Molecular and biochemical parasitology最新文献

Background

Chinese citizens traveling abroad bring back imported malaria cases to China. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. To complement existing diagnostic methods, we aimed to develop a new loop-mediated isothermal amplification (LAMP) assay to detect and identify Plasmodium falciparum in Chinese travelers returning from Africa.

Methods

We developed a miniaturized LAMP assay to amplify the actin I gene of P. falciparum. Each reaction consumed only 25% of the reagents used in a conventional LAMP assay and the same amount of DNA templates used in nested PCR. We evaluated this LAMP assay's performance and compared it to microscopy and a nested PCR assay using 466 suspected malaria cases imported from Africa. We assessed the sensitivity of the new LAMP assay using cultured P. falciparum, clinical samples, and a plasmid construct, allowing unprecedented precision when quantifying the limit of detection.

Results

The new LAMP assay was highly sensitive and detected two more malaria cases than nested PCR. Compared to nested PCR, the sensitivity and specificity of the novel LAMP assay were 100% [95% confidence interval (CI) 98.5–100%] and 99.1% (95% CI 96.7–99.9%), respectively. When evaluated using serial dilutions of the plasmid construct, the detection limit of the new LAMP was as low as 10² copies/μL, 10-fold lower than PCR. The LAMP assay detected 0.01 parasites/μL of blood (equal to 0.04 parasites/μL of DNA) using cultured P. falciparum and 1–7 parasites/μL of blood (4–28 parasites/μL of DNA) in clinical samples, which is as good as or better than previously reported and commercially licensed assays.

Conclusion

The novel LAMP assay based on the P. falciparum actin I gene was specific, sensitive, and cost-effective, as it consumes 1/4 of the reagents in a typical LAMP reaction.

The UNC-49 receptor is a Cys-loop GABA receptor that is unique to the nematode phylum. The receptor differs from mammalian GABA receptors both in amino acid sequence and pharmacology which highlights its potential as a novel anthelmintic target. Sequence differences within and near the various ligand-binding loops of the nematode receptor suggest that there could be structural differences compared to mammalian receptors that result in different pharmacological and functional features. Here we investigated three residues in the UNC-49 receptor from the parasitic nematode Haemonchus contortus: K181, E183, and T230. Analysis of these residues was conducted via site-directed mutagenesis, electrophysiology, MD simulations, and mutant cycling analysis. In the UNC-49 receptor, E183 lies in close proximity to K181 where together they appear to play a role in GABA sensitivity and pharmacology, possibly interacting via an ionic bond. While the introduction of single alanine residues at each position separately had a negative impact on GABA EC₅₀, the double alanine mutant (K181A/E183A) exhibited wildtype-level GABA EC₅₀ and some differences in pharmacology. Overall, this study has revealed a potentially novel role for these two residues in nematode UNC-49 GABA receptors that could aid in understanding their function.

Background

Malaria infection can result in distinct clinical outcomes from asymptomatic to severe. The association between patho-physiological changes and molecular changes in the host, and their correlation with severity of malaria progression is not fully understood.

Methods

In this study, we addressed mass spectrometry-based temporal profiling of serum metabolite levels from mice infected with Plasmodium berhgei (strain ANKA).

Results

We show global perturbations and identify changes in specific metabolites in correlation with disease progression. While metabolome-wide changes were apparent in late-stage malaria, a subset of metabolites exhibited highly correlated changes with disease progression. These metabolites changed early on following infection and either continued or maintained the change as mice developed severe disease. Some of these have the potential to be sentinel metabolites for severe malaria. Moreover, glycolytic metabolites, purine nucleotide precursors, tryptophan and its bioactive derivatives were many fold decreased in late-stage disease. Interestingly, uric acid, a metabolic waste reported to be elevated in severe human malaria, increased with disease progression, and subsequently appears to be detoxified into allantoin. This detoxification mechanism is absent in humans as they lack the enzyme uricase.

Conclusions

We have identified candidate marker metabolites that may be of relevance in the context of human malaria.

Trichomonas vaginalis is an early divergent protozoan parasite that causes trichomoniasis, the most common non-viral sexually transmitted infection. In metazoans, there is abundant and detailed research on the cell cycle and the components involved in the regulation mechanisms. Regulators such as the cyclin-dependent kinases (CDKs) and cyclins activate the highly regulated processes of cell division. While CDKs have important roles in the phosphorylation of specific substrates, cyclins are important activating-components of CDKs that allow orderly passage through the different stages of the cell cycle. Cell cycle cyclins are characterized by showing drastic changes in their concentration during the cell cycle progression. However, in protists such as T. vaginalis, some biological processes such as cell cycle regulation remain less well studied. In an attempt to gain insight into cell cycle regulation in T. vaginalis, as an initial approach we characterized four proteins with features of cyclins. The genes encoding these putative cyclins were cloned to produce the recombinant proteins TvCYC1, TvCYC2, TvCYC3, and TvCYC4. The functional activity of TvCYC2, TvCYC3, and TvCYC4 was assessed through their complementation of a yeast cln1,2,3Δ mutant strain; TvCYC1 was not able to complement this mutant. Furthermore, our results suggest that TvCYC1, TvCYC2, and TvCYC3, are able to interact with and activate the kinase activity of TvCRK1, a kinase previously characterized by our group. The present study represents the first characterization of cyclins potentially involved in cell cycle regulation in T. vaginalis.

