Transcriptome and histone epigenome of Plasmodium vivax salivary-gland sporozoites point to tight regulatory control and mechanisms for liver-stage differentiation in relapsing malaria
Graphical abstract
Introduction
Malaria is among the most significant infectious diseases impacting humans globally, with 3.3 billion people at risk of infection, 381 million suspected clinical cases and up to ∼660,000 deaths attributed to malaria in 2014 (WHO, 2015). Two major parasitic species contribute to the vast majority of human malaria, Plasmodium falciparum and Plasmodium vivax. Historically, P. falciparum has attracted the majority of global attention, due to its higher contribution to morbidity and mortality. However, P. vivax is broadly distributed, more pathogenic than previously thought, and is recognised as the key obstacle to malaria elimination in the Asia-Pacific and Americas (Feachem et al., 2010). Unlike P. falciparum, P. vivax can establish long-lasting ‘sleeper cells’ (=hypnozoites (HPZs)) in the host liver that emerge weeks, months or years after the primary infection (=relapsing malaria) (Price et al., 2009). Primaquine is the only approved drug that prevents relapse. However, the short half-life, long dosage regimens and incompatibility of primaquine with glucose-6-phosphate-dehydrogenase deficiency (which requires pre-screening of recipients; Baird, 2013) makes it unsuitable for widespread use. As a consequence, P. vivax is overtaking P. falciparum as the primary cause of malaria in a number of co-endemic regions (Sattabongkot et al., 2004). Developing new tools to diagnose, treat and/or prevent HPZ infections is considered one of the highest priorities in the malaria elimination research agenda (Mueller et al., 2009).
When Plasmodium sporozoites (SPZs) are deposited by an infected mosquito, they likely traverse the skin cells, enter the blood-stream and are trafficked to the host liver, as has been shown in rodents (Lindner et al., 2012). The SPZs’ journey from skin deposition to hepatocytes takes less than a few minutes (Shin et al., 1982). Upon reaching the liver, SPZs traverse Kupffer and endothelial cells to reach the parenchyma, moving through several hepatocytes before invading a final hepatocyte suitable for development (Mota et al., 2001, Lindner et al., 2012). With the hepatocyte, P. vivax SPZs either immediately continue development as replicating schizonts and establish a blood infection, or delay replication and persist as HPZs. Regulation of this major developmental fate decision is not understood and this represents a key gap in current knowledge of P. vivax biology and control.
It has been hypothesized that P. vivax SPZs exist within an inoculum as replicating ‘tachysporozoites’ and relapsing ‘bradysporozoites’ (Lysenko et al., 1977) and that these subpopulations may have distinct developmental fates as schizonts or HPZs, thus contributing to their relapse phenotype (Lysenko et al., 1977, Price et al., 2007, White, 2011). This observation is supported by the stability of different HPZ phenotypes (ratios of HPZ to schizont formation) in P. vivax infections of liver-chimeric mouse models (Mikolajczak et al., 2015). It is well documented that P. vivax HPZ activation patterns stratify with climate and geography (White, 2011) and recent modelling suggests transmission potential selects for HPZ phenotype (White et al., 2014). Clearly the ability for P. vivax to dynamically regulate HPZ formation and relapse phenotypes in response to high or low transmission periods under different climate conditions would confer a significant evolutionary advantage. Epigenetic programming of the SPZ is intriguing as a potential mechanism to regulate the liver-stage developmental fate.
To determine fates in the SPZ stage, control of protein expression must take place. Studies using rodent malaria parasites have identified genes (Mueller et al., 2005) that are transcribed in SPZs but translationally repressed (i.e., present as transcript but un- or under-represented as protein), via RNA-binding proteins (Silvie et al., 2014a), and ready for immediate translation after the parasites’ infection of the mammalian host cell (Mackellar et al., 2010, Mikolajczak et al., 2015). Epigenetic control of PfAP2-G through chromatin structural remodelling regulates gametocyte (dimorphic sexual stages) development in blood stages (Josling and Llinas, 2015). Studies of P. falciparum blood stages have identified the importance of histone modifications as a primary epigenetic regulator (Lopez-Rubio et al., 2009, Duffy et al., 2014) and characterized key markers of heterochromatin (H3K9me3) and euchromatin/transcriptional activation (H3K4me3 and H3K9ac). In P. falciparum SPZs, these marks are significantly reconfigured during development in the mosquito (Gomez-Diaz et al., 2017) and play a role in the silencing of genes expressed during vertebrate infection (Zanghi et al., 2018). Histone methyltransferase inhibitors stimulate Plasmodium cynomolgi HPZs to become schizonts in macaque hepatocytes (Malmquist et al., 2012, Dembele et al., 2014). Further, histone methyltransferases have been implicated in HPZ formation in studies of differential transcription in P. cynomolgi liver stages (Dembele et al., 2014, Cubi et al., 2017). It is therefore possible that translational repression and other mechanisms of epigenetic control contribute to the P. vivax SPZ fate decision and HPZ formation, persistence and activation.
Despite recent advances (Roobsoong et al., 2015), current approaches for in vitro P. vivax culture do not support routine maintenance in the laboratory and tools to directly perturb gene function are not established. Although in vitro liver stage assays and humanised mouse models are being developed (Mikolajczak et al., 2015), ‘omics’ analysis of P. vivax liver stage dormancy has, until recently (Gural et al., 2018), been impossible and even now is in its early stages. Recent characterization (Cubi et al., 2017) of liver stages (HPZs and schizonts) of P. cynomolgi (a related and relapsing parasite in macaques) provides valuable insight, but investigations in P. vivax directly are needed. The systems analysis of P. vivax SPZs that reside in the mosquito salivary glands and are poised for transmission and liver infection offers a key opportunity to gain insight into P. vivax infection. Plasmodium vivax SPZs have been explored previously by microarray (Westenberger et al., 2010) and most recently, in a single RNA-seq replicate (Kim et al., 2017) and a study on SPZ activation (Roth et al., 2018). No epigenetic data are currently available for any P. vivax life-cycle stage. Here, we present a detailed characterization of the P. vivax SPZ transcriptome and histone epigenome and use these data to better understand this key infective stage and the role of SPZ programming in invasion and infection of the human host, and development within the host liver.
Section snippets
Ethics statement
Collection of venous blood from human patients with naturally acquired P. vivax infection was approved by the Ethical Review Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand (Human Subjects Protocol number TMEC 11-033) with the informed written consent of each donor individual. All mouse tissue used in the current study was from preserved infected tissues generated previously (Mikolajczak et al., 2015) at the Seattle Children’s Research Institute (SCRI: formerly
Results and discussion
Mosquito infections were generated by membrane feeding of blood samples taken from P. vivax-infected patients in western Thailand. Approximately 3–15 million P. vivax SPZs were harvested per isolate from Anopheles dirus salivary glands. Using RNA-seq (PvSPZ-Thai1 to 9), we detected transcription for 5714 P. vivax genes (Auburn et al., 2016) at high coverage (4930 with a mean CPM ≥ 1.0; Supplementary Fig. S1 and Supplementary Table S1 and S2). Among the most highly transcribed genes detected are
Acknowledgements
The authors acknowledge funding from the National Health and Medical Research Council (NHMRC, Australia; APP1021544, 1043345 and 1092789), the Australian Research Council (ARC), the Victorian State Government Operational Infrastructure Support, Australia and Australian Government National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme, the Ian Potter Foundation, Australia the National Institute of Health, USA, the Bill and Melinda Gates
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All authors contributed equally.