Within seconds after an infected mosquito bites, the malaria parasite navigates the host skin and blood vessels to invade the liver, where it will stay embedded until thousands of infected cells burst into the bloodstream, launching malaria’s deadly blood-stage infection.
Now, for the first time, a team from Seattle Children’s Research Institute describes how malaria Plasmodium parasites prepare for this journey as they lie in waiting in the mosquito’s salivary glands.
Researchers say this knowledge may help identify new strategies to block transmission of the parasite – a critically important step needed for the eradication of malaria, a disease that continues to sicken over 300 million people and kill an estimated 435,000 people worldwide every year.
“Essentially the parasite makes a blueprint of the proteins it needs to infect the liver while still in the mosquito, far in advance of actually making the proteins once in the human,” said Dr. Stefan Kappe, the senior author on the paper published in Nature Communications and a malaria researcher in Seattle Children’s Center for Global Infectious Disease Research.
“It’s cool biology that offers new insight into how we might begin to stop the parasite from infecting the liver.”
Researchers get a closer look at the parasite inside the mosquito
An effective vaccine to protect against malaria does not exist.
To design interventions that will help win the fight against malaria, the Kappe Lab at Seattle Children’s is focused on understanding the complex biology of the malaria parasite and the host immune response to infection.
They are exploring new strategies to protect against the multiple stages of infection that will not only prevent disease in infected individuals but break the cycle of human-mosquito-human transmission.
Former postdoctoral scientist in the Kappe Lab, Dr. Scott Lindner, now at Penn State University’s Huck Center for Malaria Research, and Dr. Kristian Swearingen, a visiting scientist from Seattle’s Institute for Systems Biology, also contributed to the published paper.
Together with Kappe’s expertise in the mosquito stages of the malaria parasite called sporozoites, Lindner and Swearingen applied their proficiencies in the study of RNA molecules and proteins.
This allowed them to explore the sporozoites, which are ultimately transmitted to the human when the mosquito bites, in greater detail.
“We found that the sporozoites make and store all these plans for proteins, but then don’t actually make the protein,” Swearingen said.
“It’s the first window into a process in which the proteins are only made once the parasite negotiates the liver environment, suggesting the proteins are needed for the parasite to infect the human.”
Blueprint revealed may lead to new malaria drug, vaccine targets
Kappe explains that this strategy gives the malaria parasite the adaptability it needs to launch its attack seconds after the mosquito bites.
“The parasite has planned ahead, gathering the tools it needs to infect the human while it’s sitting in the mosquito waiting for this unpredictable event to happen,” he said.
“It may exist in this prepared state for days or even weeks until it has opportunity to strike in the human, so it has to be ready whenever this moment comes.”
According to Swearingen, the blueprint revealed in their research may ultimately lead scientists to new ways to disable the parasite.
“Any of these proteins that are critical for the parasite to invade the liver could serve as potential targets for new malaria drugs or vaccines,” he said. “With effective tools, we could see a day where malaria is completely eradicated.”
Human malaria occurs after the bite of a female Anopheles mosquito carrying Plasmodium parasites; the disease emerges mostly in tropical and sub-tropical regions around the world.
Five Plasmodium species infect humans: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi (P. falciparum being the most pathogenic and most frequently associated with mortality) (WHO, 2017).
The World Health Organization (WHO) has reported a slight increase in the amount of cases of the disease worldwide, rising from 211 million in 2015 to 216 million in 2016, despite an overall tendency to become decreased having been observed during the last few years (WHO, 2017).
Such increase has been linked to an expansion of Plasmodium‘s resistance to drugs, thereby urging the development of more effective treatment and prevention strategies, such as vaccines (WAMIN Consortium Authors et al., 2016; Amato et al., 2018). Understanding the parasite’s biology and its relationship with a host’s interaction will help in depicting its behavior during invasion and indicate effective methods for developing improved ways of avoiding its development in a particular host (Weiss et al., 2015; Patarroyo et al., 2016).
Most of malaria’s clinical symptoms are related to its ability to select, invade, and proliferate inside erythrocytes.
A pool of proteins acting as invasion ligands having affinity for receptors on erythrocyte surface would thus enable the recognition, attachment and invasion of the parasite in its merozoite form (Weiss et al., 2015).
The parasite can enable the differential expression of its invasion ligands by recognizing host cell surface receptors; these have redundancy in their function, leading to the emergence of different invasion pathways denominated invasion phenotypes (Gaur et al., 2004; Stubbs et al., 2005).
Many specific invasion phenotypes have been described for Plasmodium falciparum strains, mostly being related to the invasion-associated protein families of erythrocyte binding antigens (Pf EBAs, associated with pfeba175, pfeba140, pfeba181, and pfebl1 genes) and reticulocyte binding-like homologs (Pf Rhs, related to pfrh1, pfrh2a, pfrh2b, pfrh4, and pfrh5 genes) (Iyer et al., 2007; Tham et al., 2012).
These phenotypes have also been classically correlated with sialic acid-dependent or -independent invasion patterns (Dolan et al., 1990). Sialic acid-dependent parasites are essentially associated with greater expression of ligands needing sialic acid moieties in erythrocyte membrane receptors, such as members of the Pf EBA protein family (i.e., Pf EBA175, Pf EBA140, Pf EBA181, Pf EBL1) and some members of the Pf Rh family (i.e., Pf Rh1) (Cowman et al., 2017).
