Apicomplexan parasites like Toxoplasma gondii inject hundreds of foreign components into the host cytosol that have sophisticated mechanisms of intersecting host signaling pathways and are required for parasite survival and virulence. This implies that there has been a along evolutionary history between parasites and mammalian hosts, including rodents and humans, and suggests that there ought to be innate immune sensors designed to detect the parasite directly and engage an immune response to the parasite. As a post doc in the Boothroyd lab I have identified NLRP1 as a sensor for Toxoplasma infection that limits parasite growth and dissemination in mice, rats and humans.

Toxoplasma is contracted by ingesting parasite cysts which invade the host in the small intestine. Interestingly, differences in immune response established early in infection may play a role in determining long term out come of disease. In addition to defining the molecular mechanisms of innate immune activation, I am looking at the relationship between parasite, commensal microbiota and the host immune system in determining health status during chronic infection.

As a graduate student I studied with Dr. Gregory Barton at UC Berkeley to determine how nucleic acid sensing Toll-like receptors (TLR7 and TLR9) discriminate between host- and pathogen-derived nucleic acids. The specificity for DNA and RNA come at the inherent risk of autoimmunity. To limit this cost, activation of TLR7 and TLR9 is limited to the endosomal compartments by a requirement for processing by endosomal proteases. We think that this limits potential for recognition of self-nucleic acids which are sometimes released by dying or damaged cells into the extracellular space, linking activation to compartments where freshly release nucleic acids from captured pathogens may be more abundant.

Honors & Awards

  • Dennis and Marsha Dammerman Fellow, Damon Runyon Cancer Research Foundation (2011-2014)
  • Outstanding Postdoctoral Mentor Award, Stanford Immunology (2011-2012)

Boards, Advisory Committees, Professional Organizations

  • Organizer, Symposium for Immunity and Pathogenesis (2012 - 2012)
  • Retreat Planning Committee, Microbiology and Immunology (2013 - Present)

Professional Education

  • Doctor of Philosophy, University of California Berkeley (2010)

Stanford Advisors


All Publications

  • Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS biology Pernas, L., Adomako-Ankomah, Y., Shastri, A. J., Ewald, S. E., Treeck, M., Boyle, J. P., Boothroyd, J. C. 2014; 12 (4)


    Recent information has revealed the functional diversity and importance of mitochondria in many cellular processes including orchestrating the innate immune response. Intriguingly, several infectious agents, such as Toxoplasma, Legionella, and Chlamydia, have been reported to grow within vacuoles surrounded by host mitochondria. Although many hypotheses have been proposed for the existence of host mitochondrial association (HMA), the causes and biological consequences of HMA have remained unanswered. Here we show that HMA is present in type I and III strains of Toxoplasma but missing in type II strains, both in vitro and in vivo. Analysis of F1 progeny from a type II×III cross revealed that HMA is a Mendelian trait that we could map. We use bioinformatics to select potential candidates and experimentally identify the polymorphic parasite protein involved, mitochondrial association factor 1 (MAF1). We show that introducing the type I (HMA+) MAF1 allele into type II (HMA-) parasites results in conversion to HMA+ and deletion of MAF1 in type I parasites results in a loss of HMA. We observe that the loss and gain of HMA are associated with alterations in the transcription of host cell immune genes and the in vivo cytokine response during murine infection. Lastly, we use exogenous expression of MAF1 to show that it binds host mitochondria and thus MAF1 is the parasite protein directly responsible for HMA. Our findings suggest that association with host mitochondria may represent a novel means by which Toxoplasma tachyzoites manipulate the host. The existence of naturally occurring HMA+ and HMA- strains of Toxoplasma, Legionella, and Chlamydia indicates the existence of evolutionary niches where HMA is either advantageous or disadvantageous, likely reflecting tradeoffs in metabolism, immune regulation, and other functions of mitochondria.

