Molecular Mechanism Of Molecular Mechanism Of ParasitismParasitism

Hamid-Ur-Rahman, M.Phil Ist

Some Basic TerminologiesSome Basic Terminologies

Parasite A parasite is an organism that is entirely dependent

on another organism referred to as its host, for all or part of its lifecycle and metabolic requirements.

Host An organism which harbors the parasite and

provides the nourishment and shelter to the latter Parasitism A relationship in which a parasite benefits and the

host provides the benefit. The host gets nothing in return and always suffers from some injury.

Parasitism is differentiated from parasitoidism, a relationship in which the host is always killed by the parasite; parasitoidism occurs in some Hymenoptera (ants, wasps, and bees), Diptera (flies), and a few Lepidoptera (butterflies and moths): the female lays her eggs in or on the host, upon which the larvae feed on hatching.

Brood parasitism A form of parasitism called brood parasitism is

practiced by the cuckoo and the cowbird, which do not build nests of their own but deposit their eggs in the nests of other species and abandon them there. Though the cowbird’s parasitism does not necessarily harm its host’s brood, the cuckoo may remove one or more host eggs to avoid detection, and the young cuckoo may heave the host’s eggs and nestlings from the nest.

Fig. ParasitiodismFemale wasp lay egg on Tomato horn worm caterpillar

Fig.Brood Parasitism

Social Parasitism Another form of parasitism, such as that practiced by

some ants on ants of other species, is known as social parasitism.

Hyperparasitism Parasites may also become parasitized; such a

relationship, known as hyperparasitism, may be exemplified by a protozoan (the hyperparasite) living in the digestive tract of a flea living on a dog.

Parasitism can take the form of isolated cheating or exploitation among more generalized mutualistic interactions. For example, broad classes of plants and fungi exchange carbon and nutrients in common mutualistic mycorrhizal relationships; however, some plant species known as myco-heterotrophs "cheat" by taking carbon from a fungus rather than donating it.

Fig. Social Parasitism

Fig. HyperasatismA leaf-mining caterpillar (left) is blackened and dead. It was killed by a wasp larva

The initial interaction between parasite and host often involves invasion of a parasite into host tissue or host cells.

Single cell protozoan parasites often evade the host immune response by residing within host cells.

They must enter the host cell with minimal trauma to ensure that they preserve an environment suitable for their replication and not trigger a lethal host immune response

This intracellular invasion paradigm is also shared by a helminth parasite, Trichinella spiralis.

Most other helminths reside in the extracellular space.

Often an invasive larval form will penetrate skin or the mucosa of the gastrointestinal tract to gain entry into the host. Migration can be fairly extensive.

To complete this invasion process, helminth parasites have evolved sensory organs for finding and navigating within the host, attachment mechanisms utilizing specialized mouth structures or glue-like secretions, and hydrolytic enzymes to digest macromolecular barriers in the extracellular space.

INVASION INTO CELLS How to Enter a Cell: Phagocytosis or Invasion???? Intracellular parasitism implies entry of a pathogen into

the cytoplasm of a host cell. Various strategies have been evolved by microorganisms to achieve this goal.

Because some host cells may have the ability to internalize foreign material by phagocytosis, distinction between parasite invasion or uptake is often a challenging issue.

Phagocytosis is a peculiar type of endocytosis that is usually performed by specialized cells.

It involves binding of a particle to the cell plasmalemma through various ligand-receptor interactions (integrins, Fc receptors, hydrophobic interactions), zippering of the membrane around the particle and internalization into the resulting vacuole.

The vacuole membrane is directly derived from the plasmalemma of the cell and will be enriched in some molecules (receptors) if these were involved in the binding.

The vacuole enters the endocytic pathway and fuses eventually with lysosomes after acidification by a proton ATPase located in its membrane. Various markers of the successive compartments are known,

Fig. Shows Process of Phagocytosis

especially the lysosomal membrane glycoproteins (LGP) that are markers of late endosomes and lysosomes.

Host cell invasion by many microorganisms does not utilize the phagocytosis route and the vacuole that is formed either does not enter the endocytic pathway or is destroyed before the phagolysosome stage.

An artificial gradation can be described starting from Sporozoa that appear to have created an internalization procedure entirely distinct from phagocytosis; through Trypanosoma cruzi that may use the phagocytic system to get into the cell but then escape into the cytoplasm; to Leishmania promastigotes that are internalized and live into a mostly typical phagolysosome.

