The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. We have recently identified a T. cruzi-released protein related to thiol-disulfide oxidoreductase family, called Tc52, which is crucial for parasite survival and virulence. In vitro, Tc52 in combination with IFN-γ activates human macrophages. In vivo, active immunization with Tc52 relieves the immunosuppression associated to acute infection and elicits a specific immune response. As dendritic cells (DC) have a central role in the initiation of immune responses, we investigated whether Tc52 may modulate DC activity. We show that Tc52 induces human DC maturation. Tc52-treated immature DC acquire CD83 and CD86 expression, produce inflammatory chemokines (IL-8, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1α), and present potent costimulatory properties. Tc52 binds to DC by a mechanism with the characteristics of a saturable receptor system and signals via Toll-like receptor 2. While Tc52-mediated signaling involves its reduced glutathione-binding site, another portion of the molecule is involved in Tc52 binding to DC. Finally, we report that immunization with Tc52 protects mice in vivo against lethal infection with T. cruzi. Together these data evidence complex molecular interactions between the T. cruzi-derived molecule, Tc52, and DC, and suggest that Tc52 and related class of proteins might represent a new type of pathogen-associated molecular patterns. Moreover, the immune protection data suggest that Tc52 is among candidate molecules that may be used to design an optimal multicomponent vaccine to control T. cruzi infection.

A l'occasion de l'analyse écoépidémiologique d'un mésofoyer marocain de leishmaniose cutanée à Leishmania tropica, 7 907 Phlébotomes ♀, capturés au piège CDC, ont été disséqués, de l'été à l'automne 1989. Parmi les espèces du genre Phlebo tomus, seul P. sergenti était porteur de promastigotes. Quatrevingt-neuf souches, appartenant au complexe L. tropica, ont été ainsi isolées. La fréquence de l'infestation vectorielle, nulle en juin, est passée à 1,3 % en août, pour atteindre 9,9 % en octobre, situant la période à risque en fin de saison chaude. Sur les 89 souches In a Moroccan focus of cutaneous leishmaniasis caused by Leish mania tropica, 7 907 female sandflies captured with CDC traps were dissected from summer to autumn 1989. Among species of the genus Phlebotomus, only P. sergenti harbored promastigotes. Eighty-nine strains belonging to the complex L. tropica were iso lated. The frequency of vector infection was zero in June, rose to 1.3 % in August, and reached 9.9 % in October, which indi cates that the period of high risk is at the end of the hot season. Out of 89 strains isolated, 74 were completely typed and corres ponded to the following four zymodemes: MON-102 (one strain), MON-107 (56 strains), MON-122 (two strains), and MON-123 (15 strains). Only the first two were observed in humans. The dis tribution of zymodemes MON-102 and MON-107 was very diffe rent in humans, dogs, and the vector. In one of the sites sur veyed, which was strongly dominated by MON-107, the absence of human cases involving this zymodeme suggests the existence of a wild reservoir.A la suite de la confirmation biochimique du premier cas marocain de leishmaniose cutanée humaine (LCH) à Leishmania tropica (5), trois enquêtes entomologiques ont pu être réalisées dans la province d'Azilal, de juin à octobre 1989. Les prospections, effectuées en période d'activité vec torielle, ont permis la capture et la dissection de 7 907 Phlé botomes ♀. Quatre-vingt-neuf souches, appartenant toutes au complexe chimiotaxonomique L. tropica, ont été iso lées du seul Phlebotomus sergenti. Soixante-quatorze d'entre elles ont été identifiées jusqu'au zymodème, confirmant le rôle vecteur de cette espèce.

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In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (e.g. logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [35S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunofluorescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.

