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Laura Knoll

Picture of Laura  KnollProfessor of Medical Microbiology & Immunology
3303 Microbial Sciences Building
1550 Linden Drive
Office: (608) 262-3161
Laboratory: 262-4242
Email: ljknoll@wisc.edu
Overview · Personnel · Publications
  • Wilson SK, Knoll LJ (2017) Patatin-like phospholipases in microbial infections with emerging roles in fatty acid metabolism and immune regulation by Apicomplexa. Mol. Microbiol. 107(1):34-46 (PMC5739999) View Abstract · Pubmed Record

    Emerging lipidomic technologies have enabled researchers to dissect the complex roles of phospholipases in lipid metabolism, cellular signaling and immune regulation. Host phospholipase products are involved in stimulating and resolving the inflammatory response to pathogens. While many pathogen-derived phospholipases also manipulate the immune response, they have recently been shown to be involved in lipid remodeling and scavenging during replication. Animal and plant hosts as well as many pathogens contain a family of patatin-like phospholipases, which have been shown to have phospholipase Aactivity. Proteins containing patatin-like phospholipase domains have been identified in protozoan parasites within the Apicomplexa phylum. These parasites are the causative agents of some of the most widespread human diseases. Malaria, caused by Plasmodium spp., kills nearly half a million people worldwide each year. Toxoplasma and Cryptosporidium infect millions of people each year with lethal consequences in immunocompromised populations. Parasite-derived patatin-like phospholipases are likely effective drug targets and progress in the tools available to the Apicomplexan field will allow for a closer look at the interplay of lipid metabolism and immune regulation during host infection.

  • Milligan-Myhre K, Wilson SK, Knoll LJ (2016) Developmental change in translation initiation alters the localization of a common microbial protein necessary for Toxoplasma chronic infection. Mol. Microbiol. 102(6):1086-1098 (PMC5161674) View Abstract · Pubmed Record

    The Toxoplasma gondii cyst stage is resistant to drug therapy. To identify potential targets for new therapeutics, we screened insertional mutants of T. gondii for a reduced ability to form cysts in the brains of mice. In one of these mutants, named 38C3, the mutagenesis plasmid inserted into the mRNA of a protein that is highly conserved in microbes but is not present in humans. The mutation in 38C3 causes reduced brain cyst production during chronic infection, but does not affect acute virulence, so the disrupted gene and protein are called T. gondii Brain Colonization Protein 1 (TgBCP1). TgBCP1 has three potential in frame start codons that produce 51, 33 or 25 kDa proteins. In rapidly replicating tachyzoites, translation initiates at the third methionine, producing the 25 kDa form that is conserved in many bacteria and protozoans. Brain cysts exclusively express the 51 kDa form of TgBCP1, which is secreted from the parasites and localizes to the cyst wall. Only expression of the long form of TgBCP1 restored cyst formation in the 38C3 mutant. TgBCP1 is essential for cyst formation and is the first example of a developmental regulation in translation initiation site preference for a T. gondii protein.

  • Pittman KJ, Cervantes PW, Knoll LJ (2016) Z-DNA Binding Protein Mediates Host Control of Toxoplasma gondii Infection. Infect. Immun. 84(10):3063-70 (PMC5038082) View Abstract · Pubmed Record

    Intrinsic to Toxoplasma gondii infection is the parasite-induced modulation of the host immune response, which ensures establishment of a chronic lifelong infection. This manipulation of the host immune response allows T. gondii to not only dampen the ability of the host to eliminate the parasite but also trigger parasite differentiation to the slow-growing, encysted bradyzoite form. We previously used RNA sequencing (RNA-seq) to profile the transcriptomes of mice and T. gondii during acute and chronic stages of infection. One of the most abundant host transcripts during acute and chronic infection was Z-DNA binding protein 1 (ZBP1). In this study, we determined that ZBP1 functions to control T. gondii growth. In activated macrophages isolated from ZBP1 deletion (ZBP1(-/-)) mice, T. gondii has an increased rate of replication and a decreased rate of degradation. We also identified a novel function for ZBP1 as a regulator of nitric oxide (NO) production in activated macrophages, even in the absence of T. gondii infection. Upon stimulation, T. gondii-infected ZBP1(-/-) macrophages display increased proinflammatory cytokines compared to wild-type macrophages under the same conditions. These in vitro phenotypes were recapitulated in vivo, with ZBP1(-/-) mice having increased susceptibility to oral challenge, higher cyst burdens during chronic infection, and elevated inflammatory cytokine responses. Taken together, these results highlight a role for ZBP1 in assisting host control of T. gondii infection.

  • Knoll LJ (2016) Functional Analysis of the Rhoptry Kinome during Chronic Toxoplasma gondii Infection. MBio 7(3): (PMC4916387) View Abstract · Pubmed Record

    Toxoplasma gondii is one of the most common parasitic infections of humans worldwide. Once exposed, humans remain infected with T. gondii for life, and there are no therapeutics capable of eliminating a chronic infection. In the search for novel drug targets, T. gondii is known to contain several unique secretory organelles, one of which is called the rhoptries. Rhoptry organelles contain and secrete numerous proteins with kinase domains, but the roles of most of these kinases during infection remain unknown. In a recent mBio article, B. A. Fox et al. [mBio 7(3):e00193-16, 2016, http://dx.doi.org/10.1128/mBio.00193-16] performed a tour de force deletion analysis of 31 rhoptry kinases and examined their roles in the development of chronic infection. While rhoptry kinase deletion strains that displayed an acute infection defect also showed a reduction in chronic infection cyst burden, two rhoptry kinase deletion strains had decreased cyst burden without any change in acute virulence. These results indicate the necessity of the rhoptry kinases for the establishment and perhaps maintenance of chronic infection. They also highlight the potential of these kinases as drug targets to clear chronic infection or as candidates to generate a nonpersisting vaccine.

