Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.
Animals ; Aspartic Acid Endopeptidases ; Biology ; Cathepsin D ; Cathepsins ; Clonorchiasis ; Clonorchis sinensis ; Eggs ; Helminths ; Humans ; Intestines ; Metacercariae ; Ovum ; Parasites ; Peptide Hydrolases ; Rats ; Reproduction

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.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major candidate for the blood-stage malaria vaccine. Genetic polymorphisms of global pfama-1suggest that the genetic diversity of the gene can disturb effective vaccine development targeting this antigen. This study was conducted to explore the genetic diversity and gene structure of pfama-1 among P. falciparum isolates collected in the Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 19 full-length pfama-1 sequences were obtained from KP-Pakistan P. falciparum isolates, and genetic polymorphism and natural selection were investigated. KP-Pakistan pfama-1 exhibited genetic diversity, wherein 58 amino acid changes were identified, most of which were located in ectodomains, and domains I, II, and III. The amino acid changes commonly found in the ectodomain of global pfama-1 were also detected in KP-Pakistan pfama-1. Interestingly, 13 novel amino acid changes not reported in the global population were identified in KP-Pakistan pfama-1. KP-Pakistan pfama-1 shared similar levels of genetic diversity with global pfama-1. Evidence of natural selection and recombination events were also detected in KP-Pakistan pfama-1.

Plasmodium vivax variant interspersed repeats (vir) refer to the key protein used for escaping the host immune system. Knowledge in the genetic variation of vir genes can be used for the development of vaccines or diagnostic methods. Therefore, we evaluated the genetic diversity of the vir genes of P. vivax populations of several Asian countries, including Pakistan, which is a malaria-endemic country experiencing a significant rise in malaria cases in recent years. We analyzed the genetic diversity and population structure of 4 vir genes (vir 4, vir 12, vir 21, and vir 27) in the Pakistan P. vivax population and compared these features to those of the corresponding vir genes in other Asian countries. In Pakistan, vir 4 (S=198, H=9, Hd=0.889, Tajima’s D value=1.12321) was the most genetically heterogenous, while the features of vir 21 (S=8, H=7, Hd=0.664, Tajima’s D value =-0.63763) and vir 27 (S =25, H =11, Hd =0.682, Tajima’s D value=-2.10836) were relatively conserved. Additionally, vir 4 was the most genetically diverse among Asian P. vivax populations, although within population diversity was low. Meanwhile, vir 21 and vir 27 among all Asian populations were closely related genetically. Our findings on the genetic diversity of vir genes and its relationships between populations in diverse geographical locations contribute toward a better understanding of the genetic characteristics of vir. The high level of genetic diversity of vir 4 suggests that this gene can be a useful genetic marker for understanding the P. vivax population structure. Longitudinal genetic diversity studies of vir genes in P. vivax isolates obtained from more diverse geographical areas are needed to better understand the function of vir genes and their use for the development of malaria control measures, such as vaccines.

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.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major candidate for the blood-stage malaria vaccine. Genetic polymorphisms of global pfama-1suggest that the genetic diversity of the gene can disturb effective vaccine development targeting this antigen. This study was conducted to explore the genetic diversity and gene structure of pfama-1 among P. falciparum isolates collected in the Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 19 full-length pfama-1 sequences were obtained from KP-Pakistan P. falciparum isolates, and genetic polymorphism and natural selection were investigated. KP-Pakistan pfama-1 exhibited genetic diversity, wherein 58 amino acid changes were identified, most of which were located in ectodomains, and domains I, II, and III. The amino acid changes commonly found in the ectodomain of global pfama-1 were also detected in KP-Pakistan pfama-1. Interestingly, 13 novel amino acid changes not reported in the global population were identified in KP-Pakistan pfama-1. KP-Pakistan pfama-1 shared similar levels of genetic diversity with global pfama-1. Evidence of natural selection and recombination events were also detected in KP-Pakistan pfama-1.

