seen in Asian and Oceanian PfCSP. Haplotype network analysis of global PfCSP revealed that global PfCSP clustered into 103 different haplotypes with geographically-separated populations.
Myanmar and global PfCSP displayed genetic diversity. N-terminal and C-terminal non-repeat regions were relatively conserved, but the central repeat region displayed high levels of genetic polymorphism in Myanmar and global PfCSP. The observed geographic pattern of genetic differentiation and the points of evidence for natural selection and recombination suggest that the functional consequences of the polymorphism should be considered for developing a vaccine based on PfCSP.
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.
•A substantial level of genetic diversity is still maintained in Myanmar pvmsp-3α populations.•A sufficient size of P. vivax population contributing to genetic diversity remains in Myanmar.•Further study is needed to understand the genetic diversity and evolution of Myanmar P. vivax population.