Vespa et al., 1994;Biron and Gazzinelli, 1995 Aliberti et al., 1996;Fearon and Locksley, 1996; Trinchieri and Scott, 1996). In addition, the early Seder et al., 1993; Abbas et al., 1996; Fearon macrophage proinflammatory cytokines by Trypanoand Locksley, 1996). During infection with T.cruzi, the soma cruzi is considered to be important in controlling induction of parasite-specific CMI is likely to be involved the infection and the outcome of Chagas' disease. Here in at least two aspects of Chagas' disease pathophysiology we show that the potent tumour necrosis factor-α-, (Vespa et al., 1994;Fearon and Locksley, 1996; Brener interleukin-12-and nitric oxide-inducing activities of and Gazzinelli, 1997). The first is the control of parasite T.cruzi trypomastigote mucins were recovered quantireplication and its spread in the vertebrate host tissues. tatively in a highly purified and characterized glycosyl-The second is the inflammatory reaction observed in the phosphatidylinositol (GPI) anchor fraction of this infected host tissues, which is likely to be a major cause material. The bioactive trypomastigote GPI fraction of cardiac tissue damage during the acute and chronic was compared with a relatively inactive GPI fraction phases of the disease. prepared from T.cruzi epimastigote mucins. The trypoRelatively little is known about the protozoan parasite mastigote GPI structures were found to contain addimolecules that initiate the synthesis of pro-inflammatory tional galactose residues and unsaturated, instead of cytokines and nitric oxide (NO) by macrophages. Recent saturated, fatty acids in the sn-2 position of the alkylstudies have suggested a role for glycosylphosphatidylacylglycerolipid component. The latter feature is esseninositol (GPI) anchors from Plasmodium falciparum tial for the extreme potency of the trypomastigote (Schofield and Hackett, 1993;Schofield et al., 1996; GPI fraction, which is at least as active as bacterial Tachado et al., 1996Tachado et al., , 1997 and Trypanosoma brucei endotoxin and Mycoplasma lipopeptide and, therefore, (Magez et al., 1998) in this process. However, no evidence one of the most potent microbial proinflammatory of the biochemical purity of the P.falciparum GPIs was agents known.provided, making estimates of their concentrations dubiKeywords: Chagas' disease/cytokines/glycosylphosphaous. Furthermore, the possibility of Mycoplasma lipopeptidylinositol/inflammation/nitric oxide tide (Mühlradt et al., 1997) contamination (that has confounded other studies into proinflammatory factors) cannot be formally excluded. In the case of the T.brucei GPI work, highly purified and structurally characterized

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease in humans, has a complex life cycle alternating between the insect vector and the mammalian host. In the vector, it multiplies as noninfective epimastigotes that migrate to the hindgut and differentiate into infective metacyclic trypomastigotes. During the insect blood meal, the metacyclic trypomastigotes are deposited with the feces and urine near a skin wound, initiating the natural infection.T. cruzi is unable to synthesize sialic acids (SA), 1 but it expresses a unique trans-sialidase (TS), which transfers ␣2-3-linked SA from host glycoproteins and glycolipids to acceptors containing terminal ␤-galactosyl residues present on the parasite surface (reviewed in Refs. 1-4). Several studies characterizing the nature and structure of the SA acceptors have been published. These acceptors are abundant on the parasite surface and were first described as major surface glycoproteins of epimastigotes by Alves and Colli (5), who called them bands A, B, and C. Subsequently, a similar cell surface glycoprotein complex, called GP24, GP31, and GP37 was described by Ferguson et al. (6), and Previato et al. (7) first described a 43-kDa SA acceptor. More recently, they have been called 38/43 glycoconjugates (8), and the so called epimastigote lipophosphoglycan-like molecule could belong to the same family of molecules (9). In metacyclic trypomastigote forms, the SA acceptors were reported originally as the 35/50-kDa antigens (10, 11) that were subsequently defined as mucin-like glycoproteins (12). In the trypomastigote forms found in mammals, the SA acceptors were described as a group of molecules that share the stagespecific epitope 3 (Ssp-3) (13), an epitope dependent on parasite

The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.

Previous studies have established that mutations in the NDR1 gene in Arabidopsis thaliana suppress the resistance response of three resistance proteins, RPS2, RPM1, and RPS5, to Pseudomonas syringae pv. tomato (Pst) strain DC3000 containing the cognate effector genes, avrRpt2, avrRpm1, and avrpPhB, respectively. NDR1 is a plasma membrane (PM)-localized protein, and undergoes several post-translational modifications including carboxy-terminal processing and N-linked glycosylation. Expression of NDR1 under the NDR1 native promoter complements the ndr1-1 mutation, while overexpression of NDR1 results in enhanced resistance to virulent Pst. Sequence analysis and mass spectrometry suggest that NDR1 is localized to the PM via a C-terminal glycosylphosphatidyl-inositol (GPI) anchor. GPI modification would potentially place NDR1 on the outer surface of the PM, perhaps allowing NDR1 to act as a transducer of pathogen signals and/or interact directly with the pathogen.

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