Byoung-Kuk Na

Critical roles for excretory–secretory cysteine proteases during tissue invasion of Paragonimus westermani newly excysted metacercariae

Paragonimus westermani is a trematode parasite, which causes pulmonary and/or extrapulmonary granulomatous disease in humans. Successful invasion of the host tissue is critical for the survival of this tissue‐invasive parasite. The enzymatic hydrolysis of host proteins is clearly a prerequisite of this process. In this study, we have investigated the functional roles of the excretory–secretory cysteine proteases of P. westermani newly excysted metacercariae (PwNEM) in tissue invasion. The 27 and 28 kDa enzymes (PwMc27 and PwMc28) purified from PwNEM excretory–secretory products (ESP), preferentially degraded fibrillar proteins, but not globular proteins. PwMc28 significantly facilitated the invasion of PwNEM into mouse peritoneum, whereas a diffusible cysteine protease inhibitor, trans‐epoxysuccinyl‐l‐leuciloamido‐(4‐guanidino) butane (E‐64) inhibited this process dose‐dependently. Two distinct isoforms of PwMc28 (PwMc28a and PwMc28b), which exhibited two amino acid differences in their mature domains, were identified by tandem mass spectrometry and sequence analysis. Both enzymes were localized at the tegument on the anterior border and on the oral sucker, which suggests excretion–secretion via exocytosis or via the excretory canal network. The mRNA transcripts of PwMc28a and b were expressed abundantly during the active invasion/migration through the host’s tissues, suggesting their relevant function to tissue invasion/migration in the definitive host.

High frequency of genetic diversity of Plasmodium vivax field isolates in Myanmar.

Malaria is one of the most serious problems threatening human health in Myanmar. Although the morbidity and mortality rates due to malaria have been gradually declining, Myanmar still contributes to a large proportion of malarial death in the South-East Asia region. However, little is known about the nature and extent of genetic diversity of the malarial parasites circulating in Myanmar. In this study, we investigated the overall infection status of Plasmodium and the population diversity of Plasmodium vivax by analyzing three genetic markers, circumsporozoite protein (CSP), merozoite surface protein-1 (MSP-1), and merozoite surface protein-3 (MSP-3alpha), of P. vivax field isolates collected from infected individuals. In 349 blood samples collected from the individuals who exhibited clinical symptoms associated with malaria, 63.0% showed a positive result for malaria (220/349). P. vivax was detected in 58.2% (128/220) and Plasmodium falciparum was detected in 29.1% (64/220). Mixed infections with both parasites were detected in 12.7% (28/220). The 116 blood samples in which single infection of P. vivax was confirmed were selected and subjected to further genetic analysis. Genotyping of the CSP gene of P. vivax showed that VK210 type (98.3%, 114/116) is predominant in Myanmar, but a significant level of mixed infections of VK210 and VK247 types (24.1%, 28/116) was also identified. Sequence analyses of MSP-1 and MSP-3alpha genes revealed a large number of distinguishable alleles: 12 for MSP-1 and 25 for MSP-3alpha. These results collectively suggest that the P. vivax population in Myanmar is highly diverse and multiple clonal infections are prevalent in the country.

A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite.

Cysteine proteases of helminth parasites play essential roles in parasite physiology as well as in a variety of important pathobiological processes. In this study, we identified a multigene family of cathepsin F cysteine proteases in Clonorchis sinensis (CsCFs). We identified a total of 12 CsCF genes through cDNA cloning using degenerate PCR primers followed by RACE. Sequence and phylogenetic analysis of the genes suggested they belonged to the cathepsin F-like enzyme family and further clustered into three different subfamilies. Enzymatic and proteomic analysis of C. sinensis excretory and secretory products (ESP) revealed that multiple isoforms of CsCF were the major proteins present in the ESP and the proteolytic activity of the ESP is mainly attributable to the enzymes. Comparative analysis of representative enzymes for each subfamily, CsCF-4, CsCF-6, and CsCF-11, showed that they share similar biochemical properties typical for cathepsin F-like enzymes, but significant differences were also identified. The enzymes were expressed throughout various developmental stages of the parasite and the transcripts increased gradually in accordance with the maturation of the parasite. Immunolocalization analysis of CsCFs showed that they were mainly localized in the intestine and intestinal contents of the parasite. These results collectively suggested that CsCFs, which are apparently synthesized in the epithelial cells lining the parasite intestine and secreted into the intestinal lumen of the parasite, might have a cooperative role for nutrient uptake in the parasite. Furthermore, they were eventually secreted into outside of the parasite and may perform additional functions for host-parasite interactions.