Creating a New Compartment in the Host Cell: Sporozoa

A major feature of Sporozoa is to have evolved specific structures and organelles for host cell invasion; no other group except Microsporidia has achieved such a complex differentiation.

Among Sporozoa, the Plasmodium genus has been the most thoroughly investigated.

Most of our knowledge on host cell invasion by Sporozoa has been obtained from studies of erythrocyte invasion by malaria merozoites.

Studies on other members of the group that invade nucleated cells have shown that the invasion process is not significantly different from that described in Plasmodium

The very conserved organization of the invasive stage (zoite) among Sporozoa is a key to the explanation of invasion in this group.

Indeed, all zoites of Sporozoa share a cellular polarity and specialized organelles organized into an apical complex (from Apicomplexa).

The apical complex occupies the anterior part of the cell and comprises a cytoskeleton (anterior rings, subpellicular microtubules, inner membrane complex) and vesicular electron dense organelles (rhoptries, micronemes, dense granules). All the components of this structure are considered to be involved in host cell invasion.

The invasion phenomenon itself can be divided into three successive steps: recognition and attachment, internalization and vacuole development, vacuole maturation

o Recognition and attachment Surface to surface binding between zoite and host cell

is supposed to involve not only zoite surface molecules and host cell surface, but also bridging molecules exocytosed by the zoite (erythrocyte binding antigen in Plasmodium falciparum, 135kDa Duffy receptor in P. knowlesi or P. vivax, both of which are contained in micronemes) or components of the extracellular matrix (laminin in Toxoplasma).

The red cell surface receptors used by P. falciparum which include glycophorins, sialic acids and band 3 are still incompletely identified. P. vivax and P. knowlesi use different receptors among which is a 45 kDa glycoprotein that carries the Duffy determinant.

A reorientation step has been observed in Plasmodium after attachment.

It may result from a gradient of receptor distribution on merozoite surface, or from the presence of apically located higher affinity receptors.

This reorientation brings the apex in contact with the cell membrane, which is necessary for internalization. Such a passive reorientation step may not be present in other Sporozoa where gliding motility or conoid flexing could bring about the apical contact.

The recognition or receptor-ligand interaction between Plasmodium and its host cell is highly specific as illustrated by the very narrow range of invasion capabilities of these organisms.

The matter is much less clear for other Sporozoa, the prototype of which is Toxoplasma.

These can invade any type of cell in vitro except the erythrocyte, and the efficiency of invasion may be

modulated by receptor-ligand interaction or a ubiquitous membrane molecule such as cholesterol might serve as a receptor triggering invasion for these zoites.

o Internalization, parasitophorous vacuole formation

Motility and moving junction. Once a zoite has made an apical contact with the

target cell, there is a close junction between both plasmalemma.

Freeze fracture shows a rhomboidal array of intramembrane particles which is likely to be a crystalline array of lipids.

As demonstrated in P. knowlesi, all the preliminary steps can occur in the presence of cytochalasin B that inhibits both the zoite motility and invasion..

This has been observed in all Sporozoa studied so far and strongly suggests that invasion operates by using the gliding motility system of the zoites.

This is likely to involve an actin-based motor located in the inner membrane complex of the zoite .

The apical junction turns into a annulus through which the zoite glides into the developing vacuole.

Vacuole membrane The origin of the vacuole membrane is unclear. It is in

continuity with the plasmalemma of the host cell through the moving junction during the invasion process, but its structure is dramatically different.

It is almost completely devoid of intramembranous particles or host cell surface proteins.

Contribution of host cell to the vacuole membrane is therefore probably restricted to lipids.

Exocytosis of organelles The zoite contribution to the vacuole is believed to

occur through the exocytosis of specialized apical organelles named rhoptries.

Rhoptries are pedunculated organelles, the ducts of which extend toward the apex of the zoite and are open for exocytosis during the invasion.

A complex set of proteins, some of which are grouped in families, have been identified in these organelles in both P. falciparum and Toxoplasma.

Some of these are associated with the developing vacuole membrane, thus confirming the exocytic process .

Ultrastructural (Plasmodium) or biochemical (Toxoplasma) data on rhoptries suggest the presence of lipids that could also be inserted in the vacuole membrane upon exocytosis.

Enzyme activities such as proteases and phospholipases may be involved in invasion. These activities could modify cell surface proteins or lipids or the underlying cytoskeleton during formation of the vacuolar membrane.

Vacuole maturation Once the vacuole is completed a maturation step

occurs, during or shortly after invasion, that is characterized by the exocytosis of dense granules from the parasite.

