Regulation of the malaria parasite's developmental cycle

Potential targets for anti-malarial drugs and for preventing transmission of the parasite are described in articles in Genes & Cancer and Nature Communications.

By Karina Toledo  |  Agência FAPESP – Physicists frequently refer to signal transduction when describing energy conversion, but biochemists have been using the term for some decades now to describe how chemical signals determine what happens inside cells.

Brazilian researchers studying Plasmodium falciparum, the deadliest of human malaria species, have discovered a series of genes whose expression is modulated by melatonin, a hormone produced by the host.

Their findings were published in a cover article in the journal Genes & Cancer. According to the authors of the research, which was performed as part of a FAPESP Thematic Project, some of these genes are potential targets for the development of new anti-malarial drugs.

“Our group had already shown in a previous study that melatonin, the hormone that regulates the sleep-wake cycle in the host organism, can also influence the parasite’s developmental cycle. We have studied this signaling pathway, among others, in the parasite,” said Célia Garcia, a professor at the University of São Paulo’s Bioscience Institute (IB-USP) and principal investigator of the Thematic Project.

In another study, published in 2012 in the Journal of Pineal Research, Garcia’s team discovered that a strain of P. falciparum genetically modified not to produce a protein called PK7 (protein kinase 7) was incapable of regulating its developmental cycle via melatonin signaling. 

“This finding suggested to us that PK7 plays a key role in signal transduction by this pathway,” Garcia said. “We therefore began using the GM strain in our research to obtain a better understanding of how melatonin signaling occurs and hence how information is exchanged between Plasmodium and human cells.”

The paper published in Genes & Cancer describes the group’s investigation of how treatment with melatonin modifies gene expression in a wild-type strain of the parasite in comparison with the PK7-knockout strain. Their aim was to determine which genes are controlled by melatonin and additionally which of them are influenced by PK7.

The in vitro experiments used human erythrocytes (red blood cells) infected with P. falciparum. “During this stage of its lifecycle, the parasite causes all the symptoms of malaria, such as high fever, shaking chills and muscle pain,” Garcia said.

Plasmodium is an intracellular parasite with a complex multistage lifecycle. Both morphology and biochemistry differ in each stage, enabling it to interact flexibly with the host organism and survive in a variety of environments.

The researchers used large-scale RNA sequencing to analyze the parasite’s gene expression during the trophozoite stage.

“This methodology identifies short RNA sequences and compiles them into a large jigsaw puzzle to show which genes are expressed during a given physiological state,” Garcia said. “This is Big Data science: each RNA sequence analyzed contains 100 base pairs, and millions of sequences are obtained. They are then compared with Plasmodium’s genome, which contains about 23 million base pairs, to identify the gene to which each short sequence corresponds. The more 100-base pair sequences are identified for the same gene, the more that gene is being expressed.”

The analysis showed that melatonin modified the expression of 38 genes in the wild-type strain, enhancing it in 31 and lowering it in seven; in contrast, no significant changes were observed in the PK7-knockout strain. 

The differentially expressed genes included SEA-1, which encodes a protein expressed in infected red blood cells during the schizont stage.

“Antibodies against SEA-1 had already been shown to decrease the parasite’s replication by inhibiting schizont rupture. SEA-1-knockout parasites had also been found to exhibit replication defects,” Garcia said. 

Another gene altered by melatonin is CAF1, which plays a key role in regulating several genes during the intraerythrocytic stage.

“Proteins involved in host cell egress and in the invasion of new host cells are defective in CAF1-knockout parasites,” Garcia said.

Lifecycle blocking

Other possible therapeutic targets are genes associated with E3 ubiquitin ligases, proteins that are part of the ubiquitin proteasome system (UPS), Garcia added. 

The proteasome is the cell’s protein recycler. It removes damaged or invasive proteins from the cell and performs several other important cellular functions.

The next step was to repeat the experiment replacing melatonin with cyclic adenosine monophosphate (cAMP), an important second messenger in the melatonin signaling pathway.

In this case, the experiment was performed on the parasite during all three stages of its development in red blood cells. In the wild-type strain, exposure to cAMP modified the expression of 75 genes in the ring stage, 101 in the trophozoite stage, and 141 in the schizont stage.

“The idea of using cAMP came from a paper we published in 2005, in which we showed that the parasite converts the melatonin signal into an increase in cAMP and activation of PKA [protein kinase A],” Garcia said. “Several other papers besides ours show the importance of cAMP in the parasite’s invasion of red blood cells.”

The experiment pointed to an increase in expression of NEK3 in trophozoites treated with cAMP. NEK3 belongs to the NEK family of proteins, which are important for DNA replication.