  • Pittman KJ, Knoll LJ (2015) Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections. Microbiol. Mol. Biol. Rev. 79(4):387-401 (PMC4557073) View Abstract · Pubmed Record

    Toxoplasma gondii represents one of the most common parasitic infections in the world. The asexual cycle can occur within any warm-blooded animal, but the sexual cycle is restricted to the feline intestinal epithelium. T. gondii is acquired through consumption of tissue cysts in undercooked meat as well as food and water contaminated with oocysts. Once ingested, it differentiates into a rapidly replicating asexual form and disseminates throughout the body during acute infection. After stimulation of the host immune response, T. gondii differentiates into a slow-growing, asexual cyst form that is the hallmark of chronic infection. One-third of the human population is chronically infected with T. gondii cysts, which can reactivate and are especially dangerous to individuals with reduced immune surveillance. Serious complications can also occur in healthy individuals if infected with certain T. gondii strains or if infection is acquired congenitally. No drugs are available to clear the cyst form during the chronic stages of infection. This therapeutic gap is due in part to an incomplete understanding of both host and pathogen responses during the progression of T. gondii infection. While many individual aspects of T. gondii infection are well understood, viewing the interconnections between host and parasite during acute and chronic infection may lead to better approaches for future treatment. The aim of this review is to provide an overview of what is known and unknown about the complex relationship between the host and parasite during the progression of T. gondii infection, with the ultimate goal of bridging these events.

  • Pittman KJ, Aliota MT, Knoll LJ (2014) Dual transcriptional profiling of mice and Toxoplasma gondii during acute and chronic infection. BMC Genomics 15:806 (PMC4177681) View Abstract · Pubmed Record

    The obligate intracellular parasite Toxoplasma gondii establishes a life-long chronic infection within any warm-blooded host. After ingestion of an encysted parasite, T. gondii disseminates throughout the body as a rapidly replicating form during acute infection. Over time and after stimulation of the host immune response, T. gondii differentiates into a slow growing, cyst form that is the hallmark of chronic infection. Global transcriptome analysis of both host and parasite during the establishment of chronic T. gondii infection has not yet been performed. Here, we conducted a dual RNA-seq analysis of T. gondii and its rodent host to better understand host and parasite responses during acute and chronic infection. We obtained nearly one billion paired-end RNA sequences from the forebrains of uninfected, acutely and chronically infected mice, then aligned them to the genomic reference files of both T. gondii and Mus musculus. Gene ontology (GO) analysis of the 100 most highly expressed T. gondii genes showed less than half were shared between acute and chronic infection. The majority of the highly expressed genes common in both acute and chronic infection were involved in transcription and translation, underscoring that parasites in both stages are actively synthesizing proteins. Similarly, most of the T. gondii genes highly expressed during chronic infection were involved in metabolic processes, again highlighting the activity of the cyst stage at 28 days post-infection. Comparative analyses of host genes using uninfected forebrain revealed over twice as many immune regulatory genes were more abundant during chronic infection compared to acute. This demonstrates the influence of parasite development on host gene transcription as well as the influence of the host environment on parasite gene transcription. RNA-seq is a valuable tool to simultaneously analyze host and microbe transcriptomes. Our data shows that T. gondii is metabolically active and synthesizing proteins at 28 days post-infection and that a distinct subset of host genes associated with the immune response are more abundant specifically during chronic infection. These data suggest host and pathogen interplay is still present during chronic infection and provides novel T. gondii targets for future drug and vaccine development.

  • Neal LM, Knoll LJ (2014) Toxoplasma gondii profilin promotes recruitment of Ly6Chi CCR2+ inflammatory monocytes that can confer resistance to bacterial infection. PLoS Pathog. 10(6):e1004203 (PMC4055779) View Abstract · Pubmed Record

    Ly6C+ inflammatory monocytes are essential to host defense against Toxoplasma gondii, Listeria monocytogenes and other infections. During T. gondii infection impaired inflammatory monocyte emigration results in severe inflammation and failure to control parasite replication. However, the T. gondii factors that elicit these monocytes are unknown. Early studies from the Remington laboratory showed that mice with a chronic T. gondii infection survive lethal co-infections with unrelated pathogens, including L. monocytogenes, but a mechanistic analysis was not performed. Here we report that this enhanced survival against L. monocytogenes is due to early reduction of bacterial burdens and elicitation of Ly6C+ inflammatory monocytes. We demonstrate that a single TLR11/TLR12 ligand profilin (TgPRF) was sufficient to reduce bacterial burdens similar to T. gondii chronic infection. Stimulation with TgPRF was also sufficient to enhance animal survival when administered either pre- or post-Listeria infection. The ability of TgPRF to reduce L. monocytogenes burdens was dependent on TLR11 and required IFN-γ but was not dependent on IL-12 signaling. TgPRF induced rapid production of MCP-1 and resulted in trafficking of Ly6Chi CCR2+ inflammatory monocytes and Ly6G+ neutrophils into the blood and spleen. Stimulation with TgPRF reduced L. monocytogenes burdens in mice depleted with the Ly6G specific MAb 1A8, but not in Ly6C/Ly6G specific RB6-8C5 depleted or CCR2-/- mice, indicating that only inflammatory monocytes are required for TgPRF-induced reduction in bacterial burdens. These results demonstrate that stimulation of TLR11 by TgPRF is a mechanism to promote the emigration of Ly6Chi CCR2+ monocytes, and that TgPRF recruited inflammatory monocytes can provide an immunological benefit against an unrelated pathogen.

