Plasmodium falciparum enolase: stage-specific expression and sub-cellular localization

<p>Abstract</p> <p>Background</p> <p>In an earlier study, it was observed that the vaccination with <it>Plasmodium falciparum </it>enolase can confer partial protection against malaria in mice. Evidence has also build up to indicate that enolases may perform several non-glycolytic functions in pathogens. Investigating the stage-specific expression and sub-cellular localization of a protein may provide insights into its moonlighting functions.</p> <p>Methods</p> <p>Sub-cellular localization of <it>P. falciparum </it>enolase was examined using immunofluorescence assay, immuno-gold electron microscopy and western blotting.</p> <p>Results</p> <p>Enolase protein was detected at every stage in parasite life cycle examined. In asexual stages, enolase was predominantly (≥85–90%) present in soluble fraction, while in sexual stages it was mostly associated with particulate fraction. Apart from cytosol, enolase was found to be associated with nucleus, food vacuole, cytoskeleton and plasma membrane.</p> <p>Conclusion</p> <p>Diverse localization of enolase suggests that apart from catalyzing the conversion of 2-phosphoglycericacid into phosphoenolpyruvate in glycolysis, enolase may be involved in a host of other biological functions. For instance, enolase localized on the merozoite surface may be involved in red blood cell invasion; vacuolar enolase may be involved in food vacuole formation and/or development; nuclear enolase may play a role in transcription.</p

Generation and characterisation of monoclonal antibodies specific to Plasmodium falciparum enolase

Background & objectives: Glycolysis is the sole source of energy for the intraerythrocytic stages ofPlasmodium falciparum, making glycolytic enzymes putative therapeutic targets. Enolase, a singlecopy gene in P. falciparum is one such enzyme whose activity is elevated ~10–15 fold in infectedRBC’s. It holds the possibility of having multiple biological functions in the parasite and hence canbe a suitable candidate for diagnostic and chemotherapeutic purposes.Methods: We have aimed at generating parasite-specific reagents in the form of monoclonalantibodies. We have raised monoclonal antibodies against the recombinant P. falciparum enolase.Results: Two IgG monoclonals were obtained with 1:1000 titre and specific for P. falciparum enolase.Apicomplexan parasites including P. falciparum enolase has a plant like pentapeptide sequence(104EWGWS108) which is uniquely different from the host counterpart. A peptide spanning thispentapeptide region (ELDGSKNEWGWSKSK) coupled to BSA was used to raise parasite-specificantibody. Four monoclonals were obtained with 1:1000 titre and of IgM isotype.Interpretation & conclusion: All the monoclonals are specific for P. falciparum enolase and one ofthem display reactivity against native P. falciparum enolase signifying this pentapeptide to be surfaceexposed and immunogeni

Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth

Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivax P. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India

With India aiming to achieve malaria elimination by 2030, several strategies have been put in place. With that aim, mass surveillance is now being conducted in some malaria-endemic pockets. As dry season mass surveillance has been shown to have its importance in targeting the reservoir, a study was undertaken to assess the parasite load by a sensitive molecular method during one of the mass surveys conducted in the dry winter period. It was executed in two malaria-endemic villages of Dhalai District, Tripura, in northeast India, also reported as P. falciparum predominated area. The present study found an enormous burden of Rapid Diagnostic Test negative malaria cases with P. vivax along with P. vivax and P. falciparum mixed infections during the mass surveillance from febrile and afebrile cases in dry winter months (February 2021–March 2021). Of the total 150 samples tested, 72 (48%) were positive and 78 (52%) negative for malaria by PCR. Out of the 72 positives, 6 (8.33%) were P. falciparum, 40 (55.55%) P. vivax, and 26 (36.11%) mixed infections. Out of 78 malaria negative samples, 6 (7.7%) were with symptoms, while among the total malaria positive, 72 cases 7 (9.8%) were with symptoms, and 65 (90.2%) were asymptomatic. Out of 114 samples tested by both microscopy and PCR, 42 samples turned out to be submicroscopic with 4 P. falciparum, 23 P. vivax, and 15 mixed infections. Although all P. vivax submicroscopic infections were asymptomatic, three P. falciparum cases were found to be febrile. Evidence of malaria transmission was also found in the vectors in the winter month. The study ascertained the use of molecular diagnostic techniques in detecting the actual burden of malaria, especially of P. vivax, in mass surveys. As Jhum cultivators in Tripura are at high risk, screening for the malarial reservoirs in pre-Jhum months can help with malaria control and elimination

