Haruki Uemura

Expression of trypomastigote trans-sialidase in metacyclic forms of Trypanosoma cruzi increases parasite escape from its parasitophorous vacuole.

Trypanosoma cruzi actively invades mammalian cells by forming parasitophorous vacuoles (PVs). After entry, the parasite has to escape from these vacuoles in order to replicate inside the host cell cytosol. Trans‐sialidase (TS), a parasite enzyme that is used to obtain sialic acid from host glycoconjugates, has been implicated in cell invasion and PV exit, but how the enzyme acts in these processes is still unknown. Here we show that trypomastigotes derived from infected mammalian cells express and release 20 times more TS activity than axenic metacyclic trypomastigotes, which correspond to the infective forms derived from the insect vector. Both forms have the same capacity to invade mammalian cells, but cell derived trypomastigotes exit earlier from the vacuole. To test whether high TS expression is responsible for this increased exit from the PV, trypomastigote TS was expressed on the surface of metacyclic forms. Transfected and non‐transfected metacyclics attached to and invaded HeLa or CHO cells equally. In contrast, metacyclics expressing TS on the surface escaped earlier from the vacuole than non‐transfected metacyclics, or metacyclics expressing TS in their cytoplasm. Sialic acid may act as a barrier, which is removed by surface and/or secreted TS, because all types of parasites escaped earlier from the vacuoles of sialic acid‐deficient Lec 2 cells than wild‐type CHO cells. In addition, trypomastigotes and metacyclic forms expressing TS differentiated earlier into amastigotes. These results indicate that the increased expression of TS in cell‐derived trypomastigotes is responsible for the earlier exit from the PV to the cytoplasm and their subsequent differentiation into amastigotes.

Differentiation of Entamoeba histolytica and E. dispar DNA from cysts present in stool specimens by polymerase chain reaction: its field application in the Philippines

Abstract It has been established that two distinct species exist within what was originally known as Entamoeba histolytica. These are E. dispar and E. histolytica, for the nonpathogenic and pathogenic forms, respectively. Differentiation of these two organisms is of great clinical importance since they are morphologically indistinguishable and both forms can infect the human intestinal cavity to different degrees. A simple and rapid DNA-extraction method that can be used directly on formalin-fixed stool specimens has been developed. The extracted DNA was used for the identification of the species existing in the stools by polymerase chain reaction (PCR). A total of 72 randomly collected stool samples from the Philippines were analyzed. In all, 19 samples reacted with E. dispar primers, resulting in the expected 101-bp PCR products; however, none reacted with E. histolytica primers. Furthermore, sensitivity assay suggests that genomic DNA from as few as five cysts can be used as a template for PCR. These observations imply that the use of genomic DNA directly extracted from formalin-fixed stool specimens for PCR amplification is a useful tool for obtaining a sensitive and accurate diagnosis that can be applied even in epidemiology studies.

Novel Mutations in K13 Propeller Gene of Artemisinin-Resistant Plasmodium falciparum

We looked for mutations in the Plasmodium falciparum K13 propeller gene of an artemisinin-resistant parasite on islands in Lake Victoria, Kenya, where transmission in 2012–2013 was high. The 4 new types of nonsynonymous, and 5 of synonymous, mutations we detected among 539 samples analyzed provide clues to understanding artemisinin-resistant parasites.

The use of the green fluorescent protein to monitor and improve transfection in Trypanosoma cruzi

Universidade Federal de Sao Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 Sao Paulo, Brazil

Malaria Parasites Giving Rise to Recrudescence In Vitro

ABSTRACT Recrudescences were simulated in vitro with drug treatment to examine how drug-sensitive parasites survive the treatment. Various numbers of cultured parasites were treated with lethal doses of pyrimethamine or mefloquine for various lengths of time. Recrudescences were observed in parasite populations with larger initial numbers of parasites when the treatment duration was prolonged. Equal numbers of parasitized erythrocytes were treated with various concentrations of pyrimethamine or mefloquine. There was no clear linear relationship between the incidence of recrudescence and the drug concentration. Parasites that had recrudesced were continuously allowed to recrudesce in the succeeding recrudescence experiments. Both the duration from the cessation of treatment to the time at which the recrudescent parasitemia level reached 1% and the growth rate of recrudescent parasites were equal among these recrudescences. The recrudescent parasites in these experiments were as sensitive to the drugs as the parasites tested before treatment were. These results suggest that a parasite culture may contain parasites in some phases that are not killed by drug for up to 10 days, which explains the recrudescences that occur even after treatment.

Organization of trans-sialidase genes in Trypanosoma cruzi.

Trypanosoma cruzi trans-sialidase is encoded by a family of genes containing a conserved region, which corresponds to the catalytic and amino-terminal domain of the enzyme. Most, but not all genes, also encode a variable region formed by 12 amino acid repeats at the carboxy-terminus of the protein that are not required for enzymatic activity. To design gene knock-out strategies and understand how trans-sialidase expression is regulated, we have studied the genome organization of trans-sialidase genes. We show here that the different types of trans-sialidase genes are distributed in more than one chromosomal band with sizes ranging from 0.8 to 1.5 Mb pairs in several T. cruzi strains. In the Y-strain, all repeat-containing genes are localized in one chromosomal band of 1.1 Mb, while the repeat-minus genes are in two chromosomes of 0.82 and 0.79 Mb. The repeat-containing genes have similar catalytic and intergenic regions, but variable lengths of the repeated region. The trans-sialidase genes with the repeats are in tandem of up to 12 genes in at least four different clusters. Each cluster contains genes with different numbers of repeats, according to the physical maps of eight independent cosmids, and in the same cluster there are genes that code for active and inactive trans-sialidases. There are 80 +/- 30 copies of the repeat-containing genes grouped in two NotI fragments of 120 and 180 Kb. Therefore, in the Y-strain, the trans-sialidase genes containing repeats might be arranged in three to four clusters in two homologous chromosomes, each cluster having up to 12 genes with different repeat numbers.

