Transcriptome analysis of Plasmodium berghei during exo-erythrocytic development

Reto Caldelari, Sunil Dogga, Marc W. Schmid, Blandine Franke-Fayard, Chris J. Janse, Dominique Soldati-Favre, Volker Heussler

Posted on: 2 May 2019

Preprint posted on 7 February 2019

How is the Plasmodium parasite’s progeny different in the liver and the blood? A transcriptomic study provides clues and implications for pathology.

Selected by Mariana De Niz

Categories: bioinformatics, cell biology, genetics, microbiology

Transcriptome analysis of Plasmodium berghei during exo-erythrocytic development

Background

The Plasmodium parasite, causative of malaria, has two key stages of development in the mammalian host: a pre-erythrocytic stage (also known as liver stage), and an erythrocytic stage which involves continuous invasion of red blood cells, and is responsible for pathologies that make malaria a heavy burden for global health efforts. Only the blood stages are clinically symptomatic, resulting in malaria and its syndromes and complications. Vast research effort has been devoted over many decades to understanding erythrocytic stages of Plasmodiumdevelopment, in an attempt to find an ‘Achilles heel’ on the parasite, to render it susceptible to anti-malarial treatment and vaccines.

Pre-erythrocytic stages are a silent stage of parasite development, yet massive parasite asexual replication occurs, whereby a single parasite can produce thousands of progeny in a short amount of time, making this one of the fastest replication rates among eukaryotes. The parasite’s developmental cycle involves replication by schizogony, during which nuclear division, and organelle growth and replication, occur (Figure 1) (1). At the end of the process, segregation occurs ensuring that each progeny parasite has a set of functional organelles. The erythrocytic stages of development also involve asexual replication by schizogony and also culminate in the production of merozoites. Whether schizogony at both stages is regulated in a similar manner, is unknown.

In their work, Caldelari et al performed a genome-wide RNA-seq analysis at various temporal sub-stages of Plasmodium berghei throughout the parasite’s development in the liver. The main aim of the work was to better understand gene regulation and metabolic networks during pre-erythrocytic stage schizogony, and to determine whether differences exist between pre-erythrocytic and erythrocytic stage-derived merozoites and/or schizonts (2).

Key findings

The transcription profiles of merozoites and schizonts arising from liver stages differ substantially from those of merozoites and schizonts arising from blood stages.

A main difference in blood and liver stage parasites was found to be in metabolic processes, due to the high usage of fatty acids to generate various parasite membranes in pre-erythrocytic stages.
A significant difference existed in genes related to mechanisms of egress from the respective host cells.

Four genes particularly upregulated in blood stage schizonts were related to immune evasion, antigenic variation, and sialic-acid-dependent invasion of red blood cells. Of particular relevance is the upregulation of PBANKA_1443300 (MSP9) (3,4), as it suggests that red blood cell invasion by merozoites derived from liver, as opposed to blood, might have a mechanisms of red blood cell invasion independent of sialic acid.

Surprisingly, genes were specifically upregulated in liver stage schizonts and detached cells included sbp1, mahrp1a and mahrp1b, which are indirectly involved in parasite sequestration during blood stages of infection (5).

Among some genes predominantly expressed in developing pre-erythrocytic stages, an important finding was PBANKA_1003900, whose expression profile is as high as that of lisp1 and lisp2, although PBANKA_1003900 is currently annotated as being gametocyte-specific (6). The authors suggested re-naming it as lisp3.

Figure 1. The Plasmodium life cycle is divided into two developmental stages in the mammalian host, after infection with the parasite by the bite of a mosquito (step 1 in schematic). The first stage is the pre-erythrocytic stage which takes place in the liver and is clinically silent (steps 2-9 in schematic). During this stage, the parasite undergoes asexual replication leads to the production of thousands of merozoites by schizogony (steps 6-7) which are slowly released to the bloodstream within merosomes (step 8). These merozoites invade red blood cells (RBCs) (step 9). In RBCs, merozoites develop into rings and trophozoites, and undergo schizogonic replication (steps 10-12), and also culminate in the generation and fast release of progeny merozoites which again invade RBCs (step 13), exponentially amplifying the parasite population. Caldelari et al made important findings suggesting that merozoites derived from liver and blood, differ. (Figure adapted from De Niz et al Nature Reviews Microbiology, 15(1):37-54).

