Life Cycle of Plasmodium
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.
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).