Plasmodium vivax variant interspersed repeats (vir) refer to the key protein used for escaping the host immune system. Knowledge in the genetic variation of vir genes can be used for the development of vaccines or diagnostic methods. Therefore, we evaluated the genetic diversity of the vir genes of P. vivax populations of several Asian countries, including Pakistan, which is a malaria-endemic country experiencing a significant rise in malaria cases in recent years. We analyzed the genetic diversity and population structure of 4 vir genes (vir 4, vir 12, vir 21, and vir 27) in the Pakistan P. vivax population and compared these features to those of the corresponding vir genes in other Asian countries. In Pakistan, vir 4 (S=198, H=9, Hd=0.889, Tajima’s D value=1.12321) was the most genetically heterogenous, while the features of vir 21 (S=8, H=7, Hd=0.664, Tajima’s D value =-0.63763) and vir 27 (S =25, H =11, Hd =0.682, Tajima’s D value=-2.10836) were relatively conserved. Additionally, vir 4 was the most genetically diverse among Asian P. vivax populations, although within population diversity was low. Meanwhile, vir 21 and vir 27 among all Asian populations were closely related genetically. Our findings on the genetic diversity of vir genes and its relationships between populations in diverse geographical locations contribute toward a better understanding of the genetic characteristics of vir. The high level of genetic diversity of vir 4 suggests that this gene can be a useful genetic marker for understanding the P. vivax population structure. Longitudinal genetic diversity studies of vir genes in P. vivax isolates obtained from more diverse geographical areas are needed to better understand the function of vir genes and their use for the development of malaria control measures, such as vaccines.

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.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major candidate for the blood-stage malaria vaccine. Genetic polymorphisms of global pfama-1suggest that the genetic diversity of the gene can disturb effective vaccine development targeting this antigen. This study was conducted to explore the genetic diversity and gene structure of pfama-1 among P. falciparum isolates collected in the Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 19 full-length pfama-1 sequences were obtained from KP-Pakistan P. falciparum isolates, and genetic polymorphism and natural selection were investigated. KP-Pakistan pfama-1 exhibited genetic diversity, wherein 58 amino acid changes were identified, most of which were located in ectodomains, and domains I, II, and III. The amino acid changes commonly found in the ectodomain of global pfama-1 were also detected in KP-Pakistan pfama-1. Interestingly, 13 novel amino acid changes not reported in the global population were identified in KP-Pakistan pfama-1. KP-Pakistan pfama-1 shared similar levels of genetic diversity with global pfama-1. Evidence of natural selection and recombination events were also detected in KP-Pakistan pfama-1.

Plasmodium vivax variant interspersed repeats (vir) refer to the key protein used for escaping the host immune system. Knowledge in the genetic variation of vir genes can be used for the development of vaccines or diagnostic methods. Therefore, we evaluated the genetic diversity of the vir genes of P. vivax populations of several Asian countries, including Pakistan, which is a malaria-endemic country experiencing a significant rise in malaria cases in recent years. We analyzed the genetic diversity and population structure of 4 vir genes (vir 4, vir 12, vir 21, and vir 27) in the Pakistan P. vivax population and compared these features to those of the corresponding vir genes in other Asian countries. In Pakistan, vir 4 (S=198, H=9, Hd=0.889, Tajima’s D value=1.12321) was the most genetically heterogenous, while the features of vir 21 (S=8, H=7, Hd=0.664, Tajima’s D value =-0.63763) and vir 27 (S =25, H =11, Hd =0.682, Tajima’s D value=-2.10836) were relatively conserved. Additionally, vir 4 was the most genetically diverse among Asian P. vivax populations, although within population diversity was low. Meanwhile, vir 21 and vir 27 among all Asian populations were closely related genetically. Our findings on the genetic diversity of vir genes and its relationships between populations in diverse geographical locations contribute toward a better understanding of the genetic characteristics of vir. The high level of genetic diversity of vir 4 suggests that this gene can be a useful genetic marker for understanding the P. vivax population structure. Longitudinal genetic diversity studies of vir genes in P. vivax isolates obtained from more diverse geographical areas are needed to better understand the function of vir genes and their use for the development of malaria control measures, such as vaccines.