Identification and molecular characterization of two novel Trypanosoma cruzi genes encoding polypeptides sharing sequence motifs found in proteins involved in RNA editing reactions.

We have previously identified a Trypanosoma cruzi cDNA 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 reported that Tc52 also plays a role in T. cruzi-associated immunosuppression observed during Chagas' disease. Moreover, Tc52 gene targeting deletion strategy allowed us to demonstrate that monoallelic disruption of Tc52 resulted in the alteration of the metacyclogenesis process and the production of less virulent parasites. Sequence analysis of a 7358 bp genomic fragment containing the Tc52 encoding gene revealed two additional open reading frames (ORF-A and C). The ORFs are likely to have protein coding function by a number of criteria, including reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunofluorescence analyses. The deduced amino-acid (aa) sequence of the ORF-A localized upstream of the Tc52 gene revealed that it contains within its N-terminus (aa 1 to 170) four RGG boxes known to act as RNA binding motifs in some proteins that interact with RNA, interspersed with a high density of glycine with regular spacing of tryptophan (WX(9-10)) in which X is often a glycine. Moreover, the C-terminal part of the ORF-C (aa 253-289) contains a motif that is strikingly similar (7-35% identity, 14-46% similarity over 28aa) to a short sequence (RNP1) comprising the consensus sequence RNA binding domain (CS-RBD) found in a number of proteins that interact with RNA. The aa sequence from the ORF-C localized downstream of the Tc52 gene showed significant homology to human adenosine deaminase acting on RNA (hADAT1) that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala) and to its homologue yeast protein (Tad1p) (22-25% identity and an additional 38-40% similarity over 177aa). Moreover, highly similar motifs of the deaminase domain are present in the T. cruzi ORF-C. Furthermore, the 5' flanking regions of the genes contained repeat TATA and CAAT nucleotide sequences which resemble the motifs found upstream of the transcription initiation sites in eukaryotic promoters. Therefore, the characterization of novel T. cruzi genes encoding proteins which show similarity to components of RNA processing reactions provides new tools to investigate the gene expression regulation in these parasitic organisms. Moreover, our recent findings on the Tc52 encoding gene underline the interest of genetic manipulation of T. cruzi, not only making it possible to use more closely an in vitro approach to find out how genes function, but also to obtain 'attenuated' strains that could be used in the development of vaccinal strategies.

Cloning of a Leishmania major gene encoding for an antigen with extensive homology to ribosomal protein S3a.

Following purification by affinity chromatography, a Leishmania major S-hexylglutathione- binding protein of molecular mass 66kDa was isolated. The immune serum against the parasite 66kDa polypeptide when used to screen a L. major cDNA library could identify clones encoding for the human v-fos transformation effector homologue, namely ribosomal protein S3a, and thus was named LmS3a-related protein (LmS3arp). A 1027bp cDNA fragment was found to contain the entire parasite gene encoding for a highly basic protein of 30kDa calculated molecular mass sharing homology to various ribosomal S3a proteins from different species. Using computer methods for a multiple alignment and sequence motif search, we found that LmS3arp shares a sequence homology to class theta glutathione S-transferase mainly in a segment containing critical residues involved in glutathione binding. These new findings are discussed in the light of recent published data showing multiple function(s) of the ribosomal proteins S3a.

Phlebotomus perniciosus Newstead, 1911, infection by various zymodemes of the Leishmania infantum complex in the Granada province (southern Spain).

This study presents the results of the isoenzymatic characterization of 21 strains of Leishmania of sandfly (P. perniciosus) origin from the Torvizcón area. It forms an integral part of a larger eco-epidemiological study of the Alpujarras (Granada province, Southern Spain). The strains analysed were shown to belong to the L. infantum complex based on the results of 15 enzymes. The electrophoretic profiles for the enzymes MDH, G6PD and NP1 have permitted the identification of four zymodemes: GR-1 (5 strains), GR-2 (2 strains), GR-3 (13 strains) and GR-7 (1 strain); only one of these zymodemes, GR-1, was found in the Torvizcón area in the vertebrate host (man and dog). This is the first time zymodeme GR-7 has been described.

Experimental studies on the evolution of antimony-resistant phenotype during the in vitro life cycle of Leishmania infantum: implications for the spread of chemoresistance in endemic areas.

Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modulation of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50,90/10,10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12,28,35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote-promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasite's life cycle.

[Description of Aedes (Ochlerotatus) coluzzii n. sp. (Diptera, Culicidae), twin A species of the detritus complex]. / Description d'Aedes (Ochlerotatus) coluzzii n. sp. (Diptera, Culicidae), espèce jumelle A du complexe detritus.