  • Settles EW, Moser LA, Harris TH, Knoll LJ (2014) Toxoplasma gondii upregulates interleukin-12 to prevent Plasmodium berghei-induced experimental cerebral malaria. Infect. Immun. 82(3):1343-53 (PMC3957979) View Abstract · Pubmed Record

    A chronic infection with the parasite Toxoplasma gondii has previously been shown to protect mice against subsequent viral, bacterial, or protozoal infections. Here we have shown that a chronic T. gondii infection can prevent Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. Treatment with soluble T. gondii antigens (STAg) reduced parasite sequestration and T cell infiltration in the brains of P. berghei-infected mice. Administration of STAg also preserved blood-brain barrier function, reduced ECM symptoms, and significantly decreased mortality. STAg treatment 24 h post-P. berghei infection led to a rapid increase in serum levels of interleukin 12 (IL-12) and gamma interferon (IFN-γ). By 5 days after P. berghei infection, STAg-treated mice had reduced IFN-γ levels compared to those of mock-treated mice, suggesting that reductions in IFN-γ at the time of ECM onset protected against lethality. Using IL-10- and IL-12βR-deficient mice, we found that STAg-induced protection from ECM is IL-10 independent but IL-12 dependent. Treatment of P. berghei-infected mice with recombinant IL-12 significantly decreased parasitemia and mortality. These data suggest that IL-12, either induced by STAg or injected as a recombinant protein, mediates protection from ECM-associated pathology potentially through early induction of IFN-γ and reduction in parasitemia. These results highlight the importance of early IL-12 induction in protection against ECM.

  • Tobin Magle C, Pittman KJ, Moser LA, Boldon KM, Knoll LJ (2014) A toxoplasma patatin-like protein changes localization and alters the cytokine response during toxoplasmic encephalitis. Infect. Immun. 82(2):618-25 (PMC3911373) View Abstract · Pubmed Record

    Toxoplasma gondii is an obligate intracellular parasite that forms a lifelong infection within the central nervous system of its host. The T. gondii genome encodes six members of the patatin-like phospholipase family; related proteins are associated with host-microbe interactions in bacteria. T. gondii patatin-like protein 1 (TgPL1) was previously determined to be necessary for parasites to suppress nitric oxide and prevent degradation in activated macrophages. Here, we show that in the rapidly replicating tachyzoite stage, TgPL1 is localized within vesicles inside the parasite that are distinct from the dense granules; however, in the encysted bradyzoite stage, TgPL1 localizes to the parasitophorous vacuole (PV) and cyst wall. While we had not previously seen a defect of the TgPL1 deletion mutant (ΔTgPL1) during acute and early chronic infection, the localization change of TgPL1 in bradyzoites caused us to reevaluate the ΔTgPL1 mutant during late chronic infection and in a toxoplasmic encephalitis (TE) mouse model. Mice infected with ΔTgPL1 are more resistant to TE and have fewer inflammatory lesions than mice infected with the wild type and ΔTgPL1 genetically complemented with TgPL1. This increased resistance to TE could result from several contributing factors. First, we found that ΔTgPL1 bradyzoites did not convert back to tachyzoites readily in tissue culture. Second, a subset of cytokine levels were higher in ΔTgPL1-infected mice, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1). These studies suggest that TgPL1 plays a role in the maintenance of chronic T. gondii infection.

  • Payne AJ, Neal LM, Knoll LJ (2013) Fusidic acid is an effective treatment against Toxoplasma gondii and Listeria monocytogenes in vitro, but not in mice. Parasitol. Res. 112(11):3859-63 (PMC4096717) View Abstract · Pubmed Record

    Fusidic acid is a bacteriostatic antibiotic that inhibits the growth of bacteria by preventing the release of translation elongation factor G (EF-G) from the ribosome. The apicomplexan parasite Toxoplasma gondii has an orthologue of bacterial EF-G that can complement bacteria and is necessary for parasite virulence. Fusidic acid has been shown to be effective in tissue culture against the related pathogen Plasmodium falciparum, and current drug treatments against T. gondii are limited. We therefore investigated the therapeutic value of fusidic acid for T. gondii and found that the drug was effective in tissue culture, but not in a mouse model of infection. To determine whether this trend would occur in another intracellular pathogen that elicits a T helper 1-type immune response, we tested the efficacy of fusidic acid for the bacterium Listeria monocytogenes. Similar to its effects on T. gondii, fusidic acid inhibits the growth of L. monocytogenes in vitro, but not in mice. These findings highlight the necessity of in vivo follow-up studies to validate in vitro drug investigations.

  • Magle CT, Pittman KJ, Moser LA, Boldon KM, Knoll LJ (2013) A Toxoplasma patatin-like protein changes localization and alters the cytokine response during toxoplasmic encephalitis. Infect. Immun. : View Abstract · Pubmed Record

    Toxoplasma gondii is an obligate intracellular parasite that forms a life-long infection within the central nervous system of its host. The T. gondii genome encodes six members of the patatin-like phospholipase family; related proteins are associated with host-microbe interactions in bacteria. T. gondii patatin-like protein 1 (TgPL1) was previously determined to be necessary for parasites to suppress nitric oxide and prevent degradation in activated macrophages. Here we show that in the rapidly replicating tachyzoite stage, TgPL1 is localized within vesicles inside the parasite that are distinct from the dense granules; however, in the encysted bradyzoite stage, TgPL1 localizes to the parasitophorous vacuole (PV) and cyst wall. While we had not previously seen a defect of the TgPL1 deletion mutant (ΔTgPL1) during acute and early chronic infection, the localization change of TgPL1 in bradyzoites caused us to reevaluate the ΔTgPL1 mutant during late chronic infection and in a toxoplasmic encephalitis (TE) mouse model. Mice infected with ΔTgPL1 are more resistant to TE and have fewer inflammatory lesions than wild type and ΔTgPL1 genetically complemented with TgPL1 infected mice. This increased resistance to TE could result from several contributing factors. First, we found that ΔTgPL1 bradyzoites did not convert back to tachyzoites readily in tissue culture. Second, a subset of cytokine levels were higher in ΔTgPL1 infected mice, including interferon gamma (IFN-γ), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1). These studies suggest that the TgPL1 plays a role in the maintenance of T. gondii chronic infection.

