[Show abstract][Hide abstract] ABSTRACT: The classic anti-viral cytokine interferon (IFN)-β can be induced during parasitic infection, but relatively little is know about the cell types and signaling pathways involved. Here we show that inflammatory monocytes (IMs), but not neutrophils, produce IFN-β in response to T. gondii infection. This difference correlated with the mode of parasite entry into host cells, with phagocytic uptake predominating in IMs and active invasion predominating in neutrophils. We also show that expression of IFN-β requires phagocytic uptake of the parasite by IMs, and signaling through Toll-like receptors (TLRs) and MyD88. Finally, we show that IMs are major producers of IFN-β in mesenteric lymph nodes following in vivo oral infection of mice, and mice lacking the receptor for type I IFN-1 show higher parasite loads and reduced survival. Our data reveal a TLR and internalization-dependent pathway in IMs for IFN-β induction to a non-viral pathogen.Immunology and Cell Biology advance online publication, 26 August 2014; doi:10.1038/icb.2014.70.
[Show abstract][Hide abstract] ABSTRACT: The Red Queen hypothesis proposes that there is an evolutionary arms race between host and pathogen. One possible example
of such a phenomenon could be the recently discovered interaction between host defense proteins known as immunity-related
GTPases (IRGs) and a family of rhoptry pseudokinases (ROP5) expressed by the protozoan parasite, Toxoplasma gondii. Mouse IRGs are encoded by an extensive and rapidly evolving family of over 20 genes. Similarly, the ROP5 family is highly
polymorphic and consists of 4–10 genes, depending on the strain of Toxoplasma. IRGs are known to be avidly bound and functionally inactivated by ROP5 proteins, but the molecular basis of this interaction/inactivation
has not previously been known. Here we show that ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding
domain of an IRG and that this produces a profound allosteric change in the IRG structure. This has two dramatic effects:
1) it prevents oligomerization of the IRG, and 2) it alters the orientation of two threonine residues that are targeted by
the Toxoplasma Ser/Thr kinases, ROP17 and ROP18. ROP5s are highly specific in the IRGs that they will bind, and the fact that it is the
most highly polymorphic surface of ROP5 that binds the IRG strongly supports the notion that these two protein families are
co-evolving in a way predicted by the Red Queen hypothesis.
[Show abstract][Hide abstract] ABSTRACT: The intracellular parasite Toxoplasma gondii has multiple strategies to alter host cell function, including the injection of rhoptry proteins into the cytosol of host
cells as well as bystander populations, but the consequence of these events is unclear. Here, a reporter system using fluorescent
parasite strains that inject Cre recombinase with their rhoptry proteins (Toxoplasma-Cre) was combined with Ai6 Cre reporter mice to identify cells that have been productively infected, that have been rhoptry
injected but lack the parasite, or that have phagocytosed T. gondii. The ability to distinguish these host-parasite interactions was then utilized to dissect the events that lead to the production
of interleukin-12 p40 (IL-12p40), which is required for resistance to T. gondii. In vivo, the use of invasion-competent or invasion-inhibited (phagocytosed) parasites with IL-12p40 (YET40) reporter mice revealed
that dendritic cell (DC) and macrophage populations that phagocytose the parasite or are infected can express IL-12p40 but
are not the major source, as larger numbers of uninfected cells secrete this cytokine. Similarly, the use of Toxoplasma-Cre parasite strains indicated that dendritic cells and inflammatory monocytes untouched by the parasite and not cells injected
by the parasite are the primary source of IL-12p40. These results imply that a soluble host or parasite factor is responsible
for the bulk of IL-12p40 production in vivo, rather than cellular interactions with T. gondii that result in infection, infection and clearance, injection of rhoptry proteins, or phagocytosis of the parasite.
Infection and Immunity 07/2014; 82(10). DOI:10.1128/IAI.01643-14 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.
