Dissecting The role of Plasmodium metacaspase-2 in malaria gametogenesis and sporogony.

The family of apicomplexan specific proteins contains caspases-like proteins called "metacaspases". These enzymes are present in the malaria parasite but absent in human; therefore, these can be explored as potential drug targets. We deleted the MCA-2 gene from Plasmodium berghei genome using a gene knockout strategy to decipher its precise function. This study has identified that MCA-2 plays an important role in parasite transmission since it is critical for the formation of gametocytes and for maintaining an appropriate number of infectious sporozoites required for sporogony. It is noticeable that a significant reduction in gametocyte, oocysts, ookinete and sporozoites load along with a delay in hepatocytes invasion were observed in the MCA-2 knockout parasite. Furthermore, a study found the two MCA-2 inhibitory molecules known as C-532 and C-533, which remarkably inhibited the MCA-2 activity, abolished the in vitro parasite growth, and also impaired the transmission cycle of P. falciparum and P. berghei in An. stephensi. Our findings indicate that the deletion of MCA-2 hampers the Plasmodium development during erythrocytic and exo-erythrocytic stages, and its inhibition by C-532 and C-533 critically affects the malaria transmission biology.

A nonpeptidyl molecule modulates apoptosis-like cell death by inhibiting P. falciparum metacaspase-2.

Metacaspases are novel cysteine proteases found in apicomplexan whose function is poorly understood. Our earlier studies on Plasmodium falciparum metacaspase-2 (PfMCA-2) revealed that the caspase inhibitor, Z-FA-FMK efficiently inhibited PfMCA-2 activity and, expression, and significantly blocked in vitro progression of the parasite developmental cycle via apoptosis-like parasite death. Building on these findings, we synthesized a set of novel inhibitors based on structural modification of Z-FA-FMK with the amides of piperic acid and investigated their effect on PfMCA-2. One of these analogs, SS-5, specifically inhibited the activity and expression of PfMCA-2. The activities of some other known malarial proteases (falcipains, plasmepsins and vivapain), and human cathepsins-B, D and L, and caspase-3 and -7 were not inhibited by SS-5. SS-5 blocked the development of P. falciparum in vitro (IC50 1 µM) and caused prominent morphological distortions. Incubation with SS-5 led to persistent parasite oxidative stress accompanied by depolarization of mitochondrial potential and accumulation of intracellular Ca2+. SS-5 also inhibited the development of P. berghei in a murine model. Our results suggest that the inhibition of PfMCA-2 results in oxidative stress, leading to apoptosis-like parasite death. Thus, SS-5 offers a starting point for the optimization of new antimalarials, and PfMCA-2 could be a novel target for antimalarial drug discovery.

Esterases are responsible for malathion resistance in Anopheles stephensi: A proof using biochemical and insecticide inhibition studies.

BACKGROUND & OBJECTIVES: Increase in prevalence and intensity of insecticide-resistance in vectors of vector-borne diseases is a major threat to sustainable disease control; and, for their effective management, studies on resistance mechanisms are important to come out with suitable strategies. Esterases are major class of detoxification enzymes in mosquitoes, which confers protection against insecticides in causing resistance. This study was aimed at biochemical characterization of esterases responsible for malathion resistance in Anopheles stephensi mosquitoes, along with its validation through biochemical techniques and native-PAGE assays. METHODS: Laboratory maintained susceptible and resistant An. stephensi mosquitoes were used for assessing the activity and effect of α - and ß -esterases on malathion. Bioassay, synergist bioassay, biochemical assay and native- PAGE were employed to characterize the role of esterases in conferring malathion-resistance. RESULTS: Notably significant (p < 0.0001) enhancement in α - and ß -esterases activity was observed with 2-fold increase in resistant An. StephensiGOA compared to susceptible An. StephensiBB. native-PAGE depicted two major bands 'a' (Rf = 0.80) and 'b' (Rf = 0.72) in susceptible An. stephensiBB , while one intense band 'b' (Rf = 0.72) was visible in resistant An. stephensiGOA. Inhibition assay revealed complete inhibition of α - and ß -esterases activity in presence of 1 mM malathion in susceptible strain compared to observed partial inhibition in resistant strain on native-PAGE. INTERPRETATION & CONCLUSION: This study provides a better understanding on the role of esterase enzyme (carboxylesterase) in conferring malathion-resistance in An. stephensi mosquitoes, as evident from the native-PAGE assay results. The study results could be used in characterizing the resistance mechanisms in vectors and for suggesting alternative chemical insecticide based resistance management strategies for effective vector-borne disease control.

Additional burden of asymptomatic and sub-patent malaria infections during low transmission season in forested tribal villages in Chhattisgarh, India.

BACKGROUND: The burden of sub-patent malaria is difficult to recognize in low endemic areas due to limitation of diagnostic tools, and techniques. Polymerase chain reaction (PCR), a molecular based technique, is one of the key methods for detection of low parasite density infections. The study objective was to assess the additional burden of asymptomatic and sub-patent malaria infection among tribal populations inhabiting three endemic villages in Keshkal sub-district, Chhattisgarh, India. A cross-sectional survey was conducted in March-June 2016, during the low transmission season, to measure and compare prevalence of malaria infection using three diagnostics: rapid diagnostic test, microscopy and nested-PCR. RESULTS: Out of 437 individuals enrolled in the study, 103 (23.6%) were malaria positive by PCR and/or microscopy of whom 89.3% were Plasmodium falciparum cases, 77.7% were afebrile and 35.9% had sub-patent infections. CONCLUSIONS: A substantial number of asymptomatic and sub-patent malaria infections were identified in the survey. Hence, strategies for identifying and reducing the hidden burden of asymptomatic and sub-patent infections should focus on forest rural tribal areas using more sensitive molecular diagnostic methods to curtail malaria transmission.

Interaction of heme proteins with anionic polyfluorene: insights into physiological effects, folding events, and inhibition activity.

Because of the toxicity caused by the heme redox-active iron proteins, their elevated levels, localization, and accumulation in the brain, many forms of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, occur as a result of which the brain becomes vulnerable to oxidative stress, ultimately resulting in neuronal death. An anionic water-soluble conjugated polyfluorene derivative poly(9,9-bis(6-sulfate hexyl) fluorene-alt-1,4-phenylene) sodium salt (P1) that binds Fe³âº proteins with very high selectivity and sensitivity is reported here. The photophysical properties of P1 were modified by the interaction with ferric heme-containing proteins cytochrome c (Cc), methemoglobin (MetHb), and hemin. P1 was found to be highly sensitive toward Fe³âº heme proteins as compared to nonmetalloproteins. We observed that the respective activities of ferric heme proteins were inhibited and proteins were unfolded, due to modification in their heme microenvironment in the presence of the polymer P1. The observations reported in this article provide the first example for the use of a water-soluble conjugated polymer in applications, such as (1) to detect small quantities of iron proteins in aqueous medium/physiological condition with the highest K(sv) values of 2.27 × 108 M⁻¹ for Cc, 3.81 × 107 M⁻¹ for MetHb, and 5.31 × 107 M⁻¹ for hemin; (2) to study the physiological effects of heme metalloproteins; (3) to visualize the folding events in real time; and (4) the inhibition activity of metalloproteins can be selectively studied using a conjugated polymer based assay system rapidly without interference from nonmetalloproteins at biological pH. All this is achieved by generating optical events, taking advantage of the bright fluorescence of anionic polyfluorene P1 in this case, that can be observed and monitored by modification in the absorption and emission color in real time.