This study explores the potential of lead resistant bacterium Acinetobacter junii Pb1 for adsorption/accumulation of lead using various techniques. In the present work, growth of A. junii Pb1 was investigated in the presence of a range of Pb(II) concentrations (0, 100, 250, 500, and 1000 mg l −1). Lead was found to have no toxic effect on the growth of A. junii Pb1 at 100 and 250 mg l −1 concentrations. However, further increase in Pb(II) concentration (500 mg l −1) showed increase in lag phase, though growth remained unaffected and significant growth inhibition was observed when concentration was increased to 1000 mg l −1. Same was confirmed by the observations of flow cytometry. Further, the effect of Pb(II) on A. junii Pb1 was evaluated by using fluorescence microscopy, spectrofluorimetry, and flow cytometry. The spectrofluorimetry and fluorescence microscopy results revealed the accumulation of Pb(II) inside the bacterial cells as evident by green fluorescence due to lead binding fluorescent probe, Leadmium Green AM dye. Flow cytometry observations indicate an increase in cell size and granularity of exposure to lead. Thus, present work provides a new understanding of Pb(II) tolerance in A. junii Pb1 and its potential use in remediation of lead from contaminated soil.
Background
Targeting multiple key antigens that mediate distinct P. falciparum erythrocyte invasion pathways is an attractive approach for the development of blood-stage malaria vaccines. However, the challenge is to identify antigen cocktails that elicit potent strain-transcending parasite-neutralizing antibodies efficacious at low IgG concentrations feasible to achieve through vaccination. Previous reports have screened inhibitory antibodies primarily against well-adapted laboratory parasite clones. However, validation of the parasite-neutralizing efficacy against clinical isolates with minimal in vitro cultivation is equally significant to better ascertain their prospective in vivo potency.
Method
We evaluated the parasite-neutralizing activity of different antibodies individually and in combinations against laboratory adapted clones and clinical isolates. Clinical isolates were collected from central India and Mozambique (Africa), characterized for their invasion properties and genetic diversity of invasion ligands.
Results
In our portfolio, we evaluated 25 triple antibody combinations and identified the MSP-Fu+CyRPA+RH5 antibody combination to elicit maximal parasite neutralization against P. falciparum clinical isolates with variable properties that underwent minimal in vitro cultivation.
Conclusion
The MSP-Fu+CyRPA+RH5 combination exhibited highly robust parasite-neutralization against P. falciparum clones and clinical-isolates, thus substantiating them as promising candidate antigens and establishing a proof of principle for the development of a combinatorial P. falciparum blood-stage malaria vaccine.
Malaria remains a major health problem worldwide. All clinical symptoms of malaria are attributed to the asexual blood stages of the parasite life cycle. Proteins resident in apical organelles and present on the surface of P. falciparum merozoites are considered promising candidates for the development of blood stage malaria vaccines. In the present study, we have identified and characterized a microneme associated antigen, PfMA [PlasmoDB Gene ID: PF3D7_0316000, PFC0700c]. The gene was selected by applying a set of screening criteria such as transcriptional upregulation at late schizogony, inter-species conservation and the presence of signal sequence or transmembrane domains. The gene sequence of PfMA was found to be conserved amongst various
Plasmodium
species. We experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion. PfMA was localized by immunofluorescence and immuno-electron microscopy to be in the micronemes, an apical organelle of merozoites. The functional role of the PfMA protein in erythrocyte invasion was identified as a parasite adhesin involved in direct attachment with the target erythrocyte. PfMA was demonstrated to bind erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner and its antibodies inhibited P. falciparum erythrocyte invasion. Invasion of erythrocytes is a complex multistep process that involves a number of redundant ligand-receptor interactions many of which still remain unknown and even uncharacterized. Our work has identified and characterized a novel P. falciparum adhesin involved in erythrocyte invasion.
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