A COOH-terminal domain regulates the activity of Leishmania proton pumps LDH1A and LDH1B.

Leishmania donovani requires actively transporting proton efflux pumps to survive the acidic environment of macrophage phagolysosomal vacuoles and to maintain an electrogenic H(+) gradient for nutrient uptake. The L. donovani genome contains a differentially expressed pair of genes, LDH1A and LDH1B, with homology to yeast H(+)-ATPases that are 98% identical in sequence with amino acid differences concentrated at the COOH-terminus (15 of last 37 differ), a region implicated in regulation of yeast and plant proton pumps. Functional complementation of a Saccharomyces cerevisiae strain deficient in endogenous H(+)-ATPase activity, support of yeast growth at low pH, and ability to acidify media demonstrate that LDH1A and LDH1B encode proton pumps. LDH1A and LDH1B encode a COOH-terminal autoinhibitory domain as COOH-truncated peptides support increased rates of growth in yeast, enhanced media acidification, increased enzyme activity (V(max)) and decreased K(m). This regulatory domain mediates differing function properties; LDH1A, but not LDH1B, supports yeast growth at pH 3 and LDH1A shows a greater ability to acidify media. Deletion of the last eight amino acids from LDH1B permits growth at pH 3 and increases media acidification, swapping of the COOH-tails between LDH1A and LDH1B results in LDH1A (with LDH1B tail) unable to support yeast growth at pH 3 and LDH1B (with LDH1A tail) now able to support growth at pH 3. Replacement of the COOH-terminal eight amino acids of LDH1B with those from LDH1A also confers the ability to support growth at pH 3. The complementation system for the Leishmania proton pumps in yeast described here provides a means to dissect the functional properties of the two isoforms, a convenient supply of protein for structural analysis and a model amenable to screening proton pump inhibitors for potential anti-leishmanial therapeutics.

Identification of surface-membrane P-type ATPases resembling fungal K(+)- and Na(+)-ATPases, in Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani.

Genomic DNA fragments encoding nine, novel, P-type ATPases in trypanosomatid organisms were amplified in PCR, using degenerate oligonucleotide primers that recognize the ATP-binding and -phosphorylation sites present in all P-type ATPases. Subsequent phylogenetic analysis, based on the presence of conserved motifs in predicted peptide sequences for six Trypanosoma brucei, T. cruzi or Leishmania donovani PCR fragments, identified calcium-, proton- and phospholipid-translocating ATPases. DNA fragments that predict proteins homologous to the fungal, type-IID, P-type, ATPase pumps that transport Na(+) or K(+) ions were also present in T. brucei (TBCA1; 1022 nucleotides representing 340 amino acids), T. cruzi (TCNA1; 1022 nucleotides representing 340 amino acids) and L. donovani (LDCA1; 1031 nucleotides representing 343 amino acids). Southern blots showed that the Na(+)-ATPases were each present as a single-copy gene. The LDCA1 fragment was used to clone the complete LDCA1 gene from an L. donovani genomic-DNA library. The LDCA1 gene encodes a protein, of 1047 amino acids, with a predicted molecular mass of 115,501 Da. The results of analyses based on northern blots and the rapid amplification of cDNA ends (RACE) indicated that LDCA1 was expressed in promastigotes and amastigotes from axenic cultures and in animal-derived amastigotes. TBCA1 was expressed, as a 5.0-kb transcript, in procyclic culture stages and bloodstream trypomastigotes, with the 5.0-kb message up-regulated six-fold in the trypomastigote stage. Western blots probed with an antibody to the partial TBCA1 peptide identified a 150-kDa protein that was detected, by immunofluorescence, on the surface membrane of procyclic T. brucei.

Characterization of P-type ATPase 3 in Plasmodium falciparum.