Reports of a wide variation in space and time of the frequency of autogeny in Aedes (Ochlerotatus) detritus (Haliday, 1833) of the Camargue (Delta of the Rhône, Bouches-du-Rhône, Gard, France) led us to make an enzymatic analysis of male and female adults from different larval biotopes. The study showed the existence of two genetically distinct, sympatric populations which are morphologically indistinguishable. By diagnostic enzymes monomorph Got-2, Gpd, the grouping of the individual into two subgroups satisfies a Hardy-Weinberg equilibrium for the polymorphic enzymes. It is concluded that Ae. detritus is composed of a complex of two sibling species provisionally designated by the letters A (Got-2RR, GpdCC) and B (Got-2LL, GpdBB). In the present article, we retrace the history of the binomen Ae. detritus since the original description (sub nom. Culex detritus) to the split into the detritus complex. Certain ecophysiological (steno-eurygamy) and chorological (bioclimatic gradients N-S) criteria show that the sibling species B should be assigned to the taxon described in UK (Holywood, County Down, Ireland) by A.H. Haliday. Species A is here named Aedes (Ochlerotatus) Coluzzii n. sp.

Changes in ecdysteroid and juvenile hormone titers correlated to the initiation of vitellogenesis in two Aedes species (Diptera, Culicidae).

Ecdysteroid and juvenile hormone titers were determined in the whole body of females of Aedes detritus and A. caspius. Since both hormones were assayed from the same extract, this method allowed determination of their simultaneous variations during egg formation, i.e., from the time the females emerged until the onset of oviposition. A drastic hormonal increase was observed at the beginning of vitellogenesis. This increase occurred as two high and sharp peaks, the first of ecdysteroids and the second, which took place 8 hr later, of juvenile hormones. The two peaks together lasted less than 12 hr, with the highest level at about 3 X 10(-7) mumol/mg fresh tissue. After the juvenile hormone peak, the oocytes entered into stage III/b, the time at which the intensive phase of vitellin accumulation in the eggs begins.

[Influence of larval nutrition on the fecundity of autogenous and anautogenous females inside the Aedes (Ochlerotatus) detritus (Haliday, 1833) complex [Diptera-Culicidae]. (author's transl)]. / Influence de la nourriture larvaire sur la fécondité des femelles autogènes et anautogènes dans le complexe Aedes (Ochlerotatus) detritus (Haliday, 1833) [Diptera - Culicidae].

Using two sibling species of the Aedes detritus (Haliday, 1833) complex, one autogenous (A) and the other anautogenous (B), the role played by the larval environment on vitellogenesis was confirmed. A poor larval diet reduced the fecondity equally of both autogenous and anautogenous females. With the latter however blood meal ensured the maturation of a not negligible number of ovocytes.

[Autogenesis and cerebral neurosecretion in Aedes detritus (Haliday, 1833) (Diptera - Culicidae]. / Autogenèse et neurosécrétion cérébrale chez Aedes detritus (Haliday, 1833) (Diptera-Culicidae)

In anautogenous females of the Dipteran Culicidae Aedes detritus (Haliday, 1833), the neurosecretory products synthesized by the A cells of the pars intercerebralis are stored in the pericarya in absence of blood meal; on the contrary, in the autogenous females they are steadily released from emergency till the stages 3-4 of vitellogenesis; a slight storage occurs by the end of vitellogenesis. These facts suggest the important meaning of these cells in the regulation processes of vitellogenesis in Mosquitoes.

[Ecology of leishmaniasis in the south of France. 15. The gonotrophic cycles in nature of Phlebotomus ariasi and P. mascittii in the Cévennes. Epidemiological significance (author's transl)]. / Ecologie des leishmanioses dans la Sud de la France. 15. Déroulement des cycles gonotrophiques chez Phlebotoms ariasi Tonnoir, 1921 et Phlebotomus mascittii Grassi, 1908 en Cévennes. Corollaire épidémiologique.

The number of gonotrophic cycles of phlebotomine sandflies in a leishmaniasis focus in the Cévennes was determined by the examination of the ovaries. The method was to count the number of dilatations on the ovariolar stalks which showed how many times the females had laid eggs. It was found that females of P. ariasi undergo at least three gonotrophic cycles. From mid-June at the beginning of the season, until the middle of August the proportion of parous females was low. After this time until the end of the season in mid-September, the frequency of 2 parous and 3 parous individuals steadily rose. It is concluded that the end of the summer is the period of maximum risk for the transmission of leishmaniasis in this focus.

[Presence in Camargue of two sympatric, sexually isolated forms of Aedes (Ochlerotatus) detritus (Haliday, 1833) [Diptera-Culicidae] (author's transl)]. / Existence chez Aedes (Ochlerotatus) detritus (Haliday, 1833) (Diptera-Culicidae) de Camargue de deux formes sympatriques et sexuellement isolées (espèces jumelles).

