Comparative transcriptomics reveal differential gene expression in Plasmodium vivax geographical isolates and implications on erythrocyte invasion mechanisms

Plasmodium vivax uses Duffy binding protein (PvDBP1) to bind to the Duffy Antigen-Chemokine Receptor (DARC) to invade human erythrocytes. Individuals who lack DARC expression (Duffy-negative) are thought to be resistance to P. vivax. In recent years, P. vivax malaria is becoming more prevalent in Africa with a portion of these cases detected in Duffy-negatives. Apart from DBP1, members of the reticulocyte binding protein (RBP) and tryptophan-rich antigen (TRAg) families may also play a role in erythrocyte invasion. While the transcriptomes of the Southeast Asian and South American P. vivax are well documented, the gene expression profile of P. vivax in Africa and more specifically the expression level of several erythrocyte binding gene candidates as compared to DBP1 are largely unknown. This paper characterized the first P. vivax transcriptome in Africa and compared with those from the Southeast Asian and South American isolates. The expression of 4,404 gene transcripts belong to 12 functional groups including 43 specific erythrocyte binding gene candidates were examined. Overall, there were 10–26% differences in the gene expression profile amongst the geographical isolates, with the Ethiopian and Cambodian P. vivax being most similar. Majority of the gene transcripts involved in protein transportation, housekeeping, and host interaction were highly transcribed in the Ethiopian P. vivax. Erythrocyte binding genes including PvRBP2a and PvRBP3 expressed six-fold higher than PvDBP1and 60-fold higher than PvEBP/DBP2. Other genes including PvRBP1a, PvMSP3.8, PvMSP3.9, PvTRAG2, PvTRAG14, and PvTRAG22 also showed relatively high expression. Differential expression was observed among geographical isolates, e.g., PvDBP1 and PvEBP/DBP2 were highly expressed in the Cambodian but not the Brazilian and Ethiopian isolates, whereas PvRBP2a and PvRBP2b showed higher expression in the Ethiopian and Cambodian than the Brazilian isolates. Compared to Pvs25, the standard biomarker for detecting female gametocytes, PvAP2-G (PVP01_1440800), GAP (PVP01_1403000), and Pvs47 (PVP01_1208000) were highly expressed across geographical samples. These findings provide an important baseline for future comparisons of P. vivax transcriptomes from Duffy-negative infections and highlight potential biomarkers for improved gametocyte detection.

148 received prior antimalarial treatment. A total of 10mL whole blood was preserved in sodium 149 heparin tubes at the time of collection. Red blood cell pellets were isolated and cryo-preserved 150 with two times glycerolyte 57 and stored in liquid nitrogen. Prior to culture, samples were 151 thawed by adding 0.2V of 12% NaCl solution drop-by-drop followed by a 5-minute room 152 temperature incubation. Ten-times volume of 1.6% NaCl solution was then added drop-by-drop 153 to the mixture and the samples were centrifuged at 1000 rcf for 10 minutes to isolate the red 154 blood cell pellet. This process was repeated with a 10x volume of 0.9% NaCl. Following 155 centrifugation, the supernatant was removed via aspiration, and 18mL of sterile IMDM (also . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure 1A). To minimize oxidative stress, each culture was checked more than 164 two times and returned to a 5% oxygen environment immediately after checking.

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Cultured pellets were isolated via centrifugation and placed in 10x volume trizol for RNA

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  180 To further confirm samples were majority schizont stage, sequence reads of each sample were 181 deconvoluted in CIBERSORTx (42) based on P. berghei homologs (43). We used the published 182 matrix to determine the frequency of expression for each gene calculated for rings, trophozoites, 183 and schizonts, respectively. Transcripts that were expressed 30% or more were sorted into their 184 respective stages (Supplementary Figure 1B). All reads were annotated using the Rsubread 185 package and classified into 12 different categories by function. We then examined the top 30 186 transcribed genes using the counts per million (CPM) metric.

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Our previously published whole genome sequence data identified several mutations and (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 16, 2023.    Figure 1A).

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Overall, about 64% (4,404 out of 6,830) of the genes were detected with transcription in 229 the Ethiopian P. vivax (Supplementary Table 2

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We further compared the expressional pattern of these 43 genes in the Ethiopian P. vivax . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure 6B). Of the 16 PvTRAg genes, PvTRAg14 and PvTRAg22 showed 320 significantly higher expression in the Ethiopian isolates compared to the others (P<0.05; Figure   321 6C). Eight other members including PvTRAg2b, PvTRAg7, PvTRAg19, PvTRAg20, PvTRAg21, 322 PvTRAg23, PvTRAg24, and PvTRAg38 showed significantly higher expression in the Brazilian 323 isolates than the others (P<0.05; Figure 6C). The remaining nine putatively functional ligands 324 showed relatively similar expression levels, except for PvMA, PvRhopH3, and PvTrx-mero that 325 were highly expressed in the Brazilian isolates (P<0.05; Figure 6D). 332 Pvs47 (PVP01_1208000) from female and male gametocytes, respectively, showed the highest 333 expression across the Ethiopian, Cambodian, and Brazilian isolates, and were consistently higher 334 than Pvs25 (Figure 7). This expression pattern suggests the potential utility of these three genes 335 as better gametocyte biomarkers across geographical isolates. Other genes indicated differential 336 expression patterns among isolates, e.g., the female gametocyte gene PVP01_0904300 (CPW-337 WPC family protein) showed consistently high levels of expression in both the Ethiopian and 338 Cambodian isolates, though much lower in the Brazilian ones. On the other hand, . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 16, 2023. ; https://doi.org/10.1101/2023.02.16.528793 doi: bioRxiv preprint 339 PVP01_1302200 (high mobility group protein B1) and PVP01_1262200 (fructose 1,6-340 bisphosphate aldolase) from the female and male gametocytes showed the highest expression 341 levels in Brazilian P. vivax but not the Ethiopian and Cambodian ones.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 16, 2023. ; https://doi.org/10.1101/2023.02.16.528793 doi: bioRxiv preprint transcripts for the Ethiopian P. vivax by gene function. The numbers shown represent the 681 number of transcripts along with the overall percentage compared to all detected transcripts.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 16, 2023. ; https://doi.org/10.1101/2023.02.16.528793 doi: bioRxiv preprint

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Transcripts that were not detected were removed from the analysis. Only transcripts involved