We report the nucleotide sequence, derived amino acid sequence and expression profile of P-type ATPase 3 (PfATPase3) from Plasmodium falciparum. An open reading frame of 7362 nucleotides, interrupted by a single intron of 168 nt, encoded a protein product of 2394 amino acids with a predicted MW of 282791 Da. Hydropathy analysis of PfATPase3 revealed six amino-terminal and six carboxyl-terminal membrane spanning regions (M1-12) flanking a large hydrophilic domain with a smaller hydrophilic loop between M4 and M5. Based on a phylogenetic comparison of conserved domains present in P-type ATPases from other organisms, PfATPase3 resembled a Type-V ATPase for which the transport affinity is unknown. The PfATPase3 topology was interrupted by four regions, termed 'inserts', unique to malarial P-type ATPases, which were high in asparagine residues and charged amino acids (inserts I1-I4). Inserts I1 and I3 also contained repeated amino acid motifs. The number and composition of repeated amino acid motifs in insert I3 were variable in seven P. falciparum strains tested. PfATPase3 was 80.2% similar to the non-insert portions of P. yoelii ATPase3, although their inserts differed in length and composition. PfATPase3 mRNA was most abundant relative to beta-tubulin during the latter half of the erythrocytic cycle and was also present in gametocytes. Using affinity-purified antibody to a 14 amino acid PfATPase3 epitope, a 260 kDa protein was detected by Western analysis. Based on immunofluorescence, the PfATPase3 protein was located intracellularly in gametocytes and, to a lesser extent, in late erythrocytic stages.

Trypanosoma brucei infection induces apoptosis and up-regulates neuroleukin expression in the cerebellum.

Human infection with Trypanosoma brucei may result in meningo-encephalitis, neuronal demyelination, blood-brain-barrier dysfunction, peri-vascular infiltration, astrocytosis and neuronal apoptosis. Prevention of the short- or long-term, parasite-induced, neuronal assault requires a better understanding of the host's responses to the infection at the molecular level. Northern analysis, cDNA micro-arrays, reverse-transcriptase-PCR (RT-PCR), SDS-PAGE and immunohistology were therefore used to investigate global gene and protein expression in the brains of mice infected with T. brucei. Temporal and spatial expression of neuroleukin (NLK), a predominant neurotrophin which is associated with neuronal protection and regeneration during neuronal assault in the brain, was then assessed. Expression of 20 of the 588 genes investigated (representing pro- and anti-inflammatory immuno-modulators, growth factors, neurotransmitters, and pro- and anti-apoptosis factors) was significantly altered (P < 0.05). TUNEL analysis revealed extensive apoptosis at peak parasitaemia, mainly in the cerebellum. RT-PCR analysis of two regulators of apoptosis, Bcl-x(L) (anti-apoptotic) and Bax (pro-apoptotic), revealed equivalent increases in levels of expression. NLK expression was up-regulated in punctated fashion in brain and was mainly localized to abnormal (stellate) catecholamine neurons (CN) in the locus coeruleus (LC) of infected [and, to a lesser degree, the normal (polygonal) cells of uninfected] brainstem. Expression of NLK receptor (NLK-R) was inversely correlated with that of NLK. At peak parasitaemia, trypanosome infection apparently induces cerebellar apoptosis and a corresponding increase in NLK expression. NLK may be modulating inflammation and is probably involved in protecting CN and the cerebellum against apoptosis.

The Trypanosoma cruzi genome contains ion motive ATPase genes which closely resemble Leishmania proton pumps.

DNA fragments homologous to members of the family of P-type ion-motive ATPases were identified in Trypanosoma cruzi by polymerase chain reaction (PCR) amplification. The sequence of one fragment, which closely resembled (87% identity) the tandemly linked proton pumps in Leishmania, was used to characterize the H(+)-ATPase genes in T. cruzi. The T. cruzi proton pump locus contains four tandemly repeated genes (TCH1-4) separated by 1.1 kb intergenic regions. The nucleotide sequence of one cloned gene of the tandem array contains a 2775 nt open reading frame encoding a predicted 101908-Da protein of 925 amino acids. The TCH genes are expressed as 3.8 and 4.9 kb polyadenylated transcripts in the epimastigote stage; expression of both transcripts is reduced in metacyclic trypomastigotes. Results of 5' and 3' RACE transcript mapping indicate that the 3.8 kb message is generated from within the tandemly repeated locus. The 3.8 kb TCH transcript has the T. cruzi mini-exon appended to a short (40 nt) 5' untranslated region (UTR) and has a 927 nt 3' UTR. The full peptide sequence of the T. cruzi proton pump is 80% identical to the Leishmania pump but lacks the extended carboxyl tail present in the Leishmania ATPase. An antibody that recognizes the 110-kDa Leishmania donovani proton pump cross-reacts with a 100-kDa protein in lysates of T. cruzi epimastigotes.

