Abstract
Differential expression of ligands in the human malaria parasite Plasmodium falciparum enables it to recognize different receptors on the erythrocyte surface, thereby providing alternative invasion pathways. Switching of invasion from using sialated to nonsialated erythrocyte receptors has been linked to the transcriptional activation of a single parasite ligand. We have used quantitative proteomics to show that in addition to this single known change, there are a significant number of changes in the expression of merozoite proteins that are regulated independent of transcription during invasion pathway switching. These results demonstrate a so far unrecognized mechanism by which the malaria parasite is able to adapt to variations in the host cell environment by post-transcriptional regulation.
Highlights
Plasmodium falciparum is the most virulent species causing malaria in humans that affects and kills millions of people worldwide
It is clear that post-transcriptional regulation of protein expression plays an important role in invasion pathway switching; it demonstrates that the parasite invasion machinery undergoes much larger changes than previously thought when it adapts to changes in the availability of erythrocyte receptors
Comparison of W2mef and W2mef/NM Merozoite Protein Expression during Invasion Pathway Switching—For the investigation of expressional changes, we took advantage of W2mef parasites utilizing either sialic acid-dependent (W2mef) or -independent (W2mef/NM) invasion pathways by selecting parasites to grow in NM-treated erythrocytes
Summary
Parasite Cultivation, Isolation, and Adaptation to NM-treated Erythrocytes—P. falciparum W2mef clone was obtained from the MR4 Malaria Resource Centre. The defined parameters were as follows: (i) sample type: iTRAQ 4-plex (peptide labeled); (ii) cysteine alkylation: methylmethanethiosulfate; (iii) digestion: trypsin; (iv) instrument: QSTAR ESI; (v) special factors: none; (vi) species: none; (vii) specify processing: quantitate; (viii) ID focus: biological modifications, amino acid substitutions; (ix) database: a concatenated target-decoy database combining PlasmoDB P. falciparum (version 6.4), IPI human (version 3.34) and 156 commonly observed contaminants (146,588 sequences; 65884993 residues); and (x) search effort: thorough. The iTRAQ results of the three independent experiments were exported from ProteinPilot as protein summary reports in Microsoft Excel file format; only P. falciparum proteins are used for further analysis (supplemental Data Set 1). The geometric mean of the iTRAQ reporter ratio and standard deviation (log ratios) of each identified protein from the six sets of iTRAQ ratio of the three independent experiments were calculated (supplemental Data Set 2). Secondary antibodies were diluted in blocking buffer, incubated for 1 h at room temperature, and washed three times in TBST 0.05% for 10 min.
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