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Vernon B. Carruthers

Ph.D, University of Western Ontario, 1991

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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 in only a small fraction of the ~2 billion people infected worldwide, the diagnosis and treatment of toxoplasmosis are suboptimal and the disease remains a significant and neglected 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 lytic cycle that ultimately destroys the infected cell, causing direct tissue pathology and indirect inflammatory damage. Our collaborators and we have shown that Toxoplasma uses a battery of adhesive protein complexes to recognize and bind host cells prior to invasion. Many of these adhesins reside in specialized secretory organelles called micronemes (Greek: small threads), which are discharged when the parasite has identified a suitable site for cell invasion. We have shown that parasites conditionally deficient in one particular complex (MIC2-M2AP) are invasion incompetent, partially defective in gliding motility, and fail to kill mice even at high doses. Our team has revealed how adhesive complexes such as MIC2-M2AP are assembled and shuttled to invasion organelles. Our most recent work involves determining the function of novel invasion-related proteins that are conserved amongst Toxoplasma's kin including the malaria parasite. Understanding the functions of such conserved invasion proteins has the potential to widely impact the development of vaccines or novel therapeutics.

The team is also exposing the functions of parasite proteases including most notably a digestive enzyme termed cathepsin protease L. We showed that cathepsin protease L is the first marker of a novel and dynamic parasite digestive organelle we termed the vacuolar compartment or VAC. We are using selective protease inhibitors and genetic mutants to determine the protease substrate range, specific role in invasion and replication and to test the efficacy of inhibitory compounds for ameliorating latent infection. Of note, these studies are revealing for the first time that the parasite internalizes and digests material from the host cell cytoplasm during intracellular replication. We are investigating the extent to which the parasite uses this uptake pathway to satisfy its nutritional demands, thwart host immune effectors and support the long-term survival of the parasite in neural cysts.

Our group is also keening interested in understanding mechanisms underlying parasite egress from host cells after replication therein. We have shown that a cytolytic protein (perforin-like protein 1) is crucial for efficient egress and is necessary for lethal infection in experimentally infected mice. Recent findings suggest that the cytolytic protein is activated by low pH to aid in parasite egress and that, conversely, its activity is suppressed during parasite invasion to ensure membrane integrity and proper entry. The activity of the cytolytic protein is also dictated by exposure to acidic phospholipids, which act as receptors that dictate the directionality of cytolytic activity for egress. Members of the team are also revealing the contributions of a secreted protease and phospholipase to parasite egress. Together, these studies challenge the previous notion of passive egress and suggest that Toxoplasma escapes from cells by secreting several effector proteins that function to disrupt physical barriers enveloping the parasite.

Having established a mouse model of latent Toxoplasma infection, we are also testing novel experimental compounds for efficacy in diminishing the chronic infection characterized by cysts within the CNS of the mice. Novel artemisinin derivatives from Gary Posner's lab (Johns Hopkins U.) and endochin-
like quinolones from Michael Riscoe's lab (Oregon Health and Science U.) are showing substantial efficacy in the treatment model. It is anticipated that combinational treatment schemes will approach or achieve complete elimination of the latent infection, a feat that is considered to be amongst the most important and challenging goals in the field.

Finally, our work is revealing new clues to the impact of latent T. gondii infection in the central nervous system. The team has shown that the host response to CNS infection involves the upregulation of numerous inflammatory markers and neuroactive substances that may alter host behavior. These studies have the potential to help explain the epidemiological links between T. gondii infection and major mental illnesses including schizophrenia and certain forms of depression, which involve alterations in neural networks.

To navigate the above research avenues, we use a diverse array of approaches along the lines of molecular genetics, proteomics, biochemistry, pharmacology, cell biology, bioinformatics and structural biology. By addressing questions with multiple approaches yields we seek to obtain a robust understanding of Toxoplasma infection biology and disseminate the findings for conceptual integration into other infectious systems.

Recent Publications:

Pszenny, V., Zhou, X.W., Davis, P.H., Hunter, C., Carruthers, V.B., and Roos, D.S. (2012) Targeted disruption of Toxoplasma gondii Serine Protease Inhibitor-1 increases in vitro cyst formation and acute virulence in mice. Inf. Immun. 80:1156-1165. *Corresponding author.