  • Moser LA, Pollard AM, Knoll LJ (2013) A genome-wide siRNA screen to identify host factors necessary for growth of the parasite Toxoplasma gondii. PLoS ONE 8(6):e68129 (PMC3695992) View Abstract · Pubmed Record

    Toxoplasma gondii is an obligate intracellular parasite that is able to infect virtually any nucleated cell of all warm-blooded animals. The host cell factors important for parasite attachment, invasion, and replication are poorly understood. We screened a siRNA library targeting 18,200 individual human genes in order to identify host proteins with a role in T. gondii growth. Our screen identified 19 genes whose inhibition by siRNA consistently and significantly lowered parasite replication. The gene ontology categories for those 19 genes represented a wide variety of functions with several genes implicated in regulation of the cell cycle, ion channels and receptors, G-protein coupled receptors, and cytoskeletal structure as well as genes involved in transcription, translation and protein degradation. Further investigation of 5 of the 19 genes demonstrated that the primary reason for the reduction in parasite growth was death of the host cell. Our results suggest that once T. gondii has invaded and established an infection, global changes in the host cell may be necessary to reduce parasite replication. While siRNA screens have been used, albeit rarely, in other parasite systems, this is the first report to describe a high-throughput siRNA screen for host proteins that affect T. gondii replication.

  • Hsiao CH, Luisa Hiller N, Haldar K, Knoll LJ (2013) A HT/PEXEL motif in Toxoplasma dense granule proteins is a signal for protein cleavage but not export into the host cell. Traffic 14(5):519-31 (PMC3622808) View Abstract · Pubmed Record

    Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL-like motif at the N-terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin-tagged versions of these uncharacterized proteins show co-localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM.

  • Tobin CM, Knoll LJ (2012) A patatin-like protein protects Toxoplasma gondii from degradation in a nitric oxide-dependent manner. Infect. Immun. 80(1):55-61 (PMC3255658) View Abstract · Pubmed Record

    Toxoplasma gondii is an obligate intracellular parasite that uses immune cells to disseminate throughout its host. T. gondii can persist and even slowly replicate in activated host macrophages by reducing the antimicrobial effects of molecules such as nitric oxide (NO). A T. gondii patatin-like protein called TgPL1 was previously shown to be important for survival in activated macrophages. Here we show that a T. gondii mutant with a deletion of the TgPL1 gene (ΔTgPL1) is degraded in activated macrophages. This degradation phenotype is abolished by the removal of NO by the use of an inducible NO synthase (iNOS) inhibitor or iNOS-deficient macrophages. The exogenous addition of NO to macrophages results in reduced parasite growth but not the degradation of ΔTgPL1 parasites. These results suggest that NO is necessary but not sufficient for the degradation of ΔTgPL1 parasites in activated macrophages. While some patatin-like proteins have phospholipase A2 (PLA2) activity, recombinant TgPL1 purified from Escherichia coli does not have phospholipase activity. This result was not surprising, as TgPL1 contains a G-to-S change at the predicted catalytic serine residue. An epitope-tagged version of TgPL1 partially colocalized with a dense granule protein in the parasitophorous vacuole space. These results may suggest that TgPL1 moves to the parasitophorous vacuole to protect parasites from nitric oxide by an undetermined mechanism.

  • Payne TM, Payne AJ, Knoll LJ (2011) A Toxoplasma gondii mutant highlights the importance of translational regulation in the apicoplast during animal infection. Mol. Microbiol. 82(5):1204-16 View Abstract · Pubmed Record

    Toxoplasma gondii is an obligate intracellular parasite of all warm-blooded animals. We previously described a forward genetic screen to identify T. gondii mutants defective in the establishment of a chronic infection. One of the mutants isolated was disrupted in the 3' untranslated region (3'UTR) of an orthologue of bacterial translation elongation factor G (EFG). The mutant does not have a growth defect in tissue culture. Genetic complementation of this mutant with the genomic locus of TgEFG restores virulence in an acute infection mouse model. Epitope tagged TgEFG localized to the apicoplast, via a non-canonical targeting signal, where it functions as an elongation factor for translation in the apicoplast. Comparisons of TgEFG expression constructs with wild-type or mutant 3'UTRs showed that a wild-type 3'UTR is necessary for translation of TgEFG. In tissue culture, the TgEFG transcript is equally abundant in wild-type and mutant parasites; however, during an animal infection, the TgEFG transcript is increased more than threefold in the mutant. These results highlight that in tissue culture, translation in the apicoplast can be diminished, but during an animal infection, translation in the apicoplast must be fully functional.