The proteins of the dense granules are targeted to either the vacuolar space , a vacuolar membranous network and the vacuole membrane or the inner side of the host cell membrane

Vacuole maturation reflects the development of metabolic exchanges between parasite and host cell

The vacuole is entirely isolated from the endosomal traffic of the host cell and the parasite must rely on transmembrane transport to get its nutrients.

The contribution of the parasite to the vacuole includes the necessary transporters or channels in that membrane.

In P. falciparum the existence of direct channels between the vacuole and the extracellular space has been claimed.

A peculiar type of vacuole maturation occurs in Piroplasma where the vacuole disappears after the exocytosis of dense granules .

The organism develops directly in the cytoplasm of the host cell. The mechanism of vacuole lysis is not known.

This type of intracellular behavior is morphologically shared with Trypanosoma cruzi , but whether any molecular function is shared remains to be investigated.

o Escaping from a Phagosome into the Cytoplasm: Trypanosoma cruzi

This organism has an intracellular phase in the vertebrate host only and invasion is performed by trypomastigotes derived from the insect gut or previous round of intracellular multiplication.

Although these organisms are highly motile, their active involvement in the invasion has long been controversial.

Fibronectin augments the internalization of T. cruzi in phagocytic and non-phagocytic cells .

It acts as a molecular bridge between parasite and host through attachment to a beta 1 integrin.

Attachment is energy dependent on the part of the parasite and does not need host cell metabolism since fixed cells can be attached and invaded

Although entry of T. cruzi in phagocytes is believed to be mediated by phagocytosis, typical phagocytic receptors (FcR, CR3) do not seem to be involved .

Penetration in non-phagocytic cells seems to be an active process that is not blocked by cytochalasin D, which inhibits actin-based systems .

During the first 60 min, T. cruzi resides in a vacuole that acquires lysosomal glycoproteins in its membrane which means that it fuses with endocytic vesicles en route to lysosomes.

Fig. Showing, Mechanisms of T.cruzi Entry in to the cell

The vacuole acidifies and a pore-forming protein, antigenically similar to C9, the membranolytic component of complement, is released in the vacuole.

The vacuole membrane becomes discontinuous and disappears leaving the parasite directly in the cytoplasm where it transforms into amastigotes.

o Living in a Phagosome- Leishmania Infectious forms of Leishmania (metacyclic

promastigotes) possess two types of surface molecules involved in attachment and invasion.

These are the lipophosphoglycan (LPG) and the major surface protease GP63.

Both of these ligands can independently mediate attachment of the parasite to macrophages

Leishmania do not invade nonphagocytic cells.

Internalization may require synergy of both parasite ligands as has been shown with artificial systems of beads coated with both molecules separately or associated.

The cellular receptors involved in binding and internalization may be multiple but the complement component CR3 seems to be a major receptor that binds both LPG and GP63.

The vacuole containing the parasite is acidified and its membrane becomes LGP-positive but MPR (mannose-6-phosphate receptor) negative.

This corresponds to a lysosomal compartment. Only some extracellular ligands appear in the vacuole via receptor-mediated endocytosis; this means that it fuses only with some of the receptor-mediated endocytic pathways

Fig.Living in a Phagosome-Leishmania

The survival and development of Leishmania sp. within the hostile environment represented by the lysosomal contents is supposed to be mediated by several protective mechanisms including the inhibition of the macrophage respiratory burst by LPG and the activity of parasitic enzymes (proteases, superoxide dismutase and acid phosphatase) that may counteract the activity of lysosomal enzymes.

o A Multicellular Organism Within a Cell: Trichinella

The L1 larvae of this nematode are born in the lamina propria of the gut, travel in the blood and eventually enter a striated skeletal muscle cell.

The mechanism of host cell penetration is not known but what has been extensively studied is the transformation of the host cell by the parasite after entry.

The multinucleated muscle fiber loses the myofibrils that are replaced by smooth membranes and mitochondria; the nuclei enlarge and develop prominent nucleoli; the cell glycocalix is replaced by a thick collagen coat and around the nurse cell thus formed, angiogenesis is triggered and a circulatory plexus develops.

This transformation reflects a dramatic alteration of the host cell. This is believed to be triggered by the worm which sends information modifying gene expression.

The exact nature of the message is not known but proteins secreted in the cell by the larva are likely to play a role in this modulation.

A 43 kDa glycoprotein exocytosed from granules found in specialized cells of the worm (stichocytes) is

Fig. Trichinella in Muscle Cell

targeted to the nuclei of the host cell where it can be immunodetected and may play a role in modulation of host genomic expression; other secreted parasite molecules could also be involved in this transformation.

o Injection into a Cell: Microsporidia These parasites are quite original with respect to

invasion since they inject themselves through the plasmalemma of the host cell.