Trichomonas vaginalis: analysis of a heat-inducible member of the cytosolic heat-shock-protein 70 multigene family.

A 2253-nucleotide (nt) transcript for a Trichomonas vaginalis heat-shock protein 70, TVCHSP70, has been isolated that encodes for a protein of 659 amino acids with a predicted molecular weight of 71.3 kDa. TVCHSP70 has a short (10-nt) 5' untranslated region (UTR), and the 263-nt 3' UTR is the longest reported for a Trichomonas peptide. Amino-acid sequence analysis and phylogenetic comparison identifies TVCHSP70 as a member of the heat-inducible cytoplasmic HSP70 gene family. Southern-blot data indicate that T. vaginalis contains at least four members of the cytoplasmic HSP70 gene family. Members of the TVCHSP70 family are expressed as 2.3-kb transcripts at low levels during 37 degrees C culture, and their expression is significantly up-regulated at 43 degrees C. Slot-blot analysis of seven T. vaginalis clinical isolates demonstrated a 3- to 44-fold up-regulation of TVCHSP70 under conditions of heat shock (43 degrees C) or oxidative stress (500 microm H2O2) as compared with controls (37 degrees C).

Antigenicity of Trichomonas vaginalis heat-shock proteins in human infections.

Patients infected with Trichomonas vaginalis mount humoral and cellular immune responses that often do not protect against reinfection. The oxidative stressors produced by leukocytes may trigger a heat-shock-like response in T. vaginalis trophozoites, helping the parasite to survive host immune defenses. The antigenicity of T. vaginalis heat-shock proteins (HSPs) was examined by immunoprecipitation of T. vaginalis heat-induced proteins with sera from infected patients and controls. When T. vaginalis was heat-shocked, HSPs of 169-167 and 140-137 kDa were specifically recognized by sera from infected male and female patients. However, the majority of T. vaginalis HSPs were also immunoprecipitated by control sera; all sera recognized 72- to 71-kDa, 47- to 45-kDa, 38- to 37-kDa, 35-kDa, and 31-kDa heat-induced proteins. At least 15 proteins from non-heat-shocked T. vaginalis were immunoprecipitated by sera from infected patients and controls, indicating that natural or cross-reacting antibodies could participate in host responses to trichomoniasis. Molecules of 158, 135, 89, and 74-72 kDa were immunoprecipitated from some non-heat-shocked parasites only by patients' sera. A 38-kDa T. vaginalis protein was immunoprecipitated only by sera from infected females and may be specific for infection in women.

Molecular typing of Trichomonas vaginalis isolates by HSP70 restriction fragment length polymorphism.

Subtyping isolates of Trichomonas vaginalis is an essential tool for understanding the epidemiology of this common sexually-transmitted disease. Restriction fragment length polymorphism (RFLP) analysis employing a probe from the heat-inducible cytoplasmic HSP70 gene family hybridized with EcoR I-digested genomic DNA was used in the molecular typing of Trichomonas isolates. Analysis of five American Type Culture Collection (ATCC) reference strains and 31 Jackson, Mississippi, isolates from six male and 21 female patients, revealed 10 distinct RFLP pattern subtypes of Trichomonas. The subtypes were temporally stable and cosmopolitan. The RFLP profiles seen in Maryland, Ohio, Massachusetts, and New York ATCC strains were identical to those of some Mississippi isolates, even though the samples were isolated 10-35 years apart. There was no correlation between metronidazole resistance and RFLP subtype with resistant isolates from eight patients distributed among six different subtypes.

