MRC National Institute for Medical Research, London
Science for Health
Parasite invasion and replication
29 December 2010
Many pathogenic microorganisms live inside the cells of the host organism they are infecting. Apicomplexan parasites are an important group of single-celled pathogens which includes Toxoplasma gondii, the causative agent of toxoplasmosis, and the malaria parasite. These parasites actively invade host cells and then divide within them. T. gondii can invade virtually any cell while the malaria parasite invades only liver cells and red blood cells in its human host. In all cases, the parasite begins to divide immediately after invasion but the signals or cues that ‘inform’ the parasite it is safely inside a cell are unknown.
Prior to invasion the parasite discharges transmembrane proteins onto its surface to mediate invasion. These are shed by intramembrane cleavage, a process associated with invasion but otherwise poorly understood. A protein called apical membrane antigen 1 (AMA1) is crucial for invasion in both Toxoplasma and the malaria parasite.
Mike Blackman (pictured), in NIMR’s Division of Parasitology, collaborating with Dominique Soldati-Favre at the University of Geneva, studied this process in Toxoplasma, which is much more readily manipulated in the laboratory than the closely-related malaria parasite. Exploiting up-to-date developments in genetic manipulation of Toxoplasma they used a conditional expression system to show that over-expression of a dominant-negative form of the rhomboid enzyme (called ROM4) produced a severe defect in parasite replication, without affecting invasion. This defect could be completely overcome by expression of the cytoplasmic ‘tail’ of AMA1 from either Toxoplasma or the human malaria parasite Plasmodium falciparum. The results suggest that the action of ROM4 on AMA1 provides the signal to the intracellular parasite to begin replication.
As demonstrated previously for many other pathogens, a better understanding of the molecular basis of pathogenesis can lead to the design of new or improved drugs or vaccines. Progression of the diseases caused by apicomplexan parasites absolutely requires that they multiply within infected individuals, but prior to this work we had no clues as to how they are triggered to start replicating following invasion. This work provides the very first information on the regulation of this important aspect of the parasite life cycle. Importantly, both AMA1 and the rhomboid enzyme (a protease called ROM4) are found in virtually all apicomplexan parasites, suggesting a common mechanism of action across this large group of microorganisms.
Regulated intramembrane proteolysis (RIP), in which proteases in biological membranes clip other membrane proteins, is well-recognised as a powerful and precise mechanism for signal transduction, triggering a signaling cascade by release of sequestered protein domains. Whereas all previous studies examining rhomboid-mediated RIP have concluded that rhomboids exclusively release signals to the exterior of the cell, this study has revealed a mechanism by which a cytosolic rhomboid cleavage product is implicated in the resulting signal. This study therefore represents the first evidence for a direct, intracellular signaling role for a rhomboid protease analogous to that performed by other classes of proteases that mediate RIP. The Apicomplexan life cycle consists of consecutive transmissive and replicative phases. Premature differentiation into replicative forms would be potentially lethal, so commitment to cell division needs to be tightly regulated in time and space. This study highlights a novel role for one of the most conserved apicomplexan microneme proteins and shows that this group of parasites has opted to use invasion molecules as signaling factors for replicative growth.
Apicomplexan parasites are widespread and cause a range of different diseases. Infection of immunocompromised patients (e.g. transplant patients or patients with AIDS) with Toxoplasma can be lethal, and even in healthy women infection with Toxoplasma during pregnancy can cause severe congenital defects in the newborn child. Cryptosporidiosis, caused by Cryptosporidium, causes outbreaks of acute though generally self-limiting diarrhea. Malaria is a huge global health threat, resulting in over 1 million deaths per annum, largely in children below the age of 5. Our work suggests new ways to combat these and other diseases caused by this group of parasites; for example, drugs that inhibit the rhomboid enzyme ROM4 would be expected to block parasite growth and halt progression of the disease.
Mike Blackman
ddROM4S-A parasites arrest late in the cell cycle
Click image to view at full-size
Indirect immunofluorescence assay of ddROM4S-A parasites after 24 h ± Shld-1 showing the mitochondrion (α-F1 ATPase beta subunit, green) broken down (A); replication arrested after a single round of division as determined by staining of the nascent apical cones of the mother and daughter parasites (α-ISP1, green) only in non-treated parasites (B, arrows); and defective karyocytokinesis with the nuclei enlarged and uncondensed (C, arrows). In red, parasites are labeled with α-GAP45.
Original article
Joana M. Santos, David J. P. Ferguson, Michael J. Blackman, Dominique Soldati-Favre (2010).
Intramembrane cleavage of AMA1 triggers Toxoplasma to switch from an invasive to a replicative mode
Science, epub ahead of print. Publisher abstract.
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