Caenorhabditis elegans is a uniquely powerful tool to aid understanding of fundamental nematode biology. While C. elegans boasts an unrivalled array of functional genomics tools and phenotype bioassays the inherent differences between free-living and parasitic nematodes underscores the need to develop these approaches in tractable parasite models. Advances in functional genomics approaches, including RNA interference and CRISPR/Cas9 gene editing, in the parasitic nematodes Strongyloides ratti and Strongyloides stercoralis provide a unique and timely opportunity to probe basic parasite biology and reveal novel anthelmintic targets in species that are both experimentally and therapeutically relevant pathogens. While Strongyloides functional genomics tools have progressed rapidly, the complementary range of bioassays required to elucidate phenotypic outcomes post-functional genomics remain more limited in scope. To adequately support the exploitation of functional genomic pipelines for studies of gene function in Strongyloides a comprehensive set of species- and parasite-specific quantitative bioassays are required to assess nematode behaviours post-genetic manipulation. Here we review the scope of the current Strongyloides bioassay toolbox, how established Strongyloides bioassays have advanced knowledge of parasite biology, opportunities for Strongyloides bioassay development and, the need for investment in tractable model parasite platforms such as Strongyloides to drive the discovery of novel targets for parasite control.

Schistosomiasis is a common parasitic disease. Hepatosplenic schistosomiasis, caused by Schistosoma japonicum and Schistosoma mansoni, involves pathological changes, including worm egg-induced hepatic granuloma and fibrosis, which can markedly affect the liver’s physiological functions. Although the drug praziquantel (PZQ) is used to treat schistosomiasis, drugs against schistosomiasis-induced liver fibrosis are rare in the clinical setting. Therefore, developing effective strategies to prevent and treat schistosomiasis-induced liver fibrosis is crucial. Previous studies have shown that miRNAs are involved in various liver diseases. In this study, we found a gradual increase in miR-181b expression in the murine liver as S. japonicum infection progressed, while the expression of Smad7 decreased. Down-regulating miR-181b significantly alleviated S. japonicum-induced hepatic granuloma and liver fibrosis. In vitro experiments showed that treatment with TGF-β1 upregulated miR-181b levels in the hepatic stellate cell (HSC) line LX2 in a concentration- and time-dependent manner. Downregulation of miR-181b significantly decreased collagen type I alpha 1 chain (COL1A1) expression in TGF-β1-stimulated LX2 cells. These findings indicate that miR-181b promotes HSC activation by down-regulating Smad7 expression, activating the TGF-β1/Smad signaling pathway, and leading to excess collagen expression and deposition. Our findings suggest that miR-181b might be a potentially novel therapeutic target for schistosomiasis-induced liver fibrosis.

Giardia duodenalis is a flagellated protozoan that inhabits vertebrate host intestines, causing the disease known as giardiasis. Similar to other parasites, G. duodenalis must take advantage of environmental resources to survive, such as inorganic phosphate (P_i) availability. P_i is an anionic molecule and an essential nutrient for all organisms because it participates in the biosynthesis of biomolecules, energy storage, and cellular structure formation. The first step in Pi metabolism is its uptake through specific transporters on the plasma membrane. We identified a symporter H⁺:P_i-type ORF sequence in the G. duodenalis genome (GenBank ID: GL50803_5164), named GdPho84, which is homologous to Saccharomyces cerevisiae PHO84. In trophozoites, P_i transport was linear for up to 15 min, and the cell density was 3 × 10⁷ cells/ml. Physiological variations in pH (6.4–8.0) did not influence P_i uptake. This P_i transporter had a high affinity, with K_0.5 = 67.7 ± 7.1 µM P_i. SCH28080 (inhibitor of H⁺, K⁺-ATPase), bafilomycin A₁ (inhibitor of vacuolar H⁺-ATPase), and FCCP (H⁺ ionophore) were able to inhibit P_i transport, indicating that an H⁺ gradient in the cell powered uphill P_i movement. PAA, an H⁺-dependent P_i transport inhibitor, reduced cell proliferation, P_i transport activity, and GdPHO48 mRNA levels. P_i starvation stimulated membrane potential-sensitive P_i uptake coupled to H⁺ fluxes, increased GdPho84 expression, and reduced intracellular ATP levels. These events indicate that these cells had an increased capacity to internalize P_i as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized P_i can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits P_i influx. Together, these results reinforce the hypothesis that P_i is a crucial nutrient for G. duodenalis energy metabolism.

Anisakiasis is a zoonotic fish-born parasitic disease caused by anisakid nematodes. Paraffin-embedded blocks containing biopsy samples taken from patients suffering gastritis with unknown causes were investigated by real-time PCR, in the Bushehr region, Iran; where human anisakiasis has not been reported, so far. A total of 50 paraffin-embedded blocks were randomly selected from 250 archived blocks of the patients with gastritis. A SYBER green-based real-time PCR targeting the ITS1 region was developed for the identification of Anisakis genus. An 86 bp partial fragment of the Anisakis spp. ITS1 gene was amplified successfully. A total of 3 out of 50 samples (6 %) had positive amplification in the samples and their pathology reports showed a significant finding of moderate chronic gastritis with or without ulcers. In conclusion, the developed qPCR could be used for detecting Anisakis spp. larval DNA in human biopsy blocks. This study showed the hidden human cases of anisakiasis in the Bushehr for the first time.