  • Milligan-Myhre KC, Rooney PJ, Knoll LJ (2011) Examination of a virulence mutant uncovers the ribosome biogenesis regulatory protein of Toxoplasma gondii. J. Parasitol. 97(6):1173-7 View Abstract · Pubmed Record

    Several insertional mutants identified in a screen for Toxoplasma gondii that were defective in establishing a chronic infection had a common site of plasmid insertion. This insertion site was determined to be 43 bp upstream of the transcription initiation site of a gene whose predicted product has homology to ribosome biogenesis regulatory protein Rrs1p, an essential protein required for ribosome biogenesis in Saccharomyces cerevisiae. Northern blot analysis of this locus, termed TgRRS1 , showed that in the C3 mutant, the full-length transcript is down-regulated and at least 1 new smaller transcript is present. Restoration of the intact predicted promoter and locus to TgRRS1 insertional mutant strain C3 did not restore brain cyst formation to the levels of the parent strain. Epitope-tagged TgRRS1 was found to localize to the parasite nucleolus, in an area corresponding to the granular component region. TgRRS1 can serve as a marker for the sub-nucleolar granular component region of T. gondii.

  • Payne TM, Lund PJ, Knoll LJ (2011) A transmembrane domain containing pellicle protein of Toxoplasma gondii enhances virulence and invasion after extracellular stress. Mol. Biochem. Parasitol. 179(2):107-10 (PMC3156857) View Abstract · Pubmed Record

    To identify Toxoplasma gondii genes important in the establishment of a persistent infection, we previously used signature-tagged mutagenesis to identify mutants with reduced cyst numbers in the brains of mice. One of the mutants, 95C5, has an insertion within a predicted six transmembrane domain protein, which localizes to the parasite pellicle, thus we named it transmembrane pellicle protein 1 (TgTPP1). Although the 95C5 mutant was found be reduced in its ability to form brain cysts, it is defective during acute infection. Addition of TgTPP1 expressed from its endogenous promoter restored the acute lethality of the 95C5 mutant to parental levels. The 95C5 mutant does not have a growth defect in standard tissue culture conditions; however, we found a significant defect in host cell penetration after extracellular stress. Overall, TgTPP1 may function during acute infection by enhancing the parasites ability to invade after extracellular stress.

  • O'Brien KB, Schultz-Cherry S, Knoll LJ (2011) Parasite-mediated upregulation of NK cell-derived gamma interferon protects against severe highly pathogenic H5N1 influenza virus infection. J. Virol. 85(17):8680-8 (PMC3165849) View Abstract · Pubmed Record

    Outbreaks of influenza A viruses are associated with significant human morbidity worldwide. Given the increasing resistance to the available influenza drugs, new therapies for the treatment of influenza virus infection are needed. An alternative approach is to identify products that enhance a protective immune response. In these studies, we demonstrate that infecting mice with the Th1-inducing parasite Toxoplasma gondii prior to highly pathogenic avian H5N1 influenza virus infection led to decreased lung viral titers and enhanced survival. A noninfectious fraction of T. gondii soluble antigens (STAg) elicited an immune response similar to that elicited by live parasites, and administration of STAg 2 days after H5N1 influenza virus infection enhanced survival, lowered viral titers, and reduced clinical disease. STAg administration protected H5N1 virus-infected mice lacking lymphocytes, suggesting that while the adaptive immune response was not required for enhanced survival, it was necessary for STAg-mediated viral clearance. Mechanistically, we found that administration of STAg led to increased production of gamma interferon (IFN-γ) from natural killer (NK) cells, which were both necessary and sufficient for survival. Further, administration of exogenous IFN-γ alone enhanced survival from H5N1 influenza virus infection, although not to the same level as STAg treatment. These studies demonstrate that a noninfectious T. gondii extract enhances the protective immune response against severe H5N1 influenza virus infections even when a single dose is administered 2 days postinfection.

  • Rooney PJ, Neal LM, Knoll LJ (2011) Involvement of a Toxoplasma gondii chromatin remodeling complex ortholog in developmental regulation. PLoS ONE 6(5):e19570 (PMC3104990) View Abstract · Pubmed Record

    The asexual cycle of the parasite Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

  • Rooney PJ, Ayong L, Tobin CM, Moreno SN, Knoll LJ (2011) TgVTC2 is involved in polyphosphate accumulation in Toxoplasma gondii. Mol. Biochem. Parasitol. 176(2):121-6 (PMC3042031) View Abstract · Pubmed Record

    Polyphosphate is found in every cell, having roles in diverse processes, including differentiation and response to stress. In this study, we characterize a Toxoplasma gondii mutant containing an insertion within the carboxy-terminal end of a homolog of Saccharomyces cerevisiae Vtc2p, a component of the polyphosphate synthetic machinery. Locus TgVTC2 encodes a 140kDa protein containing conserved SPX, VTC and transmembrane domains. TgVTC2 localizes in punctate spots within the cytoplasm that do not co-localize with known markers. The TgVTC2 mutant showed dramatically reduced polyphosphate accumulation, a defect restored by introduction of TgVTC2 to the mutant. Insertion within TgVTC2 resulted in increased transcript levels for two loci, including a putative FIKK kinase. These transcript levels were restored to wild-type levels upon complementation with the TgVTC2 locus. The TgVTC2 locus was refractory to knockout, and may be essential. Analysis of this TgVTC2 mutant will facilitate dissection of the T. gondii polyphosphate synthesis pathway.