“In schizonts, cAMP triggered increased expression of RPA1, SEA1 and NDK, as well as the myb2 transcription factor,” Garcia said. “All these genes are involved in the cell cycle and DNA replication. Increased expression in the more mature trophozoite and schizont stages must explain why the parasite’s lifecycle is accelerated by treatment with cAMP.”

Several other genes that participate in metabolic pathways also showed increased levels of expression, and these are considered potential targets for blocking the parasite’s lifecycle in red blood cells.

“Each of the genes described in the paper is involved in metabolic and physiological activities,” Garcia said. “From the academic standpoint, the results give us a better understanding of the parasite’s basic biology: how it regulates its lifecycle, and how it is able to develop in certain circumstances. In addition, some of these targets can be investigated further as adjuvants of drugs that disrupt the parasite’s lifecycle and facilitate the action of the human defense system.”

The article “Signaling transcript profile of the asexual intraerythrocytic development cycle of Plasmodium falciparum induced by melatonin and cAMP”, published in Genes & Cancer, can be read at impactjournals.com/Genes&Cancer/index.php?pii=118. 

New targets against malaria

After the hepatic stage of Plasmodium development, the asexual stage begins, in which thousands of merozoites invade the host’s red blood cells. This is followed by the ring, trophozoite and schizont stages.

The onset of the asexual stage is triggered by an unknown mechanism that bursts the host cell, releasing the merozoites into the bloodstream. They reinvade erythrocytes, and a new cycle begins. Some of the merozoites differentiate inside red blood cells into female and male gametocytes, which form gametes when ingested by the Anopheles mosquito. This is the start of the sexual stage.

“Gamete fertilization gives rise to a motile ookinete, which invades the mosquito’s midgut and divides to form sporozoites. The sporozoites migrate to the mosquito’s salivary glands, from which they are injected into a human host when it takes a blood meal,” Garcia explained.

For the sporozoites to travel to the salivary glands and be transmitted to a human host, the capsule containing the oocysts must burst. 

A study by Garcia in collaboration with Greek and Italian researchers identified two proteins present in oocyst capsules at the moment of rupture. The findings were published in Nature Communications on December 16, 2016. 

The scientists also observed that rupture did not occur in oocysts without the two proteins, which they called oocyst rupture protein (ORP) 1 and 2. ORP1 is found in the oocyst capsule from the initial stage, whereas ORP2 relocates from the oocyst cytoplasm to the capsule when mature sporozoites form.

Both proteins are considered potential new targets for blocking transmission of the parasite by mosquitoes.

“The study originated from our group’s discovery that the PfNF-YB transcription factor, which is well understood in mammalian cells, is expressed in different stages of the parasite’s intraerythrocytic stage and is located in the nucleus in late stages of the cycle, when it acts as a transcription factor by regulating gene expression or interacting with other proteins,” Garcia said. The first results were published in 2013 in the Journal of Pineal Research.

To understand the function of PfNF-YB in the sexual cycle, studies of this protein focused on the stages of the parasite’s development that occur in the mosquito, i.e., the ookinete, oocyst and sporozoite stages. Sporozoites develop inside the oocyst, and mature sporozoites travel to the mosquito’s salivary glands and are transmitted to the host via the mosquito bite.

“In P. berghei, the protein was named ORP1 because it is located in the oocyst capsule. Oocyst rupture is blocked by knocking out the gene that encodes ORP1. Part of the oocyst’s membrane comes from the midgut epithelium and part is synthesized by the parasite,” Garcia explained.

Experiments showed that wild-type parasites did not have oocysts containing sporozoites on the twelfth or twentieth day after infection, as they had already migrated to the salivary glands.

“However, knockout parasites still had several sporozoites in their midgut,” Garcia said. “The function of PfNF-YB or ORP is not linked to DNA during the sexual stage, and it is not located in the nucleus, even though it does contain well-conserved NF-YB and NF-YC domains.”

The researchers also characterized a second ORP2, which is located in the cytoplasm and migrates to the oocyst capsule when the mature sporozoite is formed. Knocking out both ORP1 and ORP2 inhibited oocyst rupture.

According to the article, the identification of ORP1 and ORP2 as essential proteins for transmission of malaria parasites provides new insight into aspects of the transmission of sporozoites via the mosquito bite and suggests that interventions targeting oocyst components could be effective ways to blocking transmission.

“In our view, understanding the basis for cellular process signaling in the different stages of the parasite’s lifecycle will help in identifying new targets. The study shows the relevance of understanding the various functions of proteins in the parasite during each stage of its lifecycle in the host’s red blood cells and in the mosquito,” Garcia said.

The article “Release of Plasmodium sporozoites requires proteins with histone-fold dimerization domains” (10.1038/NCOMMS13846), published in Nature Communications, can be read at nature.com/articles/ncomms13846.