John C. Meade

Artemisinins Inhibit Trypanosoma cruzi and Trypanosoma brucei rhodesiense In Vitro Growth

ABSTRACT 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.

Structure and expression of a tandem gene pair in Leishmania donovani that encodes a protein structurally homologous to eucaryotic cation-transporting ATPases.

An oligonucleotide probe was used to clone a cation-transporting ATPase gene from the genome of Leishmania donovani. The nucleotide sequence of the gene contained a 2,922-base-pair open reading frame that was predicted to encode a 107,406-dalton protein composed of 974 amino acids. The predicted L. donovani protein contained all the structural and functional domains expected to be present in a cation-transporting ATPase of the aspartyl phosphate class. The nucleotide sequence encoding the ATPase gene was duplicated in tandem in the parasite genome. Partial sequenation of the second member of the tandem repeat, which lay 2 kilobase pairs downstream of the ATPase gene, indicated that it was either identical to the first gene or very closely related to it. RNA homologous to either the ATPase gene or its adjacent relative was 5 kilobases in size and was approximately equally abundant in both promastigote and amastigote forms of the organism.

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.

A tandem pair of Leishmania donovani cation transporting ATPase genes encode isoforms that are differentially expressed.

The second gene (ATPase 1b) of a tandem pair of cation transporting ATPases from Leishmania donovani was cloned and sequenced. The sequence of this gene was very similar to its upstream neighbor (ATPase 1a). Both genes contained a 2922 base open reading frame capable of encoding a protein of 974 amino acids. The genes differed at 34 nucleotide base positions, predicting 20 amino acid differences between the two peptides. These changes were clustered at the carboxy terminus with 15 changes occurring in the COOH-terminal 37 amino acids. However, these changes did not alter the highly charged nature of the carboxy terminus observed in ATPase 1a. The sequence was also conserved for 73 bases upstream of ATPase 1a and 1b but downstream conservation was limited to 15 bases beyond the termination codon. RNA from ATPase 1a was 5.2 kb and was present in both developmental forms of Leishmania. By contrast the ATPase 1b gene expressed a 5.75 kb transcript which was much more abundant in the amastigote form of Leishmania than in the promastigote form.

Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells.

Cultured rat cells deficient in endogenous thymidine kinase activity (tk) were stably transformed with a recombination-indicator DNA substrate constructed in vitro by rearrangement of the herpes simplex virus tk gene sequences into a partially redundant permutation of the functional gene. The recombination-indicator DNA did not express tk, but was designed to allow formation of a functional tk gene via homologous recombination. A clonal cell line (519) was isolated that harbored several permuted herpes simplex virus tk genes. 519 cells spontaneously produced progeny that survived in medium containing hypoxanthine, aminopterin, and thymidine. Acquisition of resistance to hypoxanthine, aminopterin, and thymidine was accompanied by the rearrangement of the defective tk gene to functional configuration. The rearrangement apparently occurred by unequal exchange between one permuted tk gene and a replicated copy of itself. Recombination was between 500-base-pair tracts of DNA sequence homology that were separated by 3.4 kilobases. Exchanges occurred spontaneously at a frequency of approximately 5 X 10(-6) events per cell per generation. Recombination also mediated reversion to the tk- phenotype; however, the predominant mechanism by which cells escaped death in the presence of drugs rendered toxic by thymidine kinase was not recombination, but rather inactivation of the intact tk gene.

Genetic Characterization of Trichomonas vaginalis Isolates by Use of Multilocus Sequence Typing

ABSTRACT 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.

Cloning and Characterization of an ATPase Gene from Pneumocystis carinii which Closely Resembles Fungal H+ ATPases

ABSTRACT. 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.

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 hosts 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.

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.

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.