Invasive stages of Plasmodium spp

 Life Cycle of Plasmodium

The life cycle of Plasmodium spp, a causative agent of malaria is divided into three cycles; sporogonic, exoerythrocytic and erythrocytic cycles. The Anopheles mosquito is the definitive host while humans or animals are the only intermediate hosts for the parasite. During a blood meal, the mosquito injects sporozoite forms into the blood circulation of humans. The sporozoites move to the liver within a few minutes and invade hepatocytes, which they develop to produce exoerythrocytic merozoites that are released into the bloodstream again. These merozoites invade erythrocytes and grow into trophozoites and mature schizonts. Merozoites that are released from schizonts reinvade new erythrocytes. Few of trophozoites are changed into the gametocytes through the asexual blood-stage which are taken up by a feeding mosquito into their gut. These gametocytes get matured to form male and female gametes. They fertilize to form a zygote and develop to an ookinete and an oocyst which are finally developed into sporozoites within the mosquito gut. The sporozoites migrate to the salivary glands and the cycle again repeats when a mosquito bites a new host which takes around 30 days.

Figure 1: Life cycle of Plasmodium spp showing different cycles and different forms (Source: CDC)

Invasive stages:

Among the various forms of Plasmodium spp, sporozoites, merozoites and ookinetes are the invasive forms. The sporozoites and merozoites are invasive stages of hepatocytes and red blood cells respectively of the vertebrate host while ookinetes invade the mosquito gut epithelial cells.

1.      Sporozoites:

Sporozoites are the most versatile stage in the life cycle of Plasmodium which is formed in the invertebrate host; mosquito and eventually differentiate in the vertebrate host; humans and animals. Sporozoites are developed within the oocysts in the midgut epithelium over the course of 10 days or 4 weeks depending on the species and environmental temperature. Normally it takes less than 7 days if the ambient temperature is 30°C or more. Sporozoites are crescent-shaped ranging from 8 x 14 mm in diameter (Figure 2). 

 

The sporozoite and salivary gland invasion in the mosquito: The motile sporozoites egress into the circulatory fluid; hemocoel through holes from the weak area in the oocyst wall. Holes are possibly produced by a combined effort of the muscular action of the gut wall and the activity of the sporozoites. Within the hemocoel, the sporozoites are distributed throughout the body of the mosquito including the maxillary palps within a day or two of their release from oocysts. These sporozoites can’t adhere to most of the tissues however they invade salivary glands and rarely midgut wall, hemocytes or thoracic muscles. The sporozoites invade the salivary glands where they accumulate and remain until delivery. Sporozoites preferentially penetrate the medial lobe and the distal portions of the lateral lobes of the salivary glands where the salivary duct is not chitinous in Anopheles species. It is estimated that hundreds of sporozoites per oocyst reach the salivary glands. Their motility is normally restricted at salivary glands and accumulated in the salivary cavities. However, they can also move to the narrow salivary duct connecting to the proboscis until they are inoculated into the vertebrate host via blood meal.

The sporozoite and hepatocytes invasion in the human: During bloodsucking by mosquito onto the host, the sporozoites are transferred to the skin of the vertebrate host. Most of the sporozoites are deposited in the dermis of the host. They can penetrate the skin and enter into the blood vessels and lymphatic systems.  The sporozoites entering the blood vessels are carried to the liver within a few minutes while those which reach to lymph nodes have to fight against the host immune response to survive. A fraction of sporozoites are inactivated by preformed neutralizing antibodies among those who are infected with malaria before. The remaining are bound by dendritic cells and stimulate the humoral and cellular immune response of the host including B-cells, CD4+ T cell and CD8+ T cells.  

Inside the vertebrate host, sporozoites undergo dramatic changes in their surface protein structure and migrate actively to reach the hepatocytes. They use surface coat proteins such as circumsporozoite protein (CSP), thrombospondin related adhesive protein (TRAP) and P36 to interact with host receptors in the hepatocytes to facilitate the entry. CSP is the most abundant surface protein and plays important role in the development, motility and active invasion of sporozoites to hepatocytes. It is also one of the major antigens in the sporozoites which are the targets of many vaccines. TRAP proteins are located in the plasma membrane and translocated from the anterior to the posterior for the invasion, and also involved in the gliding motility. P36 protein is a 6-cysteine domain protein that directly binds to the CD81 receptor (P. falciparum and scavenger receptor BI (SR-BI) (P. vivax) of the hepatocytes. The sporozoites undergo multiplication in liver cells into thousands of merozoites.