Evaluation of Clonorchis sinensis Recombinant 7-Kilodalton Antigen for Serodiagnosis of Clonorchiasis

ABSTRACT The diagnostic applicability of the Clonorchis sinensis recombinant 7-kDa protein was evaluated. In enzyme-linked immunosorbent assays and immunoblots, the protein showed high sensitivities (81.3 and 71.9%, respectively) and specificities (92.6 and 89.7%, respectively) for sera obtained from various helminthic infections. Some paragonimiasis sera showed cross-reactions. The antigen might be valuable in the serodiagnosis of human clonorchiasis.

Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

ABSTRACT The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzymes primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

Genetic variations of the dihydrofolate reductase gene of Plasmodium vivax in Mandalay Division, Myanmar.

Dihydrofolate reductase (DHFR; EC1.5.1.3) is a known target enzyme for antifolate agents, which are used as alternative chemotherapeutics for chloroquine-resistant malaria. Mutations in the dhfr gene of Plasmodium vivax are thought to be associated with resistance to the antifolate drugs. In this study, we have analyzed genetic variations in the dhfr genes of clinical isolates of P. vivax (n=21) in Myanmar, to monitor antifolate resistance in this country. Sequence variations within the entire dhfr gene were highly restricted to codons from 57 to 117, and the GGDN tandem repeat region. Double (S58R and S117N/T) or quadruple mutations (F57L/I, S58R, T61M, and S117N/T), which may be closely related to the drug resistance, were recognized in most of the isolates (20/21 cases). Our results suggest that antifolate-resistant P. vivax is becoming widespread in Myanmar, as it also is in the neighboring countries in Southeast Asia. It appears that the drug resistance situation may be worsening in the country.

Parasitic Helminth Cystatin Inhibits DSS-Induced Intestinal Inflammation Via IL-

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-γ and TNF-α in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-α in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.

10^+F4/80^+

An extracellular proteinase of Acanthamoeba castellanii was purified and its biochemical and pathological properties were characterized. The molecular mass of the purified enzyme was ˜ 42 kDa as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Sephacryl S‐200 HR gel‐filtration chromatography. Therefore, its structure seemed to be monomeric with a single polypeptide. Its activity was inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. Its activity was optimum at 30 to 50 °C with a maximum at 50 °C; optimal pH was 8.0. As much as 70% of the enzyme activity was maintained at 50 °C for at least 12 h but was rapidly inactivated thereafter. The purified enzyme degraded collagen and rabbit corneal extract. Furthermore, it exhibited strong cytopathic effects on human corneal epithelial cells and fibroblast cells. These suggest the possible role of this enzyme in the pathogenesis of Acanthamoeba.

Macrophage Recruitment

A gene encoding cysteine proteinase from Clonorchis sinensis has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription–polymerase chain reaction using degenerate oligonucleotide primers derived from conserved active site of cysteine proteinases. The 5′ and 3′ regions of the gene were amplified using rapid amplification of cDNA ends. The cloned gene has an open reading frame of 696 bp and deduced amino acid sequence of 232. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form the catalytic triad. Analysis of the expressed protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa. Proteolytic activity of the expressed protein was inhibited by cysteine proteinase inhibitors such as l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, iodoacetic acid, and leupeptin. The expressed protein showed biochemical properties similar to those of cysteine proteinases of other parasites. The expressed protein strongly reacted with the sera from patients with clonorchiasis but not with the sera from patients with paragonimiasis, fascioliasis, cysticercosis, and sparganosis, or with sera from normal human controls. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of clonorchiasis.

Purification and characterization of an extracellular serine proteinase from Acanthamoeba castellanii.

Malaria is endemic or hypoendemic in Myanmar and the country still contributes to the high level of malaria deaths in South-East Asia. Although information on the nature and extent of population diversity within malaria parasites in the country is essential not only for understanding the epidemic situation but also to establish a proper control strategy, very little data is currently available on the extent of genetic polymorphisms of the malaria parasites in Myanmar. In this study, we analyzed the genetic polymorphism and natural selection at domain I of the apical membrane antigen-1 (AMA-1) among Plasmodium vivax Myanmar isolates. A total of 34 distinguishable haplotypes were identified among the 76 isolates sequenced. Comparison with the previously available PvAMA-1 sequences in the GenBank database revealed that 21 of them were new haplotypes that have never been reported till date. The difference between the rate of nonsynonymous (dN) and synonymous (dS) mutations was positive (dN-dS, 0.013+/-0.005), suggesting the domain I is under positive natural selection. The Tajimas D statistics was found to be -0.74652, suggesting that the gene has evolved under population size expansion and/or positive selection. The minimum recombination events were also high, indicating that recombination may occur within the domain I resulting in allelic diversity of PvAMA-1. Our results collectively suggest that PvAMA-1 displays high genetic polymorphism among Myanmar P. vivax isolates with highly diversifying selection at domain I. These results have significant implications in understanding the nature of P. vivax population circulating in Myanmar as well as providing useful information for malaria vaccine development based on this antigen.