Open questions and what I like about this preprint

It was for long thought that merozoites arising from liver and blood, were equal. It is only a very recent question to determine whether they are so. I like this work, because it opens a vast amount of questions that are barely beginning to be pondered in the malaria field. In their work, the authors showed that significant differences exist between liver-stage and blood-stage schizonts and merozoites. Altogether, that finding alone opens multiple questions including how the liver as a host organ, defines and modulates the outcome of infection in the blood stages, starting with the genetic makeup of the progeny that will give rise to the blood stages of infection. Specific to the work, open questions are:

Can modulation of genes related to metabolism and fatty acid processing be used to alter organelle segregation and membrane formation, to reduce or even abolish merozoite formation in the liver?

The authors found that an important difference exists between genes involved in egress in liver and blood schizonts. Assuming both types of egress mechanisms aim to ensure the maximal survival of merozoites, how could you alter merozoite survival by modifying egress mechanisms from the blood and liver?

Since you found that MSP9 is not upregulated in liver schizonts, but is upregulated in blood schizonts, and since you suggest that this is related to sialic-acid dependent mechanisms of red blood cell invasion, why do you think this differs specifically between liver and blood-derived merozoites, and how is this relevant to the parasite’s life cycle?

Converse to the above, you found that MAHRP1 and SBP1 were highly upregulated in liver schizonts/merozoites. You propose this is relevant in ensuring parasite sequestration and spleen remodelling immediately following liver egress, to ensure establishment of the parasite. However, the knock out parasites of both genes can survive in the blood albeit with lower replication rates, and despite being unable to sequester. Is there any other reason for MAHRP1 and SBP1 to be upregulated specifically in the liver-derived progeny?

You found that a highly expressed gene in pre-erythrocytic stages is currently annotated as being gametocyte specific. Are there progeny merozoites arising from the liver, already committed to gametocytogenesis or capable of forming gametocytes immediately in the first RBC invasion without the need of a previous cycle? Could you manipulate commitment at the liver stages of infection to altogether abolish pathology? Conversely, are there individuals who have enhanced transmission potential immediately following liver egress?

In your work, you used in vitro-derived parasites to analyse liver stages of infection. In vivo, different microenvironments can exist within the liver, including the different lobes, access to nutrients, oxygenation and vascularization. How do you think different microenvironments within the liver, influence a potential heterogeneity on the merozoite progeny? Furthermore, how does liver sensing of a whole body status (eg. pregnancy, immune status, nutrition state), influence the genetic makeup of the progeny merozoites produced during schizogony?

References

Adapted from De Niz M, Burda PC, Kaiser G, del Portillo HA, Spielmann T*, Frischknecht F*, Heussler VT*, Progress in imaging tools: insights gained into Plasmodiumbiology, Nature Reviews Microbiology, (2017), 15(1):37-54
Caldelari R, Dogga S, Schmid MW, Franke-Fayard B, Janse CJ, Soldati-Favre D, Heussler VT, Transcriptome analysis of Plasmodium bergheiduring exo-erythrocytic development, bioRxiv
Li X, Chen H, Oo TH, Daly TM, Bergman LW, Liu S-C, et al. A Co-ligand Complex Anchors Plasmodium falciparum Merozoites to the Erythrocyte Invasion Receptor Band 3. J Biol Chem. (2004), 4;279(7):5765–71.
Kariuki MM, Li X, Yamodo I, Chishti AH, Oh SS. Two Plasmodium falciparum merozoite proteins binding to erythrocyte band 3 form a direct complex. Biochem Biophys Res Commun.(2005), 4;338(4):1690–5.
De Niz M, Ullrich AK*, Heiber A*, Blancke Soares A, Pick C, Lyck R, Keller D, Kaiser G, Prado M, Flemming S, del Portillo HA, Janse CJ, Heussler VT*, Spielmann T*, The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites, Nature Communications, (2016), 7:11659.
Deligianni E, Andreadaki M, Koutsouris K, Siden-Kiamos I. Sequence and functional divergence of gametocyte-specific parasitophorous vacuole membrane proteins in Plasmodium parasites. Mol Biochem Parasitol. Elsevier; (2018), 1;220:15–8.