 

2.      Merozoites:

Merozoites are another invasive form of Plasmodium which are the smallest eukaryotic cells measuring 1-2 mm in size and are non-motile. They are ruptured from hepatocytes into the bloodstream where they invade the circulating erythrocytes. A typical merozoite structure looks like an electric bulb and contains an apical complex of secretory organelles (microneme, rhoptries and dense granules), mitochondria, nucleus and plastids. The inner membrane complex (IMC) just underlies the plasma membrane. The initial attachment of merozoites to erythrocytes involves weakly binding of merozoite surface protein-1 (MSP-1) of the parasite to a glycosylphosphatidylinositol (GPI) anchored protein.  The dramatic movement of merozoites and deformation of erythrocytes leads to the reorientation process of merozoites directing apex abutting to the host membrane.  Most of the interacting ligands are present on the apical end of merozoites. The commitment for invasion however occurs only after the binding of erythrocyte binding like proteins (EBA/EBL) and reticulocyte binding homologs (Rh proteins) to host membrane surface proteins. A number of erythrocyte binding like proteins (EBA/EBL); EBA-140, EBA-175, EBA-180, EBL-1 and reticulocyte binding homologs (Rh proteins); PfRh1, PfRh2a, PfRh2b, PfRh4, PfRh5 are associated with glycophorins, complement receptor-1 and unknown host receptors.  These protein-protein interactions facilitate the RON complex formation in which RON2 complexed with AMA-1 (Apical membrane protein). This junction triggers the release of the rhoptry bulb, providing proteins and lipids required for parasitophorous vacuole membrane to establish the space into which the merozoites can move as it invades. The entry of merozoite is powered by the actomyosin motor complex. All the surface proteins are cleaved during the invasion. Inside the vacuole, they digest hemoglobin for amino acids nutrient and side by side they detoxify the heme compound into hemozoin, a neutral non-toxic for the parasites. They rapidly multiply and develop into ring, trophozoite, and schizont stages, culminating in the formation of 16 to 32 mature merozoites. Each of these merozoites can invade a fresh erythrocyte and continue the cyclic, asexual blood-stage development.

 

Figure 3: Molecular structure of Merozoites (Source: Cowman et al., 2012) 

Ookinete

Alternative to asexual life cycle in the vertebrate host, some of the erythrocytic parasites differentiate into sexual forms called gametocytes. These intracellular erythrocytic forms take around 10 days (normally in P. falciparum) to develop into fully mature gametocytes.  The factors initiating and regulating gametogenesis are still not clear however few studies suggested that the harsh environmental condition in the vertebrate host due to antimalarial drugs and host immune response induce the gametocytes formation to escape the situation. These gametocytes show the sexual dimorphism with two distinct forms; microgametocyte and macrogametocyte. They don’t cause any harm to the host however they are the infective form for mosquito. The mature and functional gametocytes ingested by female Anopheles mosquitoes during bloodmeal are stimulated to transform into the gametes by environmental stimuli; pH, temperature and enzymatic activities in the mosquito midgut. Under the influence of changes, the gametocytes become extracellular within 8-15 min of ingestion. Soon after the exflagellation (bursting from red blood cells), the microgametes (male gametes) fertilize the macrogametes (female gametes) within the next one hour of the ingestion of blood. The fertilized macrogamete (zygote) differentiates into a single motile ookinete over the next 10-25 hrs which is an infective form for mosquito.

A mature ookinete is an elongated motile cell size ranges from 7 to 18 mm in length and 2 to 4 mm in diameter. Ookinetes use the anterior half part of the body for a linear or snake shape gliding locomotion. The upper part of ookinete is the apical complex, possessing secretory organelles called micronemes that contain the proteins involved in motility, tissue traversal and invasion.  A de novo synthesis during the transformation from zygotes to ookinete identified more than 90 proteins synthesized, most of which are involved in motility and invasion.  

Figure 4: (a) Structure of ookinete (Wikipedia) and (b) Invasion to mosquito midgut (Bennink et al., 2016

This ookinete migrates through the liquid of the alimentary bolus and passes through the defensive barrier and the microvillar network of the peritrophic matrix to invade epithelial cells in a mosquito’s stomach. The secretary organelles of ookinetes produce chitinase enzyme which seems to break down the peritrophic matrix layer. Few more enzymes involved in motility and infectivity of the ookinete are the micronemal proteins CDPK3 (calcium-dependent protein kinase 3) and CTRP (circumsporozoite and TRAP-related protein). Targeted disruption of both of these proteins make the ookinete immotile and fail to invade the midgut epithelium. After breaching the peritrophic matrix, the ookinete then penetrates the apical end of the mosquito midgut epithelium. A candidate for initial host cell membrane disruption and penetration by the ookinete is a micronemal protein with a perforin-like membrane attack complex domain called the membrane attack ookinete protein (MAOP).