Areas of Interest
Pathogenesis of parasitic infections: Mechanisms of cell invasion and survival during infection
The Carruthers lab seeks to understand 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.
Part of the lab focused on 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.
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 and supports the long-term survival of the parasite in neural cysts.
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 bumped kinase inhibitors from Dustin Maly’s lab (U. Washington) and endochin-like quinolones from Michael Riscoe’s lab (Oregon Health and Science U.) are showing substantial efficacy in the treatment model. We are also rationally designing and optimizing inhibitors for CPL that will be tested for cystocidal activity in culture and in chronically infected mice. 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.
Huynh, M.-Y., Liu, B., Henry, M., Liew, L., Matthews, S.J., Carruthers, V.B. (2015) Structural basis of Toxoplasma gondii MIC2-associated protein interaction with MIC2. J. Biol. Chem. 290(3):1432-41.
Tampaki, Z., Ramadhan, S., Mwakubambanya, R.S., Goulielmaki, E., Kaforou, S., Kim, K., Waters, A.P. Carruthers, V.B., Siden-Kiamos, I., Koussis, K., and Loukeris, T.G. (2015) Ectopic expression of an N. caninum serpin triggers developmental defects in Toxoplasma and Plasmodium. PLoS One 10(3):e0121379.
Lunghi, M., Galizi, R., Magini, A., Carruthers, V.B., Di Cristina, M. (2015) Expression of the glycolytic enyzmes enolase and lactate dehydrogenase during the chronic phase of Toxoplasma differentiation is regulated by an intron retention mechanism. Mol. Microbiol. 96(6):1159-75.
Tomasik, J., Schultz, T.L., Kluge, W., Yolken, R.H., Bahn, S., Carruthers, V.B. (2015) Shared immune and repair markers during experimental Toxoplasma chronic brain infection and schizophrenia. Schiz. Bulletin. 42;386-395 (PMC4753603). Highlighted in QuestioningAnswers.
Pszenny, V., Ehrenman, K., Romano, J.D., Schultz, A.J., Roos, D.S., Grigg, M.E., Carruthers, V.B., Coppens, I. (2015) A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress. J. Biol. Chem. 291;3725-3746.
Xiao, J., Li, Y., Gressitt, K.L., He, H., Kannan, G., Schultz, T.L., Svezhova, N., Carruthers, V.B., Pletnikov, M.V., Yolken, R.H., Severance, E.G. (2016) Cerebral complement C1q activation in chronic Toxoplasma infection. Brains Behav Immun pii: S0889-1591(16)30100-3.
Huynh, M.-Y., Carruthers, V.B. (2016) A Toxoplasma gondii ortholog of Plasmodium GAMA contributes to parasite attachment and cell invasion. mSphere Feb 10;1(1). pii: e00012-16.
Bullen, H., Jia, Y., Yamaryo-Botte, Y., Zhang, O., Jemelin, N.K., Carruthers, V.B., Botte, C., Soldati-Favre, D. (2016) Phosphatidic acid-mediated signaling is essential for microneme secretion in Toxoplasma. Cell Host Microbe 19(3):349-60.
McGovern, O.L. and Carruthers, V.B. (2016) Toxoplasma retromer is here to stay. Spotlight article for Trends. Parasitol. S1471-4922(16)30053-8.
Vidadala, R.S.R., Rivas, K.L., Ojo, K.K., Hulverson, M.A., Zambriski, J.A., Bruzual, I., Schultz, T.L., Huang, W., Zhang, Z., Scheele, S., DeRocher, A.E., Choi, R., Barrett, L.K., Siddaramaiah, L.K., Hol, W.G.J., Fan, E., Merritt, E.A., Parsons, M., Freiberg, G., Marsh, K., Kempf, D., Carruthers, V.B., Isoherranen, N., Doggett, J.S., Van Voorhis, W.C., Maly, D.J. (2016) Development of an Orally Available and CNS-Penetrant TgCDPK1 Inhibitor with Minimal hERG Activity for the Treatment of Toxoplasmosis. J Med Chem. 59(13):6531-6546.
Sidik, S.M., Huet, D., Ganesan, S., Huynh, M.H., Wang, T., Nasamu, A.S., Thiru, P., Saeij, J.P.J., Carruthers, V.B., Niles, J.C., Lourido., S. (2016) A Genome-Wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell (in press).