Diagnosis of visceral leishmaniasis by polymerase chain reaction of DNA extracted from Giemsa's solution-stained slides.

Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani and is a potentially fatal disease in endemic areas of the world. Nepal is an endemic area in which VL causes major public health problems in the lowland areas of the southeast regions. The aim of the present study was to evaluate the sensitivity of polymerase chain reaction (PCR) amplification for the detection of Leishmania DNA from Giemsas solution-stained bone marrow slides. Bone marrow samples were aspirated from a total of 115 VL suspected patients and used to prepare smears on glass slides and for the initiation of in vitro culture. Bone marrow slides were used for microscopic observation, DNA extraction, and subsequent PCR amplification. PCR analysis showed that all the positive samples were of Leishmania parasites. The PCR assay also showed a higher sensitivity (69%) than microscopic examination (57%) and culture (21%). In addition, PCR was able to detect VL in 12% of samples which were negative by microscopy. PCR of DNA extracted from Giemsas solution-stained bone marrow slides is a suitable tool for confirming diagnosis in patients with VL and may also be useful in the diagnosis of difficult cases. Bone marrow smears are easily stored and can be easily sent to research centers where PCR is available. This makes PCR a good option for diagnosis in the field.

Isolation and expression of an open reading frame encoding sialidase from Trypanosoma rangeli

Several protozoan parasites of human have been found to express enzymes capable of releasing terminal sialic acid residues from host glycans. These include enzymes similar in activity to bacterial and viral sialidases, as well as a novel type of enzyme, trans-sialidase, which can transfer sialic acid from one carbohydrate chain to another. Here we report the isolation of a gene and a gene fragment from the kinetoplastid Trypanosoma rangeli which encode products related in sequence to the trans-sialidase enzyme of T. cruzi. The gene fragment ORF is nearly identical to that of the complete gene, which encodes an enzymatically inactive protein. When the ORF of the gene fragment is fused to fragments from related genes, it encodes a product with sialidase activity. Both predicted T. rangeli protein products also have other potential structural features found in bacterial sialidases and in members of a previously described Trypanosoma trans-sialidase superfamily.

Plasmodium falciparum: differential selection of drug resistance alleles in contiguous urban and peri-urban areas of Brazzaville, Republic of Congo.

The African continent is currently experiencing rapid population growth, with rising urbanization increasing the percentage of the population living in large towns and cities. We studied the impact of the degree of urbanization on the population genetics of Plasmodium falciparum in urban and peri-urban areas in and around the city of Brazzaville, Republic of Congo. This field setting, which incorporates local health centers situated in areas of varying urbanization, is of interest as it allows the characterization of malaria parasites from areas where the human, parasite, and mosquito populations are shared, but where differences in the degree of urbanization (leading to dramatic differences in transmission intensity) cause the pattern of malaria transmission to differ greatly. We have investigated how these differences in transmission intensity affect parasite genetic diversity, including the amount of genetic polymorphism in each area, the degree of linkage disequilibrium within the populations, and the prevalence and frequency of drug resistance markers. To determine parasite population structure, heterozygosity and linkage disequilibrium, we typed eight microsatellite markers and performed haplotype analysis of the msp1 gene by PCR. Mutations known to be associated with resistance to the antimalarial drugs chloroquine and pyrimethamine were determined by sequencing the relevant portions of the crt and dhfr genes, respectively. We found that parasite genetic diversity was comparable between the two sites, with high levels of polymorphism being maintained in both areas despite dramatic differences in transmission intensity. Crucially, we found that the frequencies of genetic markers of drug resistance against pyrimethamine and chloroquine differed significantly between the sites, indicative of differing selection pressures in the two areas.

Longitudinal Survey of Plasmodium falciparum Infection in Vietnam: Characteristics of Antimalarial Resistance and Their Associated Factors

ABSTRACT Plasmodium falciparum is the main cause of human malaria and is one of the important pathogens causing high rates of morbidity and mortality. The total number of malaria patients in Vietnam has gradually decreased over the last decade. However, the spread of pathogens with drug resistance remains a significant problem. Defining the trend in genotypes related to drug resistance is essential for the control of malaria in Vietnam. We undertook a longitudinal survey of Plasmodium falciparum malaria in 2001, 2002, and 2005 to 2007. The pfcrt, pfmdr1, pfdhfr, and pfdhps genes were analyzed by sequencing; and correlations by study year, age, gender, and genotype were identified statistically. The ratio of the chloroquine resistance genotype pfcrt 76T was found to have decreased rapidly after 2002. High numbers of mutations in the pfdhfr and pfdhps genes were observed only in 2001 and 2002, while the emergence of parasites with a new K540Y mutation in the P. falciparum dihydropteroate synthetase (PfDHPS) was observed in 2002. For males and those in younger age brackets, a correlation between vulnerability to P. falciparum infection and strains with pfcrt 76K or strains with decreased numbers of mutations in pfdhfr and pfdhps was demonstrated. The parasites with pfcrt 76T exhibited a greater number of mutations in pfdhfr and pfdhps.