Plasmodium knowlesi as a model system for characterising Plasmodium vivax drug resistance candidate genes

Abstract

Plasmodium vivax causes the majority of malaria outside Africa, but is poorly understood at a cellular level partly due to technical difficulties in maintaining it in in vitro culture conditions. In the past decades, drug resistant P. vivax parasites have emerged, mainly in Southeast Asia, but while some molecular markers of resistance have been identified, none have so far been confirmed experimentally, which limits interpretation of the markers, and hence our ability to monitor and control the spread of resistance. Some of these potential markers have been identified through P. vivax genome-wide population genetic analyses, which highlighted genes under recent evolutionary selection in Southeast Asia, where chloroquine resistance is most prevalent. These genes could be involved in drug resistance, but no experimental proof currently exists to support this hypothesis. In this study, we used Plasmodium knowlesi , the most closely related species to P. vivax that can be cultured in human erythrocytes, as a model system to express P. vivax genes and test for their role in drug resistance. We adopted a strategy of episomal expression, and were able to express fourteen P. vivax genes, including two allelic variants of several hypothetical resistance genes. Their expression level and localisation were assessed, confirming cellular locations conjectured from orthologous species, and suggesting locations for several previously unlocalised proteins, including an apical location for PVX_101445. These findings establish P. knowlesi as a suitable model for P. vivax protein expression. We performed chloroquine and mefloquine drug assays, finding no significant differences in drug sensitivity: these results could be due to technical issues, or could indicate that these genes are not actually involved in drug resistance, despite being under positive selection pressure in Southeast Asia. These data confirm that in vitro P. knowlesi is a useful tool for studying P. vivax biology. Its close evolutionary relationship to P. vivax , high transfection efficiency, and the availability of markers for colocalisation, all make it a powerful model system. Our study is the first of its kind using P. knowlesi to study unknown P. vivax proteins and investigate drug resistance mechanisms.

UsingPlasmodium knowlesias a model for screeningPlasmodium vivaxblood-stage malaria vaccine targets reveals new candidates

“…This study clearly highlights several advantages of the P. knowlesi system as a model for testing P. vivax blood-stage antigens, as has been suggested in previous drug (51) and vaccine (46,52) studies. A key one is accessibility -access to ex vivo P. vivax samples is limited and samples are precious, whereas we were readily able to perform multiple in vitro assays using P. knowlesi.…”

“…This is in large part because it is currently not possible to continuously culture P. vivax blood stages in vitro, which rules out many biological assays. We have explored whether P. knowlesi, which has a close phylogenetic relationship with P. vivax (39,40) and has been adapted to in vitro culture in human erythrocytes (41,50), could be used to screen for P. vivax blood-stage vaccine candidates, as it can for drug-resistance candidates (51). Such an approach has proven viable to explore the most advanced P. vivax blood-stage vaccine candidate, PvDBP (46).…”

Elimination ofPlasmodium vivaxMalaria: Problems and Solutions

“…Population genomics studies of P. vivax populations from areas with drastically different CQ resistance have identified genomic sites under strong selection [100,101], but their significance in mediating drug resistance remains to be determined. When using Plasmodium knowlesi as an in vitro model, some of the markers did not seem to change the drug sensitivity phenotypes in transgenic parasites [102].…”

The molecular basis of antimalarial drug resistance in Plasmodium vivax

“…pvmdr1 was identified in 2005, and due to its strong sequence conservation with pfmdr1 , has subsequently become one of the primary candidate genes assessed in P. vivax drug sensitivity studies ( Barnadas et al, 2008 ; Brega et al, 2005 ; Orjuela-Sánchez et al, 2009 ). PvMDR1 localizes to the digestive vacuole when overexpressed in P. knowlesi , which is more closely related to P. vivax than P. falciparum is, suggesting that it plays a similar role to PfMDR1 in P. vivax ( Verzier et al, 2019 ).…”