Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

BackgroundWorldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences.MethodsTo better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5–12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or αPD1 ligand were studied.ResultsTwelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimer's disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation.ConclusionIn outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.

T1/ST2 is an immunoregulatory protein of the IL-1 receptor family that has recently been reported as being a component of the IL-33 receptor. IL-33 is a newly described cytokine known to amplify the Th2 response and reduce production of Th1 cytokines. The function of T1/ST2 during Toxoplasma gondii infection is as yet undescribed. Given the requirement of a balanced type 1/type 2 response for effective control of parasite number and immunopathology, it is likely that T1/ST2 may play a part in aiding this process. Accordingly, we have shown that T1/ST2 mRNA transcripts are upregulated in the brains of mice infected with T. gondii and that mice deficient in T1/ST2 demonstrated increased susceptibility to infection with T. gondii that correlated with increased pathology and greater parasite burden in the brains. Real-time PCR analysis of cerebral cytokine levels revealed increased mRNA levels of iNOS, IFN-c and TNF-a in infected T1/ST2 À/À mice. These effects were independent of changes in IL-10 production. This study provides the first evidence of a specific role for IL-33 receptor signalling in the brain as well as highlighting the requirement of this mechanism in limiting infection with an intracellular parasite.Key words: Encephalitis . IL-33 . IL-33 receptor . Th1/Th2 . Toxoplasma IntroductionThe receptor T1/ST2 is a member of the IL-1 receptor family that also includes the TLR and the IL-18R [1]. Further characterisation revealed that differential splicing of T1/ST2 mRNA led to production of two different transcripts encoding either a transmembrane form (ST2L) or a soluble, secreted form (sST2) [2]. T1/ST2 is expressed by a number of haematopoetic cells such as T cells, mast cells, macrophages, NK cells and invariant NKT cells [3][4][5][6][7]. ST2L has previously been used as a marker for Th2 cells although T1/ST2-negative Th2 cells exist with reports that ST2L may be better described as a marker of effector Th2 cells enhancing the Th2 response rather than aiding its development [3,[8][9][10]. As well as promoting a Th2 response ST2L has been shown to sequester the signalling molecules MyD88 and Mal to inhibit subsequent cytokine production following TLR ligation [10]. sST2 also plays a role in downregulating the inflammatory response [6,11]. 426T1/ST2 is the ligand-binding component of the receptor for the cytokine IL-33 which, together with the IL-1 receptor accessory protein, forms the IL-33 receptor [12,13]. IL-33 is a member of the IL-1 family with the ability to downregulate IFN-g production by Th1 cells in vitro and upregulate the production of the Th2 cytokines IL-5 and IL-13 from Th2 cells both in vitro and in vivo [12]. Therefore, the function of IL-33 reinforces the previously described role of T1/ST2 in enhancing a Th2 response while reducing a Th1 response.Protective immunity to the protozoan parasite Toxoplasma gondii relies on a delicate balance of type 1/type 2 immune responses to effectively control parasite proliferation and prevent pathology caused by an over-exuberant type-1 response [14]...

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