  • Tobin C1, Pollard A, Knoll L. (2010) Toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions. J Vis Exp. : PMC3156017 View Abstract · Pubmed Record

    Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFNgamma). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNgamma and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNgamma and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

  • Craver MP, Rooney PJ, Knoll LJ (2010) Isolation of Toxoplasma gondii development mutants identifies a potential proteophosphogylcan that enhances cyst wall formation. Mol. Biochem. Parasitol. 169(2):120-3 (PMC2791180) View Abstract · Pubmed Record

    Within warm-blooded animals, Toxoplasma gondii switches from an actively replicating form called a tachyzoite into a slow growing encysted form called a bradyzoite. To uncover the genes involved in bradyzoite development, we screened over 8000 T. gondii insertional mutants by immunofluorescence microscopy. We identified nine bradyzoite development mutants that were defective in both cyst wall formation and expression of a bradyzoite specific heat shock protein. One of these mutants, named 42F5, contained an insertion into the predicted gene TGME49_097520. The disrupted protein is serine/proline-rich with homology to proteophosphoglycans from Leishmania. T. gondii proteophosphoglycan (GU182879) expressed from the native promoter was undetectable in tachyzoites, but bradyzoites show punctate spots within the parasite and staining around the parasitophorous vacuole. Complementation of the 42F5 mutant with GU182879 expressed from either the alpha-tubulin or native promoter restores cyst wall formation. Overall, GU182879 is upregulated in bradyzoites and enhances cyst wall component expression and assembly.

  • Pollard AM, Knoll LJ, Mordue DG (2009) The role of specific Toxoplasma gondii molecules in manipulation of innate immunity. Trends Parasitol. 25(11):491-4 (PMC2771455) View Abstract · Pubmed Record

    Infection with the parasite Toxoplasma gondii stimulates an innate immune response in the host. T. gondii also induces alterations in infected monocytes and dendritic cells that probably contribute to its ability to disseminate and ultimately to establish persistent infection. Recent progress has linked specific parasite molecules to immune stimulation or the ability of the parasite to subvert intracellular signaling pathways in infected cells to evade immunity.

  • Pollard AM, Skariah S, Mordue DG, Knoll LJ (2009) A transmembrane domain-containing surface protein from Toxoplasma gondii augments replication in activated immune cells and establishment of a chronic infection. Infect. Immun. 77(9):3731-9 (PMC2737990) View Abstract · Pubmed Record

    Toxoplasma gondii mutants identified as defective in the establishment of chronic infection were screened to isolate those specifically impaired in their ability to replicate within activated macrophages. One of the identified mutants contains an insertion in the hypothetical gene TGME49_111670. Genetic complementation restores the ability of the mutant to replicate in immune cells and produce cysts in the brains of mice. While the mutant is more sensitive to nitric oxide than is its parental strain, it is not defective in its ability to suppress nitric oxide. The disrupted protein has no significant homology to proteins with known functions, but is predicted to have one transmembrane domain. Immunofluorescence shows the protein on the parasite surface, even in activated macrophages, colocalizing with a tachyzoite surface antigen, SAG1, and oriented with its C-terminal end external. Western analysis reveals that the protein is downregulated in bradyzoites. Despite the tachyzoite specificity of this protein, mice infected with the mutant succumb to acute infection similarly to those infected with the parent strain. Serum samples from mice with chronic T. gondii infection react to a polypeptide from TGME49_11670, indicating that the protein is seen by the immune system during infection. This study is the first to characterize a T. gondii surface protein that contains a transmembrane domain and show that the protein contributes to parasite replication in activated immune cells and the establishment of chronic infection.

  • Frankel MB, Knoll LJ (2009) The ins and outs of nuclear trafficking: unusual aspects in apicomplexan parasites. DNA Cell Biol. 28(6):277-84 (PMC2903460) View Abstract · Pubmed Record

    Apicomplexa is a phylum within the kingdom Protista that contains some of the most significant threats to public health. One of the members of this phylum, Toxoplasma gondii, is amenable to molecular genetic analyses allowing for the identification of factors critical for colonization and disease. A pathway found to be important for T. gondii pathogenesis is the Ran network of nuclear trafficking. Bioinformatics analysis of apicomplexan genomes shows that while Ran is well conserved, the key regulators of Ran--Regulator of Chromosome Condensation 1 and Ran GTPase activating protein--are either highly divergent or absent. Likewise, several import and export receptor molecules that are crucial for nuclear transport are either not present or have experienced genetic drift such that they are no longer recognizable by bioinformatics tools. In this minireview we describe the basics of nuclear trafficking and compare components within apicomplexans to defined systems in humans and yeast. A detailed analysis of the nuclear trafficking network in these eukaryotes is required to understand how this potentially unique cellular biological pathway contributes to host-parasite interactions.

  • Frickel EM, Sahoo N, Hopp J, Gubbels MJ, Craver MP, Knoll LJ, Ploegh HL, Grotenbreg GM (2008) Parasite stage-specific recognition of endogenous Toxoplasma gondii-derived CD8+ T cell epitopes. J. Infect. Dis. 198(11):1625-33 View Abstract · Pubmed Record

    BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. Interferon-gamma-producing CD8+ T cells provide essential protection against T. gondii infection, but the epitopes recognized have so far remained elusive. We employed caged major histocompatibility complex molecules to generate approximately 250 H-2L(d) tetramers and to distinguish T. gondii-specific CD8+ T cells in BALB/c mice. We identified 2 T. gondii-specific H-2L(d)-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2L(d)/GRA4 reactive T cells from multiple organ sources predominated 2 weeks after infection, while the reactivity of the H-2L(d)/ROP7 T cells peaked 6-8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection showed altered levels of antigen-specific T cells, depending on the T. gondii mutant used. Our results shed light on the identity and the parasite stage-specificity of 2 CD8+ T cell epitopes recognized in the acute and chronic phase of infection with T. gondii.