Protective Properties and Surface Localization of Plasmodium falciparum Enolase▿

The enolase protein of the human malarial parasite Plasmodium falciparum has recently been characterized. Apart from its glycolytic function, enolase has also been shown to possess antigenic properties and to be present on the cell wall of certain invasive organisms, such as Candida albicans. In order to assess whether enolase of P. falciparum is also antigenic, sera from residents of a region of Eastern India where malaria is endemic were tested against the recombinant P. falciparum enolase (r-Pfen) protein. About 96% of immune adult sera samples reacted with r-Pfen over and above the seronegative controls. Rabbit anti-r-Pfen antibodies inhibited the growth of in vitro cultures of P. falciparum. Mice immunized with r-Pfen showed protection against a challenge with the 17XL lethal strain of the mouse malarial parasite Plasmodium yoelii. The antibodies raised against r-Pfen were specific for Plasmodium and did not react to the host tissues. Immunofluorescence as well as electron microscopic examinations revealed localization of the enolase protein on the merozoite cell surface. These observations establish malaria enolase to be a potential protective antigen

Cloning, over-expression, purification and characterization of Plasmodium falciparum enolase

We have cloned, over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in large quantities (≈50 mg per litre of culture) in a highly purified form (&gt;95%). The purified protein gave a single band at ≈50 kDa on SDS/PAGE. MALDI-TOF analysis gave a mean ±SD mass of 51396±16 Da, which is in good agreement with the mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was ≈100 kDa, indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of ≈30 U·mg−1 and Km2PGA=0.041±0.004 mm. The Michaelis constant for the reverse reaction (KmPEP) is 0.25±0.03 mm. pH-dependent activity measurements gave a maximum at pH 7.4-7.6 irrespective of the direction of catalysis. The activity of this enzyme is inhibited by Na+, whereas K+ has a slight activating effect. The cofactor Mg2+ has an apparent activation constant of 0.18±0.02 mm. However, at higher concentrations, it has an inhibitory effect. Polyclonal antibody raised against pure recombinant P. falciparum enolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite, Plasmodium yoelii, which shares 90% identity with the P. falciparum protein

Targeting PfProhibitin 2-Hu-Hsp70A1A complex as a unique approach towards malaria vaccine development

Summary: Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here, we report a novel antigen Plasmodium prohibitin “PfPHB2” and identify its cognate partner “Hsp70A1A” in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach, we show that loss of Hsp70A1A in red blood cells (RBCs) or PfPHB2 in infected red blood cells (iRBCs), respectively, inhibit PfPHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting PfPHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant PfPHB2 binds to RBCs which is inhibited by anti-PfPHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of PfPHB2 to serve as antigen is further supported by detection of anti-PfPHB2 antibody in patient sera. Overall, this study proposes PfPHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment

Reply to Deora et al. Multiplexing for Plasmodium spp.? Think Again! Comment on &ldquo;Bhowmick et al. Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivax P. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India. Pathogens 2021, 10, 1259&rdquo;

We thank Deora et al. [...

Validation of a Mobile Health Technology Platform (FeverTracker) for Malaria Surveillance in India: Development and Usability Study

BackgroundA surveillance system is the foundation for disease prevention and control. Malaria surveillance is crucial for tracking regional and temporal patterns in disease incidence, assisting in recorded details, timely reporting, and frequency of analysis. ObjectiveIn this study, we aim to develop an integrated surveillance graphical app called FeverTracker, which has been designed to assist the community and health care workers in digital surveillance and thereby contribute toward malaria control and elimination. MethodsFeverTracker uses a geographic information system and is linked to a web app with automated data digitization, SMS text messaging, and advisory instructions, thereby allowing immediate notification of individual cases to district and state health authorities in real time. ResultsThe use of FeverTracker for malaria surveillance is evident, given the archaic paper-based surveillance tools used currently. The use of the app in 19 tribal villages of the Dhalai district in Tripura, India, assisted in the surveillance of 1880 suspected malaria patients and confirmed malaria infection in 93.4% (114/122; Plasmodium falciparum), 4.9% (6/122; P vivax), and 1.6% (2/122; P falciparum/P vivax mixed infection) of cases. Digital tools such as FeverTracker will be critical in integrating disease surveillance, and they offer instant data digitization for downstream processing. ConclusionsThe use of this technology in health care and research will strengthen the ongoing efforts to eliminate malaria. Moreover, FeverTracker provides a modifiable template for deployment in other disease systems