[Show abstract][Hide abstract] ABSTRACT: CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma, but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen, ROP5, that elicits a CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense-granule antigen GRA6 is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense-granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion affects the efficacy of parasite-specific CD8 T cell responses.
[Show abstract][Hide abstract] ABSTRACT: The obligate intracellular parasite Toxoplasma gondii is able to infect a broad range of hosts and cell types due, in part, to the diverse arsenal of effectors it secretes into the host cell. Here, using genetic crosses between Type II and Type III Toxoplasma strains and QTL mapping of the changes they induce in macrophage gene expression, we identify a novel dense granule protein, GRA25. Encoded on chromosome IX, GRA25 is a phosphoprotein that is secreted outside of the parasites and is found within the parasitophorous vacuole. In vitro experiments with a Type II Δgra25 strain showed that macrophages infected with this strain secrete lower levels of CCL2 and CXCL1 compared to those infected with the wild type or complemented control parasites. In vivo experiments showed that mice infected with a Type II Δgra25 strain are able to survive an otherwise lethal dose of Toxoplasma tachyzoites, and complementation of the mutant with an ectopic copy of GRA25 largely rescues this phenotype. Interestingly, the Type II and Type III versions of GRA25 vary in endogenous expression; however, both are able to promote parasite expansion in vivo when expressed in a Type II Δgra25 strain. These data establish GRA25 as a novel virulence factor and immune modulator.
Infection and immunity 04/2014; 82(6). DOI:10.1128/IAI.01339-13 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: In North America (NA) and Europe, the majority of toxoplasmosis cases are benign and generally asymptomatic, whereas in South
America (SA) toxoplasmosis is associated with much more severe symptoms in adults and congenitally infected children. The
reasons for these differences remain unknown; currently, there is little information from patients in either region on how
the immune system responds to infection with Toxoplasma gondii. Here, we report the relative abundance of 51 serum cytokines from acute and chronic toxoplasmosis cohorts of pregnant women
from the United States, where approximately one-half of clinical isolates are Type II, and Colombia, where clinical isolates
are generally “atypical” or Type I-like strains. Surprisingly, the results showed notably lower levels of 23 cytokines in
acutely infected patients from the United States, relative to uninfected US controls. In acutely infected Colombian patients,
however, only 8 cytokine levels differed detectably with 4 being lower and 4 higher relative to uninfected controls. Strikingly,
there were also differences in the cytokine profiles of the chronically infected patients relative to uninfected controls
in the US cohort. Hence, Toxoplasma appears to specifically impact levels of circulating cytokines, and our results may partly explain region-specific differences
in the clinical spectrum of toxoplasmosis.
The Journal of Infectious Diseases 03/2014; 210(6). DOI:10.1093/infdis/jiu189 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii infection has previously been described to cause dramatic changes in the host transcriptome by manipulating key regulators,
including STATs, NF-κB, and microRNAs. Here, we report that Toxoplasma tachyzoites also mediate rapid and sustained induction of another pivotal regulator of host cell transcription, c-Myc. This
induction is seen in cells infected with all three canonical types of Toxoplasma but not the closely related apicomplexan parasite Neospora caninum. Coinfection of cells with both Toxoplasma and Neospora still results in an increase in the level of host c-Myc, showing that c-Myc is actively upregulated by Toxoplasma infection (rather than repressed by Neospora). We further demonstrate that this upregulation may be mediated through c-Jun N-terminal protein kinase (JNK) and is unlikely
to be a nonspecific host response, as heat-killed Toxoplasma parasites do not induce this increase and neither do nonviable parasites inside the host cell. Finally, we show that the
induced c-Myc is active and that transcripts dependent on its function are upregulated, as predicted. Hence, c-Myc represents
an additional way in which Toxoplasma tachyzoites have evolved to specifically alter host cell functions during intracellular growth.
[Show abstract][Hide abstract] ABSTRACT: 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 germ-free animals. 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 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 with a host-protective innate immune response to the parasite.
Infection and immunity 11/2013; 82(1). DOI:10.1128/IAI.01170-13 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.