  • Frankel MB, Knoll LJ (2008) Functional analysis of key nuclear trafficking components reveals an atypical Ran network required for parasite pathogenesis. Mol. Microbiol. 70(2):410-20 (PMC2577059) View Abstract · Pubmed Record

    Protozoan parasites represent major public health challenges. Many aspects of their cell biology are distinct from their animal hosts, providing potential therapeutic targets. Toxoplasma gondii is a protozoan parasite that contains a divergent regulator of chromosome condensation 1 (TgRCC1) that is required for virulence and efficient nuclear trafficking. RCC1 proteins function as a guanine exchange factor for Ras-related nuclear protein (Ran), an abundant GTPase responsible for the majority of nucleocytoplasmic transport. Here we show that while there are dramatic differences from well-conserved RCC1 proteins, TgRCC1 associates with chromatin, interacts with Ran and complements a mammalian temperature-sensitive RCC1 mutant cell line. During the investigation of TgRCC1, we observed several unprecedented phenotypes for TgRan, despite a high level of sequence conservation. The cellular distribution of TgRan is found throughout the parasite cell, whereas Ran in late branching eukaryotes is predominantly nuclear. Additionally, T. gondii tolerates at least low-level expression of dominant lethal Ran mutants. Wild type parasites expressing dominant negative TgRan grew similarly to wild type in standard tissue culture conditions, but were attenuated in serum-starved host cells and mice. These growth characteristics paralleled the TgRCC1 mutant and highlight the importance of the nuclear transport pathway for virulence of eukaryotic pathogens.

  • Pollard AM, Onatolu KN, Hiller L, Haldar K, Knoll LJ (2008) Highly polymorphic family of glycosylphosphatidylinositol-anchored surface antigens with evidence of developmental regulation in Toxoplasma gondii. Infect. Immun. 76(1):103-10 (PMC2223667) View Abstract · Pubmed Record

    The life cycle of the apicomplexan parasite Toxoplasma gondii requires that an infectious cyst develop and be maintained throughout the life of the host. The molecules displayed on the parasite surface are important in controlling the immune response to the parasite. T. gondii has a superfamily of glycosylphosphatidylinositol (GPI)-anchored surface antigens, termed the surface antigen (SAG) and SAG-related surface antigens, that are developmentally regulated during infection. Using a clustering algorithm, we identified a new family of 31 surface proteins that are predicted to be GPI anchored but are unrelated to the SAG proteins, and thus we named these proteins SAG-unrelated surface antigens (SUSA). Analysis of the single nucleotide polymorphism density showed that the members of this family are the most polymorphic genes within the T. gondii genome. Immunofluorescence of SUSA1 and SUSA2, two members of the family, revealed that they are found on the parasite surface. We confirmed that SUSA1 and SUSA2 are GPI anchored by phospholipase cleavage. Analysis of expressed sequence tags (ESTs) revealed that SUSA1 had 22 of 23 ESTs from chronic infection. Analysis of mRNA and protein confirmed that SUSA1 is highly expressed in the chronic form of the parasite. Sera from mice with chronic T. gondii infection reacted to SUSA1, indicating that SUSA1 interacts with the host immune system during infection. This group of proteins likely represents a new family of polymorphic GPI-anchored surface antigens that are recognized by the host's immune system and whose expression is regulated during infection.

  • Lavine MD, Knoll LJ, Rooney PJ, Arrizabalaga G (2007) A Toxoplasma gondii mutant defective in responding to calcium fluxes shows reduced in vivo pathogenicity. Mol. Biochem. Parasitol. 155(2):113-22 (PMC2034501) View Abstract · Pubmed Record

    Toxoplasma gondii is an important opportunistic pathogen in immunocompromised individuals. Successful propagation in an infected host by this obligate intracellular parasite depends on its ability to enter and exit host cells. Egress from the cell can be artificially induced by causing fluxes of calcium within the parasite with the use of calcium ionophores. While this ionophore-induced egress (IIE) has been characterized in detail, it is not known whether it mimics a normal physiological process of the parasite. This is underscored by the fact that mutants in IIE do not exhibit strong defects in any of the normal growth characteristics of the parasite in tissue culture. We have isolated and characterized a T. gondii mutant that along with a delay in IIE exhibits a severe defect in establishing a successful infection in vivo. In tissue culture this mutant displays normal ability to invade, divide within cells and convert into the latent encysted bradyzoite form. Nevertheless, mice infected with this mutant are less likely to die and carry less brain cysts than those infected with wild type parasites. Thus, our results suggest that normal response to calcium fluxes plays an important role during in vivo development of T. gondii.

  • Van TT, Kim SK, Camps M, Boothroyd JC, Knoll LJ (2007) The BSR4 protein is up-regulated in Toxoplasma gondii bradyzoites, however the dominant surface antigen recognised by the P36 monoclonal antibody is SRS9. Int. J. Parasitol. 37(8-9):877-85 View Abstract · Pubmed Record

    The protozoan parasite, Toxoplasma gondii, interconverts between fast-growing tachyzoites and slow-growing bradyzoites within intermediate hosts. The surface of T. gondii is covered by the SAG1-related sequence (SRS) superfamily of glycosyl phosphatidyl inositol-anchored proteins, many of which are stage-specific. Previous transient transfection of BSR4, a member of the SRS superfamily, showed reactivity with the bradyzoite-specific P36 mAb by immunofluorescene assay. BSR4 mRNA levels were equally abundant in tachyzoites and bradyzoites, suggesting post-transcriptional regulation of the protein. In this study, we show that BSR4 protein is present in both tachyzoites and bradyzoites, but up-regulated in bradyzoites. However, stable expression of BSR4 in two BSR4-negative T. gondii strains shows minimal reactivity to the P36 mAb by Western immunoblotting, even though the BSR4 protein is abundant. We discovered that the SRS9 protein, a bradyzoite-specific member of the SRS superfamily and encoded immediately downstream of BSR4, was also ablated in the BSR4-negative strains, suggesting that SRS9 is the surface antigen recognised by the P36 mAb. Stable expression of SRS9 in the BSR4 mutant strains shows robust reactivity to the P36 mAb. Immunoprecipitation experiments confirm that the P36 mAb interacts with the SRS9 protein. These data indicate that while the BSR4 protein is up-regulated in bradyzoites, the dominant antigen that the P36 mAb recognises is SRS9.

