Volatile organic compounds associated with Plasmodium falciparum infection in vitro

Abstract

Background: In order to identify new ways to prevent transmission of vector-borne diseases such as malaria, efforts have been made to understand how insects are attracted to humans. Vector-host interaction studies have shown that several volatile compounds play an important role in attracting mosquitoes to human targets. A headspace solid-phase micro-extraction/gas chromatography-mass spectrometry (HSPME GC-MS) analysis of the volatile organic composition of extracellular vesicles (EVs) and supernatants of ultracentrifugation (SNUs) was carried out in Plasmodium falciparum-infected cultures with high and low parasitemias.

Results: A list of 18 volatile organic compounds (VOCs) was obtained from the EVs of both infected and uninfected RBCs with 1,2,3-Propanetriol, diacetate (diacetin) increased in the infected EVs, regardless of the parasitemia of the culture. The supernatant analysis, however, gave off 56 VOCs, with pentane 2,2,4-trimethyl being present in all the SNUs of uninfected erythrocytes but absent from the parasite-infected ones. Standing out in this study was hexanal, a reported insect attractant, which was the only VOC present in all samples from SNUs from infected erythrocytes and absent from uninfected ones, suggesting that it originates during parasite infection.

Conclusions: The hexanal compound, reportedly a low-level component found in healthy human samples such as breath and plasma, had not been found in previous analyses of P. falciparum-infected patients or cultures. This compound has been reported as an Anopheles gambiae attractant in plants. While the compound could be produced during infection by the malaria parasite in human erythrocytes, the A. gambiae attraction could be used by the parasite as a strategy for transmission.

Keywords: GC-MS; HSPME; Malaria; Plasmodium falciparum; Vector; Volatile organic compounds.

Fig. 1

Extracellular vesicles (EVs) are recognized by common markers. Erythrocytes and EVs were lysed with RIPA buffer. Each lane was loaded with 20 μg of protein sample, as assessed by a Bradford assay, subjected to non-denatured (a) or SDS PAGE (b, c), transferred to polyvinyl membranes and probed with antibodies against CD63 (a), PfMSP1 (b) and Glycophorin A (c). CD63 specific primary antibodies were used at 1:1000 dilution and a secondary Goat anti-Rabbit IgG HRP conjugated antibody (System Bioscience) was used at 1:20,000 dilution. MSP1 was used at 1:50 dilution with a secondary Mouse IgG HRP-conjugated antibody (R&D System) at a 1:1000 dilution. To detect Glycophorin, the E3 clone was used following the manufacturer instructions. The gel lanes were loaded as follows: M: size marker; Lane 1: uninfected erythrocytes lysate; Lane 2: EVs from uninfected erythrocytes; Lane 3: infected erythrocytes lysate; Lane 4: EVs from infected erythrocytes at low parasitemia; Lane 5: EVs from infected erythrocytes at high parasitemia; Lane 6: Human serum. Red arrows mark the bands corresponding to the bands expected

Fig. 2

Difference in abundance of VOCs in P. falciparum-infected vs uninfected cultures. The peak areas of three volatile organic compounds, a 1,2,3-propanetriol diacetate (found in the EVs) from uRBC and iRBC and b 2,2,4-trimethyl-pentane and hexanal (found in SNUs) from uRBC and iRBC were summed up across all replicates and number of experiments. The total area thus found was compared between uninfected samples and infected ones