Parasite ligands which do not need these moieties for binding to erythrocyte surface receptors prevail in sialic-acid-independent parasites (i.e., some members of the Pf Rh protein family, like Pf Rh2b and Pf Rh4) (Dolan et al., 1990; Nery et al., 2006; Ochola-Oyier et al., 2016; Cowman et al., 2017). It is also known that the parasite can switch from one invasion phenotype to another (depending on a particular host’s environment or culture conditions) by varying the expression of its key invasion ligands (Stubbs et al., 2005; Awandare et al., 2018).
Studies attempting to explain the switch mechanism involved in the parasite invasion phenotype have suggested that invasion phenotypes could result from mutations in invasion-related genes or fluctuations in such genes’ transcription (Duraisingh et al., 2003).
Some studies have also shown that modifications in the parasite’s environment can induce changes in invasion gene epigenetic regulation; nevertheless, the specific mechanisms affecting such genes’ transcription and epigenetic regulation is still not well understood (Bowyer et al., 2015).
Moreover, selective pressure by a host’s immune system and variability regarding host cell surface receptor expression could be associated with the emergence of these phenotypes (Abdi et al., 2016, 2017).
This review has considered the available knowledge regarding some parasites’ genetic aspects influencing the development of invasion phenotypes, such as transcriptional and epigenetic characteristics, looking for a better understanding of the factors involved in erythrocyte invasion leading to such diversity, taking into account the effect of some host-related factors, such as host immune response and erythrocyte surface receptors.
Malaria and Parasite Invasion
A deeper knowledge of this parasite’s biology is necessary considering that P. falciparum infection is associated with higher than average morbidity and mortality regarding the remaining species (as it could invade all erythrocyte stages and produce high parasitaemia, inducing severe anemia, acidaemia and cerebral malaria) (Imtiaz et al., 2015); its study is fundamental in elucidating the key steps of its lifecycle inside a human host (WAMIN Consortium Authors et al., 2016).
The parasite’s invasion of erythrocytes marks the erythrocytic phase of infection which begins with the parasite sensing and first attaching itself to a host erythrocyte. This is followed by reorientation and erythrocyte invasion by parasite invagination followed by formation of the parasitophorous vacuole (PV) (Cowman et al., 2017).
The parasite will transform into a ring form inside the PV, then become a trophozoite followed by a schizont form which will burst and release a fresh load of merozoite which will invade other erythrocytes, thereby maintaining the erythrocytic cycle of infection which is directly associated with malaria’s clinical symptoms (Oakley et al., 2011; WAMIN Consortium Authors et al., 2016; Mangal et al., 2017). Establishing a continuous in vitro Plasmodium falciparum parasite culture during erythrocytic phase has facilitated the study of merozoite interactions with erythrocytes (Trager and Jensen, 1976; Thompson et al., 2001; Radfar et al., 2009).
Continuous culture-related results from merozoite-erythrocyte interaction studies during the erythrocytic phase have revealed some of the specific proteins located on merozoite surface used for interaction with erythrocyte membrane proteins (erythrocyte receptors). The former (invasion ligands) enable parasite adhesion and, during the first steps of invasion, ligand selection associated with the parasite’s invasion phenotype (Nery et al., 2006; Iyer et al., 2007).
Several invasion phenotypes associated with invasion ligand expression have been described for P. falciparum, thereby enabling isolates obtained from infected patients and laboratory strains to be classified into groups (Nery et al., 2006; Ochola-Oyier et al., 2016). Parasite ligand selection has notably been seen in studies involving African populations where differences in invasion ligand expression have been found.
For example, slightly higher eba175 gene expression and lower eba181 gene expression have been observed when comparing a Ghanaian population to a Senegalese one and possible correlation of this invasion pattern to patients’ immunological responses and endemicity levels (Bowyer et al., 2015).
Population studies enable analyzing invasion phenotype occurrence and the detection of the most important invasion proteins which could be exploited for developing therapeutic strategies and preventative measures (Bowyer et al., 2015; Ochola-Oyier et al., 2016).
Many studies have shown that a selected invasion phenotype is influenced by the presence/expression of genes encoding parasite ligands, the proteins on erythrocyte surface acting as receptors for them and recognition by a host’s immune system (Josling et al., 2015; Diaz et al., 2016; Valmaseda et al., 2017).
When completely adapted to in vitro conditions, parasites do not have to change because of hostile host conditions (i.e., host immune response and different erythrocyte receptor profile); however, they may change the expression of some virulence-associated genes. Strikingly, Tarr et al. (2018) have described more relaxed transcription in parasite laboratory strains compared to clinical isolates. A slight repression in invasion-related genes has been observed in clinical isolates (Tarr et al., 2018), demonstrated by comparing pfeba175, pfeba181, pfrh1, pfrh2a, pfrh2b, pfrh4, and pfrh5 transcription between six isolates from Ghanaian patients to P. falciparum 3D7, Dd2, D10, and HB3 laboratory strains, these genes’ lower expression being observed in patient isolates compared to lab strains. Also, a similar behavior has been seen regarding var gene expression in parasites infecting naïve patients compared to different isolates from high endemic populations in Africa, as naïve patients have not had the complete repertoire of antibodies to resist parasite infection and have not been able to enforce very strong immune selectivity against erythrocyte membrane protein 1 (Pf EMP1) encoded by such var genes (Abdi et al., 2016). This has thus suggested that the parasite could have a more relaxed transcription profile and such behavior may occur for var genes as well as invasion-related genes like those associated with the Pf EBA and Pf Rh protein families.
More information:Nature Communications (2019). DOI: 10.1038/s41467-019-12936-6
Journal information: Nature Communications
Provided by Seattle Children’s Research Institute