  • Frankel MB, Mordue DG, Knoll LJ (2007) Discovery of parasite virulence genes reveals a unique regulator of chromosome condensation 1 ortholog critical for efficient nuclear trafficking. Proc. Natl. Acad. Sci. U.S.A. 104(24):10181-6 (PMC1891257) View Abstract · Pubmed Record

    Eukaryotic parasites are a leading cause of morbidity and mortality worldwide, yet little is known about the genetic basis of their virulence. Here, we present a forward genetic screen to study pathogenesis in the protozoan parasite Toxoplasma gondii. By using modified signature-tagged mutagenesis, the growth of 6,300 T. gondii insertional mutants was compared in cell culture and murine infection to identify genes required specifically in vivo. One of the 39 avirulent mutants is disrupted in a divergent ortholog of the regulator of chromosome condensation 1 (RCC1), which is critical for nuclear trafficking in model systems. Although this RCC1 mutant grows similar to wild type in standard tissue culture conditions, it is growth-impaired under nutrient limitation. Genetic complementation of mutant parasites with the T. gondii RCC1 gene fully restores both virulence in mice and growth under low-nutrient conditions. Further analysis shows that there is a significant defect in nuclear trafficking in the RCC1 mutant. These findings suggest that the rate of nuclear transport is a critical factor affecting growth in low-nutrient conditions in vivo and in vitro. Additionally, we observed that although RCC1 proteins are highly conserved in organisms from humans to yeast, no protozoan parasite encodes a characteristic RCC1. This protein divergence may represent a unique mechanism of nucleocytoplasmic transport. This study illustrates the power of this forward genetics approach to identify atypical virulence mechanisms.

  • Craver MP, Knoll LJ (2007) Increased efficiency of homologous recombination in Toxoplasma gondii dense granule protein 3 demonstrates that GRA3 is not necessary in cell culture but does contribute to virulence. Mol. Biochem. Parasitol. 153(2):149-57 View Abstract · Pubmed Record

    Toxoplasma gondii possesses unique secretory organelles, which synchronously release proteins during and after invasion. One of these organelles, the dense granules, secrete proteins after invasion which are thought to be important in development of the parasite throughout all stages of its life cycle. Dense granule protein 3 (GRA3) is a 30 kDa protein localized to the intravacuolar network and parasitophorous vacuole membrane (PVM). Like many dense granule proteins, GRA3 has no homology to proteins with described functions. However, it has been hypothesized to be involved in nutrient acquisition for the parasite due to its localization on the PVM. To begin to investigate the importance of GRA3, the locus was disrupted by homologous replacement with a chloramphenicol resistance gene in a type II strain. Two DeltaGRA3 strains were obtained after two independent electroporations with efficiency greater than 80%. No differences between wild-type and DeltaGRA3 were detected in cell culture growth rate or bradyzoite formation. Location of other parasite dense granule proteins and association with host cell organelles were also not affected in DeltaGRA3. Interestingly, at an infectious dose approximately four-fold above the lethal dose 50% for wild-type parasites, all mice infected with DeltaGRA3-2 infected mice survived acute infection. Complementation of GRA3 expression in the DeltaGRA3-2 strain restored virulence to wild-type levels, and increased the virulence of the DeltaGRA3-1, confirming that the GRA3 protein plays a role during acute infection in a type II strain.

  • Mordue DG, Scott-Weathers CF, Tobin CM, Knoll LJ (2007) A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages. Mol. Microbiol. 63(2):482-96 View Abstract · Pubmed Record

    The apicomplexan parasite Toxoplasma gondii is able to suppress nitric oxide production in activated macrophages. A screen of over 6000 T. gondii insertional mutants identified two clones, which were consistently unable to suppress nitric oxide production from activated macrophages. One strain, called 89B7, grew at the same rate as wild-type parasites in naïve macrophages, but unlike wild type, the mutant was degraded in activated macrophages. This degradation was marked by a reduction in the number of parasites within vacuoles over time, the loss of GRA4 and SAG1 protein staining by immunofluorescence assay, and the vesiculation and breakdown of the internal parasite ultrastructure by electron microscopy. The mutagenesis plasmid in the 89B7 clone disrupts the promoter of a 3.4 kb mRNA that encodes a predicted 68 kDa protein with a cleavable signal peptide and a patatin-like phospholipase domain. Genetic complementation with the genomic locus of this patatin-like protein restores the parasites ability to suppress nitric oxide and replicate in activated macrophages. A haemagglutinin-tagged version of this patatin-like protein shows punctate localization into atypical T. gondii structures within the parasite. This is the first study that defines a specific gene product that is needed for parasite survival in activated but not naïve macrophages.

  • Van Tam T, Rooney PJ, Knoll LJ (2006) Nourseothricin acetyltransferease: a positive selectable marker for Toxoplasma gondii. J. Parasitol. 92(3):668-70 View Abstract · Pubmed Record

    Molecular analysis of parasite genomes will require new molecular genetic tools. The nat1 gene of Streptomyces noursei encodes nourseothricin acetyltransferase, conferring resistance to the aminoglycoside antibiotic nourseothricin. Electroporation of nat1 cassettes into RH or Prugniaud strains of Toxoplasma gondii allows for selection of stable nourseothricin-resistant clones.