We have recently characterized a novel Leishmania major gene encoding a polypeptide of 30 kDa that was homologous to mammalian ribosomal protein S3a and was named LmS3a-related protein (LmS3arp). The protein was found to be expressed by all the Leishmania species so far examined (L. infantum, L. amazonensis, and L. mexicana). In the present study we have extended our approach to the analysis of

SummaryWe have identified previously a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we have reported that Tc52 also played a role in T. cruzi-associated immunosuppression observed during Chagas' disease. In an effort to understand further the biological role of Tc52, we used a gene-targeted deletion strategy to create T. cruzi mutants. Although T. cruzi tolerates deletion of one wild-type Tc52 allele, deletion of both genes is a lethal event, indicating that at least one active Tc52 gene is required for parasite survival. Monoallelic disruption of Tc52 (Tc52 þ/¹ ) resulted in the production of T. cruzi lines that express less Tc52 mRNA and produced lower amounts of Tc52 protein compared with wild-type cells. In axenic cultures, growth rates of epimastigote forms bearing an interrupted allele were not different from those of wild-type parasites. Furthermore, monoallelic disruption of the Tc52 gene did not modify the growth rate of epimastigotes or their sensitivity to inhibition by benznidazole and nifurtimox, the two drugs used to treat Chagasic patients. Moreover, the antimonial drug SbIII, which is known, at least in Leishmania parasites, to be conjugated to a thiol and extruded by an ATP-coupled pump, had a similar effect on wild-type and mutant parasites, being equally sensitive. Hence, parasite drug sensitivity was also observed in clones overexpressing the Tc52 protein as well as in those carrying an antisense plasmid construct. Surprisingly, a significant impairment of the ability of epimastigotes carrying a Tc52 single gene replacement or antisense construct to differentiate into metacyclic trypomastigotes and to proliferate in vitro and in vivo was observed, whereas no significant enhancement of these biological properties was seen in the case of parasites that overexpress Tc52 protein. Moreover, functional complementation of Tc52

An intense suppression of T cell proliferation to mitogens and to antigens is observed in a large number of parasitic infections. The impairment of T cell proliferation also occurred during the acute phase of Chagas' disease, caused by the intracellular protozoan parasite Trypanosoma cruzi. A wealth of evidence has accumulated that illustrates the ability of T. cruzi released molecules to influence directly a variety of diverse immunological functions. In this paper, we review the data concerning the immunoregulatory effects of T. cruzi Tc24 (a B cell activator antigen) and Tc52 (an immunosuppressive protein) released molecules on the host immune system. The gene targeting approach developed to further explore the biological function(s) of Tc52 molecule, revealed interesting unexpected functional properties. Indeed, in addition to its immunusuppressive activity a direct or indirect involvement of Tc52 gene product alone or in combination with other cellular components in T. cruzi differentiation control mechanisms have been evidenced. Moreover, targeted Tc52 replacement allowed the obtention of parasite mutants exhibiting low virulence in vitro and in vivo. Thus, the generation of a complete deficiency state of virulence factors by gene targeting should provide a means to assess the importance of these factors in the pathophysiological processes and disease progression. It is hoped that such approaches might allow rational design of tools to control T. cruzi infections. INTRODUCTIONThe ability of parasites to survive in the immune hosts depends upon a variety of escape mechanisms. One of these is the inhibition or the suppression of the immune response of their hosts. Several possible explanations have been put forward, such as antigenic competition, acquired tolerance, as well as the possible blocking role of soluble antigens or circulating immune complexes. The release by the parasites themselves of excretory-secretory products which have potent immunosuppressive activity represents another possible explanation. Moreover, a large number of reports have described the presence of suppressor cells (T lymphocytes and macrophages) in humans and animals infected with various parasites.In the case of Chagas' disease which remains a medical problem of relative important proportions in Latin America studies of the pathogenesis and immunology of the disease aiming to disclose the molecular mechanisms underlying the immunological alterations associated with T. cruzi infection are currently conducted in several laboratories over the world.Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligate intracellular parasite causing chronic infections in human and a large number of other mammalian species [1]. This protozoan parasite is transmitted to man and other vertebrate hosts in the faeces of haematophagous bugs of the Reduviid family. The complex life cycle of T. cruzi includes different stages in the insect vector and the vertebrate host. There are two parasite stages in the vector: epimastigotes and me...

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