Novel core promoter elements and a cognate transcription factor in the divergent unicellular eukaryote Trichomonas vaginalis.

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5' untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif.

Metronidazole resistance in Trichomonas vaginalis from highland women in Papua New Guinea.

BACKGROUND: The prevalence of the sexually transmissible protozoan parasite Trichomonas vaginalis in the highlands of Papua New Guinea (PNG) has been reported to be as high as 46% and although not previously studied in Papua New Guinea, clinical resistance against metronidazole (Mz), the drug most commonly used to treat trichomoniasis, is well documented worldwide. This study was primarily aimed at assessing resistance to Mz in T. vaginalis strains from the Goroka region. METHODS: Consenting patients presenting at the Goroka Base Hospital Sexually Transmitted Diseases (STD) Clinic and local women were asked to provide two vaginal swabs: one for culturing of the parasite; and one for polymerase chain reaction detection of T. vaginalis, Chlamydia trachomatis and Neisseria gonorrhoeae. T. vaginalis isolates were assayed for Mz susceptibility and a selection was genotyped. RESULTS: The prevalence of T. vaginalis was determined to be 32.9% by culture and polymerase chain reaction of swabs among 82 local women and patients from the STD clinic. An unexpectedly high level of in vitro Mz resistance was determined with 17.4% of isolates displaying unexpectedly high resistance to Mz. The ability to identify isolates of T. vaginalis by genotyping was confirmed and the results revealed a more homogeneous T. vaginalis population in Papua New Guinea compared with isolates from elsewhere. CONCLUSION: T. vaginalis is highly prevalent in the Goroka region and in vitro Mz resistance data suggest that clinical resistance may become an issue.

A functionally divergent hydrogenosomal peptidase with protomitochondrial ancestry.

Matrix proteins of mitochondria, hydrogenosomes and mitosomes are typically targeted and translocated into their respective organelles using N-terminal presequences that are subsequently cleaved by a peptidase. Here we characterize a approximately 47 kDa metallopeptidase, from the hydrogenosome-bearing, unicellular eukaryote Trichomonas vaginalis, that contains the active site motif (HXXEHX(76)E) characteristic of the beta subunit of the mitochondrial processing peptidase (MPP) and localizes to hydrogenosomes. The purified recombinant protein, named hydrogenosomal processing peptidase (HPP), is capable of cleaving a hydrogenosomal presequence in vitro, in contrast to MPP which requires both an alpha and beta subunit for activity. T. vaginalis HPP forms an approximately 100 kDa homodimer in vitro and also exists in an approximately 100 kDa complex in vivo. Our phylogenetic analyses support a common origin for HPP and betaMPP and demonstrate that gene duplication gave rise to alphaMPP and betaMPP before the divergence of T. vaginalis and mitochondria-bearing lineages. These data, together with published analyses of MPPs and putative mitosomal processing peptidases, lead us to propose that the length of targeting presequences and the subunit composition of organellar processing peptidases evolved in concert. Specifically, longer mitochondrial presequences may have evolved to require an alpha/beta heterodimer for accurate cleavage, while shorter hydrogenosomal and mitosomal presequences did not.

Genotyping Trichomonas vaginalis.

A genotyping method has been developed to distinguish each Trichomonas vaginalis isolate and has provided the first genome mapping studies of this protist with an estimated 180Mb genome. The technique was developed using high molecular weight DNA prepared from five laboratory isolates from Australia and USA and 20 clinical isolates from South Africa. Inhibition of the notorious T. vaginalis endogenous nucleases by addition of potent inhibitors was essential to the success of this study. Chromosomal DNA larger than 2.2Mb was macrorestricted to a minimum segment size of approximately 50kb, separated by pulsed field gel electrophoresis and hybridised with a variety of gene probes. Each isolate generated a unique pattern that was distinguished by each of the probes. Four single copy gene probes (fd, hmp35, ibp39 and pfoD) were identified but probes which identified several bands (pfoB and alpha-scs) per isolate were most informative for genotyping. The pyruvate:ferredoxin oxidoreductase B gene probe identified two to seven copies of pfoB (or its closely related homologue pfoA) per genome in different isolates and is an obvious candidate probe to identify epidemiological linkage between infections by this genotyping method. Cleavage of the genomes into smaller fragments failed to distinguish isolates from diverse locations indicating the proximal regions of genes are conserved.

Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex.

Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism. Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an [Fe]-hydrogenase which produces hydrogen. ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria. PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria. Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor. The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear. Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not. Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones. Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues. These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont.

Mitochondrial-type assembly of FeS centers in the hydrogenosomes of the amitochondriate eukaryote Trichomonas vaginalis.

Mitochondria are the site of assembly of FeS centers of mitochondrial and cytosolic FeS proteins. Various microaerophilic or anaerobic unicellular eukaryotes lack typical mitochondria ("amitochondriate" protists). In some of these organisms, a metabolically different organelle, the hydrogenosome, is found, which is thought to derive from the same proteobacterial ancestor as mitochondria. Here, we show that hydrogenosomes of Trichomonas vaginalis, a human genitourinary parasite, contain a key enzyme of FeS center biosynthesis, cysteine desulfurase (TviscS-2), which is phylogenetically related to its mitochondrial homologs. Hydrogenosomes catalyze the enzymatic assembly and insertion of FeS centers into apoproteins, as shown by the reconstruction of the apoform of [2Fe-2S]ferredoxin and the incorporation of 35S from labeled cysteine. Our results indicate that the biosynthesis of FeS proteins is performed by a homologous system in mitochondriate and amitochondriate eukaryotes and that this process is inherited from the proteobacterial ancestor of mitochondria.

Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance.

Ferredoxin, Fd, is often deficient in metronidazole-resistant strains of Trichomonas vaginalis and is thought to be necessary for drug activation. To directly test whether Fd is essential for metronidazole susceptibility, gene replacement technology has been developed for T. vaginalis. The selectable marker gene neomycin phosphotransferase (NEO) flanked by approximately 2.6 and approximately 2.0 kBp of the Fd 5' and 3' flanking regions (pKO-FD-NEO) was introduced into cells on linear DNA and selected for NEO gene expression. Stable transformants were shown to contain the NEO gene in the Fd locus and to have completely lost the Fd gene. Northern and immunoblot analyses confirm the loss of Fd mRNA and protein in pKO-FD-NEO cells. Analyses of the activity of hydrogenosomal proteins in Fd KO cells show a fourfold increase in hydrogenase activity and a 95% decrease in pyruvate/ferredoxin oxidoreductase (PFO) activity. In contrast, PFO and hydrogenase mRNA levels are unchanged. Surprisingly, Fd KO cells are not resistant to metronidazole under aerobic or anaerobic conditions. These cells are capable of producing molecular hydrogen, albeit at 50% the level of the parental strain, demonstrating that the Fd gene product eliminated in KO cells is neither necessary for hydrogen production nor metronidazole activation. Together these data indicate the presence of unidentified Fds or flavodoxins capable of drug activation or an unidentified mechanism that does not require either PFO or Fd for metronidazole activation.

Trichomonas vaginalis Hmp35, a putative pore-forming hydrogenosomal membrane protein, can form a complex in yeast mitochondria.

An abundant integral membrane protein, Hmp35, has been isolated from hydrogenosomes of Trichomonas vaginalis. This protein has no known homologue and exists as a stable 300-kDa complex, termed HMP35, in membranes of the hydrogenosome. By using blue native gel electrophoresis, we found the HMP35 complex to be stable in 2 m NaCl and up to 5 m urea. The endogenous Hmp35 protein was largely protease-resistant. The protein has a predominantly beta-sheet structure and predicted transmembrane domains that may form a pore. Interestingly, the protein has a high number of cysteine residues, some of which are arranged in motifs that resemble the RING finger, suggesting that they could be coordinating zinc or another divalent cation. Our data show that Hmp35 forms one intramolecular but no intermolecular disulfide bonds. We have isolated the HMP35 complex by expressing a His-tagged Hmp35 protein in vivo followed by purification with nickel-agarose beads. The purified 300-kDa complex consists of mostly Hmp35 with lesser amounts of 12-, 25-27-, and 32-kDa proteins. The stoichiometry of proteins in the complex indicates that Hmp35 exists as an oligomer. Hmp35 can be targeted heterologously into yeast mitochondria, despite the lack of homology with any yeast protein, demonstrating the compatibility of mitochondrial and hydrogenosomal protein translocation machineries.