Genomic organization, transcription, splicing and gene regulation in Leishmania.

The parasitic protozoan Leishmania is the aetiological agent of a spectrum of clinical diseases, ranging from disfiguring skin lesions to life-threatening visceral infection, and is a serious health problem in tropical and subtropical areas world-wide. Leishmania parasites undergo a dramatic transformation as they move between the different environments of an extracellular insect stage and an intracellular form in the vertebrate host. In an attempt to develop new strategies for the treatment of leishmaniasis, the techniques of molecular genetics have been utilised to elucidate the mechanisms which direct and control this cyclical differentiation. This review discusses current knowledge concerning the organization and regulation of the Leishmania nuclear genome and includes a discussion of chromosomal organization, genomic arrangement, transcription, transcript processing by trans-splicing and polyadenylation, and post-transcriptional regulation. The salient features as well as the supporting evidence for each topic are briefly reviewed.

Molecular characterization of a sarcoplasmic-endoplasmic reticulum Ca+2 ATPase gene from Trichomonas vaginalis.

DNA fragments homologous to P-type cation translocating ATPase genes were identified in Trichomonas vaginalis by polymerase chain reaction (PCR) amplification. The genomic locus corresponding to one PCR fragment, TVCA1, contains a 3,055 base-pair open reading frame encoding a 108,162 dalton protein composed of 981 amino acids. TVCA1 lacks introns, is present in a single copy, and is expressed as a 3.1 kb transcript with short 5' and 3' untranslated regions. Separate primer extension experiments map the 5' end of the TVCA1 transcript to 12 and 16 nucleotide bases (nt) upstream of the methionine initiation codon. The message polyadenylation site is located 62 nt downstream of the protein termination codon at a CA dinucleotide. The TVCA1 protein sequence shares 57-58% similarity with rabbit, schistosome, trypanosome and malarial sarcoplasmic-endoplasmic reticulum calcium (SERCA) pumps, and significantly lower similarity with plasma membrane calcium pumps and cation translocating ATPases of other ion specificities. Structural and functional domains identified in P-type ATPases as well as 61/68 residues specifically implicated in SERCA pump activity are conserved in TVCA1. However, TVCA1 lacks binding sites for phospholamban regulation, thapsigargin inhibition and the calmodulin dependent protein kinase site phosphorylation present in other SERCA pumps.

Cloning and characterization of an ATPase gene from Pneumocystis carinii which closely resembles fungal H+ ATPases.

A gene encoding a P-type cation translocating ATPase was cloned from a genomic library of rat-derived Pneumocystis carinii. The nucleotide sequence of the gene contains a 2781 base-pair open reading frame that is predicted to encode a 101,401 dalton protein composed of 927 amino acids. The P. carinii ATPase protein (pcal) is 69-75% identical when compared with eight proton pumps from six fungal species. The Pneumocystis ATPase is less than 34% identical to ATPase proteins from protozoans, vertebrates or the Ca++ ATPases of yeast. The P. carinii ATPase contains 115 of 121 residues previously identified as characteristic of H+ ATPases. Alignment of the Pneumocystis and fungal proton pumps reveals five homologous domains specific for fungal H+ ATPases.

A cell-specific nuclear receptor plays essential roles in adrenal and gonadal development.

Recent analyses of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1), in their coordinate, cell-selective expression. SF-1 was proposed to regulate the steroid hydroxylases by interacting with shared promoter elements in their 5'-flanking regions. During mouse embryonic development, SF-1 was expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting a key role in steroidogenic cell differentiation. Finally, disruption of the gene encoding SF-1 revealed its essential function in the development of the adrenal glands and gonads and in pituitary gonadotrope function. These studies suggest that SF-1 acts at multiple levels of the reproductive axis to maintain reproductive competence.

A cell-specific nuclear receptor regulates the steroid hydroxylases.

