P-type transport ATPases in Leishmania and Trypanosoma.

P-type ATPases are critical to the maintenance and regulation of cellular ion homeostasis and membrane lipid asymmetry due to their ability to move ions and phospholipids against a concentration gradient by utilizing the energy of ATP hydrolysis. P-type ATPases are particularly relevant in human pathogenic trypanosomatids which are exposed to abrupt and dramatic changes in their external environment during their life cycles. This review describes the complete inventory of ion-motive, P-type ATPase genes in the human pathogenic Trypanosomatidae; eight Leishmania species (L. aethiopica, L. braziliensis, L. donovani, L. infantum, L. major, L. mexicana, L. panamensis, L. tropica), Trypanosoma cruzi and three Trypanosoma brucei subspecies (Trypanosoma brucei brucei TREU927, Trypanosoma brucei Lister strain 427, Trypanosoma brucei gambiense DAL972). The P-type ATPase complement in these trypanosomatids includes the P1B (metal pumps), P2A (SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPases), P2B (PMCA, plasma membrane calcium ATPases), P2D (Na+ pumps), P3A (H+ pumps), P4 (aminophospholipid translocators), and P5B (no assigned specificity) subfamilies. These subfamilies represent the P-type ATPase transport functions necessary for survival in the Trypanosomatidae as P-type ATPases for each of these seven subfamilies are found in all Leishmania and Trypanosoma species included in this analysis. These P-type ATPase subfamilies are correlated with current molecular and biochemical knowledge of their function in trypanosomatid growth, adaptation, infectivity, and survival.

TITLE: Les ATPases de transport de type P chez Leishmania et Trypanosoma. ABSTRACT: Les ATPases de type P sont essentielles au maintien et à la régulation de l'homéostasie des ions cellulaires et de l'asymétrie des lipides membranaires en raison de leur capacité à déplacer les ions et les phospholipides contre un gradient de concentration en utilisant l'énergie de l'hydrolyse de l'ATP. Les ATPases de type P sont particulièrement utiles chez les trypanosomatidés pathogènes pour l'homme, qui sont exposés à des changements abrupts et dramatiques de leur environnement externe au cours de leur cycle de vie. Cette revue décrit l'inventaire complet des gènes d'ATPase de type P à motif ionique chez les Trypanosomatidae pathogènes pour l'homme ; huit espèces de Leishmania (L. aethiopica, L. braziliensis, L. donovani, L. infantum, L. major, L. mexicana, L. panamensis, L. tropica), Trypanosoma cruzi et trois sous-espèces de Trypanosoma brucei (Trypanosoma brucei brucei TREU927, Trypanosoma brucei Lister souche 427, Trypanosoma brucei gambiense DAL972). Le complément ATPase de type P dans ces trypanosomatidés comprend les sous-familles P1B (pompes métalliques), P2A (SERCA, ATPases calciques du réticulum sarcoplasmo-endoplasmique), P2B (PMCA, ATPases calciques de la membrane plasmique), P2D (pompes Na+), P3A (pompes H+), P4 (translocateurs des aminophospholipides) et P5B (sans spécificité attribuée). Ces sous-familles représentent les fonctions de transport des ATPases de type P nécessaires à la survie des trypanosomatidés, car les ATPases de type P de chacune de ces sept sous-familles sont présentes chez toutes les espèces de Leishmania et de Trypanosoma incluses dans cette analyse. Ces sous-familles d'ATPases de type P sont corrélées aux connaissances moléculaires et biochimiques actuelles sur leur fonction dans la croissance, l'adaptation, l'infectivité et la survie des trypanosomatidés.

Genetic diversity in Trichomonas vaginalis.

Recent advances in genetic characterisation of Trichomonas vaginalis isolates show that the extensive clinical variability in trichomoniasis and its disease sequelae are matched by significant genetic diversity in the organism itself, suggesting a connection between the genetic identity of isolates and their clinical manifestations. Indeed, a high degree of genetic heterogeneity in T vaginalis isolates has been observed using multiple genotyping techniques. A unique two-type population structure that is both local and global in distribution has been identified, and there is evidence of recombination within each group, although sexual recombination between the groups appears to be constrained. There is conflicting evidence in these studies for correlations between T vaginalis genetic identity and clinical presentation, metronidazole susceptibility, and the presence of T vaginalis virus, underscoring the need for adoption of a common standard for genotyping the parasite. Moving forward, microsatellite genotyping and multilocus sequence typing are the most robust techniques for future investigations of T vaginalis genotype-phenotype associations.

Genetic characterization of Trichomonas vaginalis isolates by use of multilocus sequence typing.

In this study, we introduce a multilocus sequence typing (MLST) scheme, comprised of seven single-copy housekeeping genes, to genetically characterize Trichomonas vaginalis. Sixty-eight historical and recent isolates of T. vaginalis were sampled from the American Type Culture Collection and female patients at area health care facilities, respectively, to assess the usefulness of this typing method. Forty-three polymorphic nucleotide sites, 51 different alleles, and 60 sequence types were distinguished among the 68 isolates, revealing a diverse T. vaginalis population. Moreover, this discriminatory MLST scheme retains the ability to identify epidemiologically linked isolates such as those collected from sexual partners. Population genetic and phylogenetic analyses determined that T. vaginalis population structure is strongly influenced by recombination and is composed of two separate populations that may be nonclonal. MLST is useful for investigating the epidemiology, genetic diversity, and population structure of T. vaginalis.