    View details for DOI 10.1371/journal.pbio.1001845

    View details for PubMedID 24781109

  • NLRP1 Is an Inflammasome Sensor for Toxoplasma gondii INFECTION AND IMMUNITY Ewald, S. E., Chavarria-Smith, J., Boothroyd, J. C. 2014; 82 (1): 460-468


    The obligate intracellular parasite Toxoplasma gondii is able to infect nearly all nucleated cell types of warm-blooded animals. This is achieved through the injection of hundreds of parasite effectors into the host cell cytosol, allowing the parasite to establish a vacuolar niche for growth, replication, and persistence. Here we show that Toxoplasma infection actives an inflammasome response in mice and rats, an innate immune sensing system designed to survey the host cytosol for foreign components leading to inflammation and cell death. Oral infection with Toxoplasma triggers an inflammasome response that is protective to the host, limiting parasite load and dissemination. Toxoplasma infection is sufficient to generate an inflammasome response in germfree animals. Interleukin 1β (IL-1β) secretion by macrophage requires the effector caspases 1 and 11, the adapter ASC, and NLRP1, the sensor previously described to initiate the inflammasome response to Bacillus anthracis lethal factor. The allele of NLRP1b derived from 129 mice is sufficient to enhance the B6 bone marrow-derived macrophage (BMDM) inflammasome response to Toxoplasma independent of the lethal factor proteolysis site. Moreover, N-terminal processing of NLRP1b, the only mechanism of activation known to date, is not observed in response to Toxoplasma infection. Cumulatively, these data indicate that NLRP1 is an innate immune sensor for Toxoplasma infection, activated via a novel mechanism that corresponds to a host-protective innate immune response to the parasite.

    View details for DOI 10.1128/IAI.01170-13

    View details for Web of Science ID 000328899600047

  • The Myeloid Receptor PILR beta Mediates the Balance of Inflammatory Responses through Regulation of IL-27 Production PLOS ONE Tato, C. M., Joyce-Shaikh, B., Banerjee, A., Chen, Y., Sathe, M., Ewald, S. E., Liu, M., Gorman, D., McClanahan, T. K., Phillips, J. H., Heyworth, P. G., Cua, D. J. 2012; 7 (3)


    Paired immunoglobulin-like receptors beta, PILR?, and alpha, PILR?, are related to the Siglec family of receptors and are expressed primarily on cells of the myeloid lineage. PILR? is a DAP12 binding partner expressed on both human and mouse myeloid cells. The potential ligand, CD99, is found on many cell types, such as epithelial cells where it plays a role in migration of immune cells to sites of inflammation. Pilrb deficient mice were challenged with the parasite Toxoplasma gondii in two different models of infection induced inflammation; one involving the establishment of chronic encephalitis and a second mimicking inflammatory bowel disease in order to understand the potential role of this receptor in persistent inflammatory responses. It was found that in the absence of activating signals from PILR?, antigen-presenting cells (APCs) produced increased amounts of IL-27, p28 and promoted IL-10 production in effector T cells. The sustained production of IL-27 led ultimately to enhanced survival after challenge due to dampened immune pathology in the gut. Similar protection was also observed in the CNS during chronic T. gondii infection after i.p. challenge again providing evidence that PILR? is important for regulating aberrant inflammatory responses.

    View details for DOI 10.1371/journal.pone.0031680

    View details for Web of Science ID 000303894900003

    View details for PubMedID 22479310

  • Transmembrane Mutations in Toll-like Receptor 9 Bypass the Requirement for Ectodomain Proteolysis and Induce Fatal Inflammation IMMUNITY Mouchess, M. L., Arpaia, N., Souza, G., Barbalat, R., Ewald, S. E., Lau, L., Barton, G. M. 2011; 35 (5): 721-732


    Recognition of nucleic acids as a signature of infection by Toll-like receptors (TLRs) 7 and 9 exposes the host to potential self-recognition and autoimmunity. It has been proposed that intracellular compartmentalization is largely responsible for reliable self versus nonself discrimination by these receptors. We have previously shown that TLR9 and TLR7 require processing prior to activation, which may further reinforce receptor compartmentalization and tolerance to self, yet this possibility remains untested. Here we report that residues within the TLR9 transmembrane (TM) region conferred the requirement for ectodomain proteolysis. TLR9 TM mutants responded to extracellular DNA, and mice expressing such receptors died from systemic inflammation and anemia. This inflammatory disease did not require lymphocytes and appeared to require recognition of self-DNA by dendritic cells. To our knowledge, these results provide the first demonstration that TLR-intrinsic mutations can lead to a break in tolerance.