Recent studies of the gene regulation of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1). SF-1 binds to shared promoter elements upstream of the steroid hydroxylases to mediate their coordinate expression in steroidogenic cells. Analyses of SF-1 expression during mouse embryonic development showed that SF-1 is expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting an intimate link between SF-1 and steroidogenic cell differentiation. Finally, in gene disruption experiments, the gene encoding SF-1 was shown to be essential for development of the adrenal glands and gonads. These results establish the essential role of this orphan nuclear receptor in the development and function of the primary steroidogenic tissues.

A family of cation ATPase-like molecules from Plasmodium falciparum.

We report the nucleotide and derived amino acid sequence of the ATPase 1 gene from Plasmodium falciparum. The amino acid sequence shares homology with the family of "P"-type cation translocating ATPases in conserved regions important for nucleotide binding, conformational change, or phosphorylation. The gene, which is present on chromosome 5, has a product longer than any other reported for a P-type ATPase. Interstrain analysis from 12 parasite isolates by the polymerase chain reaction reveals that a 330-bp nucleotide sequence encoding three cytoplasmic regions conserved in cation ATPases (regions a-c) is of constant length. By contrast, another 360-bp sequence which is one of four regions we refer to as "inserts" contains arrays of tandem repeats which show length variation between different parasite isolates. Polymorphism results from differences in the number and types of repeat motif contained in this insert. Inserts are divergent in sequence from other P-type ATPases and share features in common with many malarial antigens. Studies using RNA from the erythrocytic stages of the malarial life cycle suggest that ATPase 1 (including the sequence which encodes tandem repeats) is expressed at the large ring stage of development. Immunolocalization has identified ATPase 1 to be in the region of the parasite plasma membrane and pigment body. These findings suggest a possible model for the genesis of malarial antigens.

Analysis of the Pneumocystis carinii genome.

Chromosome-specific DNA probes were isolated to identify a particular chromosome in various karyotype band patterns of Pneumocystis. Bands in two P. carinii karyotype patterns hybridized to various DNA probes indicated that homologous chromosomes are of relatively similar sizes in the two P. carinii strains.

PCR amplification of DNA sequences from the transcription factor IID and cation transporting ATPase genes in Pneumocystis carinii.

Oligonucleotide primers were used to amplify DNA sequences from a plasma membrane cation transporting ATPase gene and a transcription factor IID (TFIID) gene from Pneumocystis carinii genomic DNA. The entire P. carinii ATPase gene was cloned from a genomic library by hybridization to the PCR-amplified DNA product. The nucleotide sequence of the gene contained a 2,799 base-pair open reading frame that encoded a 102,274 dalton protein composed of 933 amino acids. The P. carinii ATPase protein was 69-74% identical to four fungal proton pumps but less than 35% identical to protozoan and mammalian cation transporting ATPase genes or the Ca++ ATPases of Saccharomyces. The nucleotide sequence of a portion of the TFIID gene could be translated to produce a peptide of 53 amino acids in two regions of the sequence, interrupted by a 45 bp intron. The predicted TFIID amino acid sequence was identical to yeast TFIID genes in this region.

Conservation of cation-transporting ATPase genes in Leishmania.

DNA fragments isolated from Leishmania donovani ATPase genes were used to analyze the organization and expression of cation transporting ATPase genes in L. donovani, Leishmania tropica, Leishmania mexicana, Leishmania braziliensis, Trypanosoma brucei and Trypanosoma cruzi. The ATPase loci in all Leishmania species contained a tandem pair of ATPase genes arranged in head-to-tail orientation and separated by approximately 2 kb. No restriction site polymorphisms were detected in the internal portions of the Leishmania ATPase genes which contain domains conserved between the L. donovani and other eukaryotic plasma membrane ATPases. The ATPase locus of each of the four Leishmania species was mapped to a single small chromosome of approximately 750 kb. The ATPase locus of L. mexicana was differentially expressed. Promastigotes in exponential growth contained abundant transcripts from the upstream ATPase gene, while transcripts from the downstream gene were relatively scarce. Transcripts from the downstream ATPase gene increased in abundance in promastigotes allowed to reach the stationary phase of growth and were most abundant in amastigotes. The two trypanosome species were found to contain DNA fragments that hybridized strongly to the Leishmania ATPase gene.