The study of allozymic variations at four enzymatic loci (Est-2, alpha-Gpd, Got-1 and Got-2) in populations of Aedes detritus (Hal.) from 27 localities in Southern France has shown that this species is composed of two kinds of sympatric populations which do not interbreed in nature. Single specimens of Aedes detritus can be attributed to one or other type of population (sibling species) by the genotype at the alpha-Gpd or Got-2 loci (type A populations are homozygous for Got-2R allele and the frequency of the alpha-GpdC allele is higher than 98%; type B populations are homozygous for Got-2L allele and the frequency of the alpha-GpdB allele is higher than 98%). Moreover, allelic frequencies at the Est-2 and Got-1 loci are different in both types of populations. The fact that both kinds of populations coexist in the same pond shows that the isolating mechanism is not an adaptation to the larval environment, but rather involves mechanisms pecular to adults (precopulatory mechanism or interpopulation sterility).

[A new report of naturally anautogenous and stenogamic populations of Culex pipiens pipiens L. in the south of France (author's transl)]. / Nouvelle mention, pour le "Midi" méditerranéen, de populations naturelles anautogènes et sténogames de Culex pipiens pipiens L.

The presence of an anautogenous and stenogamic population of Culex pipiens pipiens in Languedoc, France, is reported. The anautogeny was confirmed through 10 generations. A Callot morphogram corresponded to the anautogenous form. These results raise again the question of the type of relationships, genetic or ecological, of the characters classically associated with autogeny.

[Coelomomyces psorophorae Couch, 1945 (Blastocladiales-Coelomomycetaceae), a parasite of Aedes detritus (Haliday, 1833) (Diptera-Culicidae) in the Camargue (author's transl)]. / Coelomomyces psorophorae Couch, 1945 (Blastocladiales- Coelomomycetaceae), parasite d'Aedes detritus (Haliday, 1833) (Diptera-Culicidae) en Camargue.

The Phycomycete, Coelomomyces psorophorae Couch, 1945, was found in the south of France in the salt marsh mosquito Aedes detritus (Haliday, 1833). The fungus invades the ovarioles but leaves the follicules intact. At high magnification, (using a scanning electron microscope), the crater-form punctuations adorning the sporangium were seen to result from openings (ostioles) of the network of small canals that traverse the external wall.

Intracellular growth and metacyclogenesis defects in Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele.

We 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+/-) 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+/- single mutant or selection of antisense revertant clones demonstrated that the phenotype observed is a direct consequence of Tc52 gene manipulation. Taken together, these results may suggest that Tc52 could participate among other factors in the phenotypic expression of T. cruzi virulence.

Dual role of the Leishmania major ribosomal protein S3a homologue in regulation of T- and B-cell activation.

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 LmS3arp activity on T- and B-cell functions in a murine model. The results presented in this report show that LmS3arp plays a dual role in the regulation of T- and B-cell reactivity. Indeed, we found that injection of the LmS3arp recombinant protein (rLmS3arp) into BALB/c mice induces preferential activation of B cells, as shown by the following criteria: (i) increased expression of CD69 molecules on immunoglobulin M (IgM)-secreting spleen cells, (ii) a considerable increase of IgM-secreting B cells, and (iii) elevated levels of IgM antibodies in the sera of injected animals. Moreover, the IgM antibodies are not specific to the Leishmania antigens but preferentially recognize heterologous antigens like myosin, thyroglobulin, DNA, and keyhole limpet hemocyanin. Furthermore, the strong polyclonal expansion of nonspecific, non-parasite-directed B-cell clones induced by rLmS3arp is concomitant with a marked inhibition of T-cell proliferation. Analysis of cytokine production revealed a significant downregulation of gamma interferon, interleukin-2 (IL-2), and IL-12 secretion. Taken together, our data suggest that rLmS3arp, through direct or indirect action toward B and T cells and cytokine secretion, could participate in the immunoregulatory processes that play a role in the balance of the Th1 and Th2 immune response.

Leishmania major: cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family.

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.

[Aedes (Ochlerotatus) surcoufi (Theobald, 1912). Restoration of the binomen; morphological analysis; position in the holarctic "excrucians" complex (author's transl)]. / Aedes (Ochlerotatus) surcoufi (Theobald, 1912). Rétablissement du binôme; analyse morphologique position au sein du complexe holarctique "excrucians

The study of the morphology of eggs, larvae, pupae and adults of Aedes excrucians from Germany, Italy and France has led the authors to rise again the binomen Aedes surcoufi (Theobald, 1912) to particularize the european populations. At the same time, the characteristics of the different species of the Aedes excrucians complex are discussed.

[A new Phlebotomus for Spain. Phlebotomus (Adlerius) mascittii Grassi, 1908]. / Un phlébotome mouveau pour l'Espagne. Phlebotomus (Adlerius) mascittii Grassi, 1908.

The authors have made the first discovery of Phlebotomus mascittii in Spain. The species was caught in Catalonia in the provinces of Gerona and Barcelona. Altogether 7 individual sandflies (4 male, 3 female) were collected (using oiled paper traps) in four separate sub-mediterranean sites (between 650 and 780 m).