The microsporidian spore is a highly organized system of a coiled hollow polar filament that everts at excystation to open a hole in a target cell and inject the microsporidium.

The parasite then develops directly in the cytoplasm of the host cell.

Triggering of the spore discharge seems to be initiated by ionic changes in the medium

INVASION BY HELMINTHS Helminths are multicellular parasites, often a

millimeter or more in length. With rare exceptions (see Trichinella), invasion of and

seclusion within host cells is not feasible. When speaking of host invasion by helminth

parasites, one considers tissue invasion or, more specifically, migration through extracellular barriers.

Different helminth parasites may have quite distinct pathways of invasion and migration in the host, and may employ different mechanisms to facilitate that invasion

The port of entry for several parasitic nematodes and trematodes is the skin.

However, even within this group, the exact mode of entry may vary significantly.

Invasive larvae of Stronglyloides, hookworm and schistosomes may enter the skin directly, without the need for an insect vector bite or accidental trauma.

Larvae of Bruoia and Wuchereria are deposited on skin from the salivary gland of the mosquito vector and follow the mosquito bite path to a lymphatic vessel.

The L3 larvae of Onchocerca, are also deposited at the vector bite site, but then migrate for a considerable distance through dermal connective tissue before reaching their final site of adult residence.

The intestinal wall is another barrier for helminth parasite invasion.

Here, the common endpoint of invasion is the lumen of a blood vessel, with subsequent dissemination to multiple organs.

Schistosome eggs must leave the lumen of mesenteric blood vessels and cross the intestinal wall in a path exactly opposite that of invasive gastrointestinal parasites.

o Pathways of Helminthes Invasion Specific Steps in the Invasion Process1). Tactic responsesFor the parasite entering the host from the

external environment, this is the process by which it finds the host.

For the parasite in the lumen of the gut or in the bloodstream, this is the process that defines where it will go and, equally important, when it will stop.

2). Attachment Prior to invasion it is necessary for parasites,

particularly those coming from the external environment, to attach to the host.

During the actual process of invasion, cyclical attachment and release from extracellular matrix or other structures may be necessary for motility

3). Digestion of macromolecular barriers Particularly in migration through skin and

connective tissue, the interactive extracellular matrix must be breached before a multicellular organism can invade

4). Evasion of host immunity Implicit in the ability to invade is the ability to

evade host responses that would block parasite migration or be directly lethal to the organism

A). Tactic responses and migration Some information is available for those helminth

parasites which enter the host from external environments.

Schistosome cercariae and larvae of Stronglyloides and hookworm respond to specific signals that identify the host as such and have been the most extensively studied.

A response to light ensures that schistosome cercariae reside in the same part of a pond or lake as the host.

Schistosome cercariae which infect humans are shed from the intermediate host snail under stimulus of light following a period of darkness.

Schistosoma douthitti, in contrast, which infects nocturnal mammals such as voles and muskrats, leaves the snail in darkness following a period of light.

Cercariae of Diplostomums pathaceum, which infect fish, are stimulated to swimming bursts by water turbulence or touch; Trichobilharzia ocellata, which infects ducks, is stimulated by shadows.

To contact host skin and invade, schistosome cercariae follow a thermal gradient and then use an elegantly adapted penetration response triggered by specific fatty acids present on the skin.

Specific free fatty acids like linoleic acid will stimulate cercariae to invade in vitro.

Whether this is receptor-mediated is not known, but cercariae can metabolize linoleic acid to eicosanoids , which are potential 'second messengers'.

Upon stimulation cercariae lose their glyocalyx and tails and release a protease from preacetabular gland cells to facilitate invasion.

Hookworm and Stronglyloides larvae enhance their chance of encountering host skin by aggregating in clusters at the highest points of blades of grass .

Hookworm larvae also recognize mammalian hosts by sensing CO2 and are stimulated to penetrate by host skin proteins .

The molecular details of this interaction have yet to be elucidated.

Cercariae of A. brauni do not recognize small molecules such as amino acids, monosaccharides or electrolytes, but do respond to hyaluronic acid and glycoproteins for attachment.

Penetration of the host is triggered by free fatty acids and mucus components present on the fish skin surface .

Monogenean parasites of fish skin lay eggs which fall to the sea or river bottom.