Genetic relatedness of Trichomonas vaginalis reference and clinical isolates.

We have determined the metronidazole susceptibility status of 20 Trichomonas vaginalis isolates from American Type Culture Collection (ATCC) and assessed the level of genetic relatedness in these isolates using 32 additional T. vaginalis clinical isolates for comparison. These ATCC isolates are commonly used as reference strains in T. vaginalis research and this information provides a rational basis for selection of reference strains for use in comparative studies of T. vaginalis phenotypic and clinical characteristics.

Genetic diversity of Trichomonas vaginalis clinical isolates determined by EcoRI restriction fragment length polymorphism of heat-shock protein 70 genes.

Restriction fragment length polymorphism (RFLP) analysis using a multilocus heat-inducible cytoplasmic heat-shock protein 70 (Hsp70) hybridization probe with EcoRI-digested genomic DNA was used in molecular typing of 129 Trichomonas vaginalis isolates. Results indicate that Trichomonas organisms exhibit considerable polymorphism in their Hsp70 RFLP patterns. Analysis of seven American Type Culture Collection reference strains and 122 clinical isolates, including 84 isolates from Jackson, Mississippi, 18 isolates from Atlanta, Georgia, and 20 isolates from throughout the United States, showed 105 distinct Hsp70 RFLP pattern subtypes for Trichomonas. Phylogenetic analysis of the Hsp70 RFLP data showed that the T. vaginalis isolates were organized into two clonal lineages. These results illustrate the substantial genomic diversity present in T. vaginalis and indicate that a large number of genetically distinct Trichomonas isolates may be responsible for human trichomoniasis in the United States.

Artemisinins inhibit Trypanosoma cruzi and Trypanosoma brucei rhodesiense in vitro growth.

Artemisinin compounds inhibit in vitro growth of cultured Trypanosoma cruzi and Trypanosoma brucei rhodesiense at concentrations in the low micromolar range. Artemisinin also inhibits calcium-dependent ATPase activity in T. cruzi membranes, suggesting a mode of action via membrane pumps. Artemisinins merit further investigation as chemotherapeutic options for these pathogens.

Functional complementation of the yeast P-type H-ATPase, PMA1, by the Pneumocystis carinii P-type H-ATPase, PCA1.

The opportunistic fungus Pneumocystis is the etiologic agent of an interstitial plasma cell pneumonia that primarily afflicts immunocompromised individuals. Like other fungi Pneumocystis maintains a H(+) plasma membrane gradient to drive nutrient uptake and regulates intracellular pH by ATP-dependent proton efflux. Previously, we identified a Pneumocystis gene, PCA1, whose predicted protein product was homologous to fungal proton pumps. In this study, we show by functional complementation in a Saccharomyces strain whose endogenous PMA1 proton pump activity is repressed that the Pneumocystis PCA1 encodes a H(+)-ATPase. The properties of PCA1 characterized in this system closely resemble those of yeast PMA1. Yeast expressing PCA1 grow at low pH and are able to acidify the external media. Maximal enzyme activity (V(max)) and efficiency of substrate utilization (K(m)) in plasma membranes were nearly identical for PCA1 and PMA1. PCA1 contains an inhibitory COOH-terminal domain; removal of the final 40 amino acids significantly increased V(max) and growth at pH 6.5. PCA1 activity was inhibited by proton pump inhibitors omeprazole and lansoprazole, but was unaffected by H(+)/K(+)-ATPase inhibitor SCH28080. Thus, H(+) homeostasis in Pneumocystis is likely regulated as in other fungi. This work also establishes a system for screening PCA1 inhibitors to identify new anti-Pneumocystis agents.

Trichomonas vaginalis: characterization of a family of P-type ATPase genes.

P-type ATPases are ion-transporting pumps that enable organisms to control cellular functions and survive changing environmental conditions by regulating internal ion concentrations. Eight P-type ATPases were identified in the amitochondriate protist Trichomonas vaginalis using polymerase chain reaction (PCR) amplification with oligonucleotide primers that recognize conserved motifs present in all P-type ATPases, the ATP phosphorylation site (DKTGTLT) and the ATP binding site (TGDGVND). Phylogenetic analysis and the presence of conserved motifs in predicted peptide sequences identify the Trichomonas ATPases as a sarcoplasmic-endoplasmic reticulum calcium pump (TVCA1); three additional Ca(2+) transporting pumps (TVCA2-4), three phospholipid translocases (TVAPLT1-3), and one P-type ATPase of unknown transport specificity (TVPATP8). Southern blot analyses indicate that the P-type ATPase genes are not linked and are present in single copy, except TVCA2 and TVCA4 which contain additional copies or closely related homologues within the genome. Transcripts of 3.1 kb for TVCA1, 3.0 kb for TVCA2, 2.9 kb for TVCA3, 4.0 kb for TVAPLT1, 4.2 kb for TVAPLT2, 3.9 kb for TVAPLT3, and 3.1 kb for TVPATP8 were detected by Northern blot analysis. No TVCA4 transcript was observed, however, RT-PCR amplification of a transcript product indicates that TVCA4 is expressed. RNA expression of the Trichomonas ATPases, except TVCA3, was constitutive over a range of environmental conditions. TVCA1, TVAPLT3 and TVPATP8 had the highest levels of RNA expression while TVAPLT1 and TVAPLT2 expression was the lowest.