ABSTRACT
Centrifugal elutriation discriminates cells according to their sedimentation coefficients, generating homogeneous samples well suited for genomic comparative approaches. It can, for instance, isolate G1 daughter cells from a Saccharomyces cerevisiae unsynchronized population, alleviating ageing and cell-cycle biases when conducting genome-wide/single-cell studies. The present report describes a straightforward and robust procedure to determine whether a cell population of virtually any yeast species can be efficiently elutriated, while offering solutions to optimize success. This approach was used to characterize elutriation parameters and S-phase progression of four yeast species (S. cerevisiae, Candida glabrata, Lachancea kluyveri and Pichia sorbitophila) and could theoretically be applied to any culture of single, individual cells.
Subject(s)
Centrifugation/methods , G1 Phase , Mycology/methods , Saccharomyces cerevisiae/cytology , Cell Division , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/isolation & purification , Yeasts/cytology , Yeasts/genetics , Yeasts/isolation & purificationABSTRACT
BACKGROUND: Protection against pregnancy associated malaria (PAM) is associated with high levels of anti-VAR2CSA antibodies. This protection is obtained by the parity dependent acquisition of anti-VAR2CSA antibodies. Distinct parity-associated molecular signatures have been identified in VAR2CSA domains. These two observations combined point to the importance of identifying VAR2CSA sequence variation, which facilitate parasitic evasion or subversion of host immune response. Highly conserved domains of VAR2CSA such as DBL5ε are likely to contain conserved epitopes, and therefore do constitute attractive targets for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: VAR2CSA DBL5ε-domain sequences obtained from cDNA of 40 placental isolates were analysed by a combination of experimental and in silico methods. Competition ELISA assays on two DBL5ε variants, using plasma samples from women from two different areas and specific mice hyperimmune plasma, indicated that DBL5ε possess conserved and cross-reactive B cell epitopes. Peptide ELISA identified conserved areas that are recognised by naturally acquired antibodies. Specific antibodies against these peptides labelled the native proteins on the surface of placental parasites. Despite high DBL5ε sequence homology among parasite isolates, sequence analyses identified motifs in DBL5ε that discriminate parasites according to donor's parity. Moreover, recombinant proteins of two VAR2CSA DBL5ε variants displayed diverse recognition patterns by plasma from malaria-exposed women, and diverse proteoglycan binding abilities. CONCLUSIONS/SIGNIFICANCE: This study provides insights into conserved and exposed B cell epitopes in DBL5ε that might be a focus for cross reactivity. The importance of sequence variation in VAR2CSA as a critical challenge for vaccine development is highlighted. VAR2CSA conformation seems to be essential to its functionality. Therefore, identification of sequence variation sites in distinct locations within VAR2CSA, affecting antigenicity and/or binding properties, is critical to the effort of developing an efficient VAR2CSA-based vaccine. Motifs associated with parasite segregation according to parity constitute one such site.
Subject(s)
Antigens, Protozoan/immunology , Placenta/parasitology , Plasmodium falciparum/immunology , Amino Acid Sequence , Animals , Antigens, Protozoan/chemistry , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Female , Humans , Malaria, Falciparum/complications , Malaria, Falciparum/immunology , Mice , Models, Molecular , Molecular Sequence Data , Pregnancy , Pregnancy Complications, Parasitic/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: Pregnant women acquire protective antibodies that cross-react with geographically diverse placental Plasmodium falciparum isolates, suggesting that surface molecules expressed on infected erythrocytes by pregnancy-associated malaria (PAM) parasites have conserved epitopes and, that designing a PAM vaccine may be envisaged. VAR2CSA is the main candidate for a pregnancy malaria vaccine, but vaccine development may be complicated by its sequence polymorphism. METHODS: The dynamics of P. falciparum genotypes during pregnancy in 32 women in relation to VAR2CSA polymorphism and immunity was determined. The polymorphism of the msp2 gene and five microsatellites was analysed in consecutive parasite isolates, and the DBL5epsilon + Interdomain 5 (Id5) part of the var2csa gene of the corresponding samples was cloned and sequenced to measure variation. RESULTS: In primigravidae, the multiplicity of infection in the placenta was associated with occurrence of low birth weight babies. Some parasite genotypes were able to persist over several weeks and, still be present in the placenta at delivery particularly when the host anti-VAR2CSA antibody level was low. Comparison of diversity among genotyping markers confirmed that some PAM parasites may harbour more than one var2csa gene copy in their genome. CONCLUSIONS: Host immunity to VAR2CSA influences the parasite dynamics during pregnancy, suggesting that the acquisition of protective immunity requires pre-exposure to a limited number of parasite variants. Presence of highly conserved residues in surface-exposed areas of the VAR2CSA immunodominant DBL5epsilon domain, suggest its potential in inducing antibodies with broad reactivity.
