Temperate Zone Plant Natural Products-A Novel Resource for Activity against Tropical Parasitic Diseases.

The use of plant-derived natural products for the treatment of tropical parasitic diseases often has ethnopharmacological origins. As such, plants grown in temperate regions remain largely untested for novel anti-parasitic activities. We describe here a screen of the PhytoQuest Phytopure library, a novel source comprising over 600 purified compounds from temperate zone plants, against in vitro culture systems for Plasmodium falciparum, Leishmania mexicana, Trypanosoma evansi and T. brucei. Initial screen revealed 6, 65, 15 and 18 compounds, respectively, that decreased each parasite's growth by at least 50% at 1-2 µM concentration. These initial hits were validated in concentration-response assays against the parasite and the human HepG2 cell line, identifying hits with EC50 < 1 µM and a selectivity index of >10. Two sesquiterpene glycosides were identified against P. falciparum, four sterols against L. mexicana, and five compounds of various scaffolds against T. brucei and T. evansi. An L. mexicana resistant line was generated for the sterol 700022, which was found to have cross-resistance to the anti-leishmanial drug miltefosine as well as to the other leishmanicidal sterols. This study highlights the potential of a temperate plant secondary metabolites as a novel source of natural products against tropical parasitic diseases.

Paragonimiasis in tuberculosis patients in Nagaland, India.

BACKGROUND: One of the infections that mimic tuberculosis (TB) is paragonimiasis (PRG), a foodborne parasitic disease caused by lung flukes of the genus Paragonimus. In the northeastern states of India, TB and PRG are endemic; however, PRG is rarely included in the differential diagnosis of TB. OBJECTIVE: To address limited evidence on the dual burden of TB and PRG in northeastern India, we aimed to document the prevalence of PRG among TB patients using sputum smear, stool examination for children <15 years and ELISA. DESIGN: A cross-sectional study of patients receiving TB treatment in the Médecins Sans Frontières (MSF)-supported TB programme in Mon district, in collaboration with the Regional Medical Research Centre (RMRC), Dibrugarh, Assam, between November 2012 and December 2013. RESULTS: Of 96 patients screened between November 2012 and December 2013, three (3%) had pulmonary PRG and were successfully treated with praziquantel. CONCLUSIONS: PRG should be considered in the TB diagnostic algorithms in PRG-TB dual burden areas. In case of TB-PRG co-infection, it is preferable to treat PRG first followed by anti-TB treatment a few days later.

Cathepsins B1 and B2 of Trichobilharzia SPP., bird schistosomes causing cercarial dermatitis.

Trichobilharzia regenti and T. szidati are schistosomes that infect birds. although T. regenti/T. szidati can only complete their life cycle in specific bird hosts (waterfowl), their larvae-cercariae are able to penetrate, transform and then migrate as schistosomula in nonspecific hosts (e.g., mouse, man). Peptidases are among the key molecules produced by these schistosomes that enable parasite invasion and survival within the host and include cysteine peptidases such as cathepsins B1 and B2. These enzymes are indispensable bio-catalysts in a number of basal biological processes and host-parasite interactions, e.g., tissue invasion/migration, nutrition and immune evasion. Similar biochemical and functional characteristics were observed for cathepsins B1 and B2 in bird schistosomes (T. regenti, T. szidati) and also for their homologs in human schistosomes (Schistosoma mansoni, S. japonicum). Therefore, data obtained in the research of bird schistosomes can also be exploited for the control of human schistosomes such as the search for targets of novel chemotherapeutic drugs and vaccines.

Cathepsins B1 and B2 in the neuropathogenic schistosome Trichobilharzia regenti: distinct gene expression profiles and presumptive roles throughout the life cycle.

The neurotropic bird schistosome Trichobilharzia regenti possesses papain-like cysteine peptidases which have also been shown to be crucial enzymes in various developmental stages of the related human parasites Schistosoma spp. In this paper, we present data obtained by real-time polymerase chain reaction on the temporal distribution of transcripts of two cathepsins in different developmental stages of T. regenti: cathepsin B1 originally described from the gut lumen of schistosomula with presumptive role in nutrient digestion and cathepsin B2 originally found in penetration glands of cercariae with probable involvement in invasion of the final host. In spite of their mutual resemblance at the sequence level, the mRNA expression profiles clearly show distinct expression of cathepsins B1 and B2 during the development from eggs to cercariae. In the case of both cathepsins, the highest level of transcription was detected in intravertebrate stages. Putative functions of cathepsins B1 and B2 in schistosome developmental stages are discussed.

The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti.

A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S(2) subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.

Peptidases of Trichobilharzia regenti (Schistosomatidae) and its molluscan host Radix peregra S. Lat. (Lymnaeidae): construction and screening of cDNA library from intramolluscan stages of the parasite.

Trichobilharzia regenti is a neurotropic bird schistosome,causing cercarial dermatitis in humans. In this study, ZAP cDNA expression library from Radix peregra s. lat. hepatopancreases containing intramolluscan stages of T. regenti was constructed and screened using PCR with specific and degenerate primers, designed according to previously described serine and cysteine peptidases of other parasite species. Full-length sequences of cathepsins B1 and L, and two serine peptidases, named RpSP1 and RpSP2, were obtained. The protein-protein BLAST analysis and parallel control reactions with template from hepatopancreases of T. regenti non-infected snails revealed that only cathepsin B1 was of parasite origin. The remaining sequences were derived from the snail intermediate host, which implies that the initial source of parasite mRNA was contaminated by snail tissue. Regardless of this contamination, the cDNA library remains an excellent molecular tool for detection and identification of bioactive molecules in T. regenti cercariae.