Hortua Triana, M.A., Garavito, M.F., Huynh, M.H., Carruthers, V.B., Serrano, M., Loffler, M., Zimmerman, B.H. (2012) Preliminary characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase of Toxoplasma gondii. Mol. Biochem. Parasitol. 184:71-81.

Sloves, P.J., Delhaye, S., Mouveaux, T., Werkmeister, E., Hovasse, A., Slomianny, C., Callebaut, I., Gaji, R.Y., Schaeffer-Reiss, C., Van Dorsselear, A., Carruthers, V.B., and Tomavo, S. (2012) A crucial role for Toxoplasma sortilin-like receptor in biogenesis of apical secretory organelles and host infection. Cell Host Microbe 11:515-527.

Doggett, J.S., Nilsen, A., Forquer, I., Jones-Brando, L., Yolken, R.H., Charman, S.A., Katneni, K., Schultz, T., Burrows, J., Hinrichs, D.J., Meunier, B., Carruthers, V.B., and Riscoe, M. (2012) Endochin-like-quinolones are highly efficacious against acute and latent experimental toxoplasmosis. Proc. Nat’l Acad. Sci. 109:15936-15941.

Saouros*, S., Dou*, Z., Henry, M., Simpson, P., Carruthers, V.B.**, and Matthews, S.** (2012) Microneme protein 5 regulates Toxoplasma subtilisin 1 activity by mimicking a subtilisin prodomain. J. Biol. Chem. 287:36029-36040. *co-first authors. **co-corresponding authors.

Dou, Z., Coppens, I., and Carruthers, V.B. (2013) Non-canonical maturation of two papain-family proteases in Toxoplasma gondii J. Biol. Chem. 288:3523-3534.

Kremer, K. Kamin, D., Rittweger, E., Wilkes, J., Flammer, Mahler, S.,H., Heng, J., Tonkin, C.J., Langsley, G., Hell, S.W., Carruthers, V.B., Ferguson, D.J.P., and Meissner, M. (2013) An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes PLoS Pathog. 9:e1003213.

Roiko, M.S. and Carruthers, V.B. (2013) Functional dissection of Toxoplasma gondii Perforin-Like Protein 1 reveals a dual domain mode of membrane binding for cytolysis and parasite egress. J. Biol. Chem. 288:8712-25.

Gaji, R.Y., Huynh, M.-H., Carruthers, V.B. (2013) A novel high throughput invasion screen identifies host actin regulators required for efficient cell entry by Toxoplasma gondii. PLoS One. 8:e64693.

Tomavo, S., Meissner, M., Carruthers, V.B. (2013) Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion. PLoS Pathogens Oct;9(10):e1003629.

Muniz-Feliciano, L., Van Grol, J. Portillo, J.-A.C, Liew, L., Liu, B., Carlin, C.R., Carruthers, V.B., Matthews, S., Subauste, C.S. Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. PloS Pathog. Dec;9(12):e1003809.

Lebrun, M., Carruthers, V.B., Cesbron-Delauw, M.-F. (2013) Toxoplasma Secretory Proteins and their roles in Cell Invasion and Intracellular Survival. in Toxoplasma gondii: The Model Apicomplexan – Perspectives and Methods, 2nd Ed, Louis Weiss and Kami Kim (Eds).

Blackman, M.J. and Carruthers, V.B. (2013) Recent insights into apicomplexan parasite egress provide new views to a kill. Curr Opin Microbiol 16:459-464. [highlighted as a Feature Article on the Malaria Nexus website].

Schultz, T.L., Bordon, C., Henken, C.P., Woodard, L.E., Posner, G.H., Yolken, R.H., Jones-Brando, L., and Carruthers, V.B. A thiazole derivative of artemisinin reduces Toxoplasma gondii cyst burden in infected mice. (J. Parasitol., in press).

Warring, S.D., Dou, Z., Carruthers, V.B., McFadden, G.L., van Dooren, G.G. Characterization of the chloroquine resistance transporter homologue in Toxoplasma gondii. (Euk. Cell, in press).