    View details for DOI 10.1016/j.immuni.2011.10.009

    View details for Web of Science ID 000297390800012

    View details for PubMedID 22078797

  • Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase JOURNAL OF EXPERIMENTAL MEDICINE Ewald, S. E., Engel, A., Lee, J., Wang, M., Bogyo, M., Barton, G. M. 2011; 208 (4): 643-651


    Toll-like receptor (TLR) 9 requires proteolytic processing in the endolysosome to initiate signaling in response to DNA. However, recent studies conflict as to which proteases are required for receptor cleavage. We show that TLR9 proteolysis is a multistep process. The first step removes the majority of the ectodomain and can be performed by asparagine endopeptidase (AEP) or cathepsin family members. This initial cleavage event is followed by a trimming event that is solely cathepsin mediated and required for optimal receptor signaling. This dual requirement for AEP and cathepsins is observed in all cell types that we have analyzed, including mouse macrophages and dendritic cells. In addition, we show that TLR7 and TLR3 are processed in an analogous manner. These results define the core proteolytic steps required for TLR9 function and suggest that receptor proteolysis may represent a general regulatory strategy for all TLRs involved in nucleic acid recognition.

    View details for DOI 10.1084/jem.20100682

    View details for Web of Science ID 000289404800002

    View details for PubMedID 21402738

  • Nucleic acid sensing Toll-like receptors in autoimmunity CURRENT OPINION IN IMMUNOLOGY Ewald, S. E., Barton, G. M. 2011; 23 (1): 3-9


    Trafficking and activation of the nucleic acid sensing TLRs is subject to unique regulatory requirements imposed by the risk of self-recognition. Like all TLRs these receptors traffick through the Golgi, however, access to the secretory pathway is controlled by a binding partner present in the ER. Receptor activation in the endolysosome is regulated through a proteolytic mechanism that requires activity of compartment-resident proteases, thereby preventing activation in other regions of the cell. Advances in our understanding of the cell biology of these receptors have been paralleled by efforts to understand their precise roles in autoimmunity. Mouse models have revealed that TLR7 and TLR9 make unique contributions to the types of self-molecules recognized in disease and possibly disease severity. Currently, methods of inhibiting TLR7 and TLR9 are being tested in clinical trials for systemic lupus erythamatosus.

    View details for DOI 10.1016/j.coi.2010.11.006

    View details for Web of Science ID 000288467700002

    View details for PubMedID 21146971

  • Nucleic Acid Recognition by the Innate Immune System ANNUAL REVIEW OF IMMUNOLOGY, VOL 29 Barbalat, R., Ewald, S. E., Mouchess, M. L., Barton, G. M. 2011; 29: 185-214


    Receptors of the innate immune system recognize conserved microbial features and provide key signals that initiate immune responses. Multiple transmembrane and cytosolic receptors have evolved to recognize RNA and DNA, including members of the Toll-like receptor and RIG-I-like receptor families and several DNA sensors. This strategy enables recognition of a broad range of pathogens; however, in some cases, this benefit is weighed against the cost of potential self recognition. Recognition of self nucleic acids by the innate immune system contributes to the pathology associated with several autoimmune or autoinflammatory diseases. In this review, we highlight our current understanding of nucleic acid sensing by innate immune receptors and discuss the regulatory mechanisms that normally prevent inappropriate responses to self.

    View details for DOI 10.1146/annurev-immunol-031210-101340

    View details for Web of Science ID 000289959200008

    View details for PubMedID 21219183

  • The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor NATURE Ewald, S. E., Lee, B. L., Lau, L., Wickliffe, K. E., Shi, G., Chapman, H. A., Barton, G. M. 2008; 456 (7222): 658-U88


    Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.

    View details for DOI 10.1038/nature07405

    View details for Web of Science ID 000261340000045

    View details for PubMedID 18820679

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