Hatching of eggs of Entobdella soleae is timed by photoperiod to occur just after dawn, when the common sole (Solea solea) begins resting on the sandy bottom.

Hatching is also stimulated by mucus from the host fish skin.

The ciliated oncomiracidium that emerges is phototactic and may also sense currents .

Thermosensing undoubtedly plays a role in the directional migration or invasion of some nematode parasites as it does in schistosome cercariae.

Caenorhabditis elegans larvae and adults migrate to the temperature at which they have been growing when they have been placed in thermal gradient.

Mutants with thermotactic abnormalities have been isolated. Many of them have lost the ability to migrate towards a specific temperature, whereas others show reversal of the usual behavior.

Many, although not all, of these thermotactic mutants are also defective in chemotaxis.

Chemotaxis was one of the first sensory behaviors noted in C. elegans

Worms are attracted to cyclic nucleotides, both anions and cations, some amino acids, and extracts of bacteria.

All of the chemotactic mutants have been morphologically mapped to specific cells with corresponding sensory abnormalities in the head of the worm.

Onchocerca cervipedis, (which infects deer)The adult Onchocerca live in the hindquarters of the deer and the microfilariae migrate across the body of the deer, up the neck, and into the ears.

The blackfly vectors recognize the upright ears of the deer and congregate there.

Movement in aqueous environments of the ciliated miracidia and fork-tailed cercariae of schistosomes, and oncomiracidia of monogenean fish parasites, is very rapid.

o Attachment Many nematode parasites, such as hookworm adults and

Anisakis adults, have evolved mouth structures which allow attachment to intestinal mucosa .

Monogenean parasites of fish skin, which must attach to a rapidly moving host and resist water shear, also have highly evolved attachment structures

Schistosome cercariae emit a sticky mucus substance from the posterior acetabular glands, which allow them to attach to each other as well as to skin

Cercariae may be induced to cluster together on skin in response to L-arginine emitted from their secretory glands during the initial stages of skin exploration .

o Digestion of Macromolecular Barriers Although it is plausible that enzymes such as

hyaluronidases, glycosidases or lipases may digest certain barriers that face invading helminths, evidence to date suggests that these functions are largely performed by proteolytic enzymes.

The best evidence is from studies of Strongyloides L3 larvae and schistosome cercariae.

In both of these cases, secreted proteases with the capacity to degrade connective tissue or basement membrane molecules have been identified.

Specific inhibitors of these proteases will inhibit invasion of skin by the larvae.

Related proteases have been identified in many other parasites, including hookworm , Anisakis , Ascaris , Toxocara and Onchocerca.

The enzymes discovered in excretory/secretory products of invasive stages of helminth parasites also might be important in anticoagulation, digestion, immune evasion, or morphologic transformation.

The most direct test of the function of these enzymes is to show that blocking the action of the protease by irreversible inhibitors, or active-site-directed antibodies, inhibits invasion.

Entamoeba histolytica---a Protozoan Parasite that Invades Extracellularly

In contrast to the other protozoa , Entamoeba histolytica is not at any stage in its life cycle an intracellular parasite.

Rather, it is more like the helminth parasites in that it invades tissue extracellular matrix, producing destructive lesions in the wall of the colon, and has the capacity to metastasize to other organs .

A second distinctive aspect of infection by E. histolytica is that invasion of the host is by no means necessary for parasite replication or transmission.

Only 10% or less of all cases of amebiasis result in invasive infections.

First, trophozoites of E. histolytica have specific surface lectins and adhesion-mediating glycoproteins which allow……

attachment to intestinal mucus, target cells or host extracellular matrix.

Attachment appears to be necessary for target cell lysis, which is mediated at least in part by a cytolytic ion channel-forming protein secreted by the trophozoites.

Pathogenic, invasive trophozoites of E. histolytica also release proteinases.

The major enzyme is a cysteine proteinase with cathepsin B-like substrate specificity and structural homology to members of the papain superfamily.

Release of this enzyme correlates with pathogenicity of clinical isolates and induces an immune reponse in infected individuals.

Direct inhibition of this enzyme by specific, irreversible cysteine protease inhibitors blocks the cytopathic effect of trophozoites

It also produces a specific cleavage in complement factor C3, producing biologically active C3 cleavage products.

Other potential virulence factors associated with invasion by E. histolytica include a metallocollagenase and phospholipases

As a group, the virulence factors identified in extracts or secretions of pathogenic E. histolytica can explain in large part the cytolysis and tissue destruction that characterize E. histolytica invasion.

Host neutrophils also contribute to the tissue destruction seen in amebic liver abscesses .