Nature Chemical Biology 08/2013; 9(10). DOI:10.1038/nchembio.1315 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function.
PLoS ONE 08/2013; 8(8):e70637. DOI:10.1371/journal.pone.0070637 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host via encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a Toxoplasma nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. Toxoplasma strains deleted for the TgNST1 gene (Δnst1) form cyst-like structures in vitro but no longer interact with lectins, suggesting that Δnst1 strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. In vivo infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δnst1 parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of Toxoplasma tissue cysts.
[Show abstract][Hide abstract] ABSTRACT: The tissue cyst formed by the bradyzoite stage of Toxoplasma gondii is essential for persistent infection of the host and oral transmission. Bradyzoite pseudokinase 1 (BPK1) is a component
of the cyst wall, but nothing has previously been known about its function. Here, we show that immunoprecipitation of BPK1
from in vitro bradyzoite cultures, 4 days postinfection, identifies at least four associating proteins: MAG1, MCP4, GRA8, and GRA9. To
determine the role of BPK1, a strain of Toxoplasma was generated with the bpk1 locus deleted. This BPK1 knockout strain (Δbpk1) was investigated in vitro and in vivo. No defect was found in terms of in vitro cyst formation and no difference in pathogenesis or cyst burden 4 weeks postinfection (wpi) was detected after intraperitoneal
(i.p.) infection with Δbpk1 tachyzoites, although the Δbpk1 cysts were significantly smaller than parental or BPK1-complemented strains at 8 wpi. Pepsin-acid treatment of 4 wpi in vivo cysts revealed that Δbpk1 parasites are significantly more sensitive to this treatment than the parental and complemented strains. Consistent with
this, 4 wpi Δbpk1 cysts showed reduced ability to cause oral infection compared to the parental and complemented strains. Together, these data
reveal that BPK1 plays a crucial role in the in vivo development and infectivity of Toxoplasma cysts.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Toxoplasma gondii, an important human pathogen, is an obligate intracellular parasite, thus getting in and out of cells is key for its survival. The process by which Toxoplasma exits cells, known as egress, is controlled by calcium fluxes and can be triggered by ionophores. In vivo, rapid egress from the host cell has been identified as a means to escape attack by the innate immune system. At the molecular level, calcium dependent events in this parasite are regulated in part by plant like calcium dependent kinases, which share no homology to human kinases and are thus ideal drug targets. In this study we revisited 4 mutant parasite lines that were independently selected for an inability to undergo egress in response to ionophores. In all four mutants we have identified the Calcium Dependent Kinase 3 as the gene responsible for the defects. We have shown that two of these mutants, which are in a genetic background that allows virulence studies, also have a strong phenotype in vivo. That is, the parasites fail to form latent stages in mice. This work provides important information that a single kinase is responsible for the formation of latent stages that are important for transmission of the parasite.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Toxoplasma gondii is an intracellular parasite that infects warm blooded animals, including humans. In these hosts, Toxoplasma establishes a chronic infection in the brain, which the parasite accomplishes in part by injecting effector proteins, which manipulate many cellular processes, into cells it invades. Two recent reports suggested that Toxoplasma may also inject effector proteins into cells it does not invade. To look for these “uninfected-injected” cells, we utilized three different reporter systems that are tied to injection of effector proteins and not to invasion. With these systems, we determined that Toxoplasma injects proteins into cells it does not invade and enough protein is injected to manipulate the uninfected cells in a manner consistent with what occurs in infected cells. Furthermore, by using one of the reporter systems in mice, we verified that these uninfected-injected cells can include systemic immune cells and neurons in the brain. Remarkably, in the brain, the uninfected-injected cells out-number the infected cells by many fold. Together, these results strongly suggest that Toxoplasma manipulates far more cells than previously realized and, given their abundance, these uninfected-injected cells may play a central role in how Toxoplasma engages the host's immune response.
[Show abstract][Hide abstract] ABSTRACT: The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.