Vernon B. Carruthers
Pathogenesis of parasitic infections: Mechanisms of cell invasion and survival during infection
The Carruthers lab seeks to understand invasion and survival strategies employed by microbial pathogens during infection. We use the protozoan Toxoplasma gondii as a model pathogen because of its genetic and biochemical tractability, well defined cellular structure, and the availability of excellent rodent models of disease. Toxoplasma replicates in a remarkable variety of cells and organs, causing encephalitis, pneumonia, myocarditis, ocular disease, and congenital birth defects during acute infection. While disease is seen only in a small fraction of the ~2 billion people infected worldwide, the diagnosis and treatment of toxoplasmosis are suboptimal and the disease remains a significant public health problem. In people with healthy immune status, the parasite typically remains in a chronic, encysted state, but the infection can erupt when immune function is compromised such as individuals with HIV/AIDS, organ transplant recipients, or cancer patients undergoing chemotherapy.
As an obligate intracellular pathogen, Toxoplasma critically relies on cell invasion as a major survival strategy to avoid host antibody defense and phagocytic clearance. Cell invasion also initiates the parasite’s lytic cycle that ultimately destroys the infected cell, causing direct tissue pathology and indirect inflammatory damage. We and our collaborators have shown that Toxoplasma uses a battery of adhesive protein complexes to recognize and bind host cells prior to invasion. Many of these adhesive proteins reside in specialized secretory organelles called micronemes (Greek: small threads) that are discharged when the parasite has identified a suitable site for cell invasion. We have shown that parasites conditionally deficient in one particular complex (TgMIC2-M2AP) are invasion incompetent, partially defective in gliding motility, and fail to kill mice even at high doses. The deficient strain also acts as a live-attenuated vaccine since surviving animals are protected from subsequent challenge with a lethal parasite strain. Our team is actively investigating how adhesive complexes such as TgMIC2-M2AP are assembled and shuttled to invasion organelles, in addition to identifying novel invasion-related proteins that may contribute to the parasite’s broad host range.
From another prospective, we also seek to understand what roles the host cell plays in cell invasion by Toxoplasma. Although the motive force for cell invasion is clearly parasite-derived, we propose that a number of host molecules are recruited and manipulated by the parasite to rapidly traverse the host actin cytoskeletal system and moments later pinch off the host plasma membrane to create the intracellular compartment in which replication ensues. Indeed, an astonishing degree of molecular coordination must be employed during the 20 seconds or so the parasite takes to invade a target cell. Our goal is to understand the orchestration of parasite and host proteins involved in this uncommonly rapid entry phenomenon.
The team has also identified parasite proteases that act upon adhesive complexes both within the parasite in preparation for cell invasion and on the parasite surface during parasite entry. In addition to facilitating invasion by proteolytically modifying adhesins, our findings imply that one such protease (TgCPL) may also function during intracellular replication since it occupies a novel and dynamic digestive compartment that is likely part of the parasite’s endocytic system. We are using selective inhibitors of these proteases to determine substrate range, their specific role in invasion and replication, and to test the efficacy of inhibitory compounds for ameliorating infection.
To navigate the above research avenues, we use a diverse array of approaches including molecular genetics, proteomics, bioinformatics, video and fluorescence microscopy, structural biology, mathematical modeling, and bioluminescent imaging.
Gaji, R., Flammer, H.P., and Carruthers, V.B. (2011) Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids. Traffic. 12;840-853.
Laliberté, J. and Carruthers V.B. (2011) Toxoplasma gondii toxolysin 4 is a putative metalloproteinase secreted from micronemes. Mol. Biochem. Parasitol. 177:49-56.
Gaji, R., Behnke, M.S., Lehmann, M.M., White, M.W., Carruthers, V.B. (2011) Cell cycle-dependent, intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression. Mol. Microbiol. 79;192-204.
Dou, Z. and Carruthers, V.B. (2011) Cathepsin proteases in Toxoplasma gondii. Adv. Exp. Med. Biol. 712:49-61.
Parussini, F., Coppens, I., Shah, P., Diamond, S.L., and Carruthers, V.B. (2010) Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exosomal system of Toxoplasma gondii. Mol. Microbiol. 76;1340-1357. ["MicroCommentary" in the same issue]
Kafsack, B.F.C. and Carruthers, V.B. (2010) Apicomplexan perforin-like proteins. Commun. Integr. Biol. 3;18-23.
Larson, E.T., Parussini, F., Huynh, M.-H., Giebel, J.D., Kelley, A.M., Zhang, L., Bogyo, M., Merritt, E.A., and Carruthers, V.B. (2009) Toxoplasma gondii Cathepsin L as the primary target of the invasion inhibitory compound LHVS. J. Biol. Chem. 284;26839-26850.
Huynh, M.-H. and Carruthers, V.B. (2009) Endogenous gene tagging in a Toxoplasma strain deficient in non-homologous recombination. Euk. Cell. 8;530-539.
Roiko, M.S. and Carruthers, V.B. (2009) New roles for perforins and proteases in apicomplexan egress. Cell. Microbiol. 11;1444-1452
Kafsack, B.F.C., Pena, J.D.O., Coppens, I., Ravindran, S., Boothroyd, J.C., and Carruthers, V.B. (2009) Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science 323;530-533.
Brydges S. D., Harper, J. M., Parussini, F. Coppens, I., Carruthers V.B. (2008) A transient forward targeting element for microneme regulated secretion in Toxoplasma gondii. Biol. Cell 100;253-264.
Laliberté , J. and Carruthers, V.B. (2008) Host cell manipulation by the human pathogen Toxoplasma gondii. Cell. Mol. Life Sci. 65:1900-1915.
Carruthers, V.B., Tomley, F.M. (2008) Receptor-ligand interaction and invasion: Microneme proteins in apicomplexans. Subcell. Biochem. 47;33-45.
Hager, K.M. and Carruthers, V.B. (2008) MARveling at parasite invasion. Trends Parasitol. 24;51-54.