Anaerobic Protozoa Research Articles

A thioredoxin reductase-class of disulphide reductase in the protozoan parasite Giardia duodenalis

We describe the purification and characterisation of a thioredoxin reductase-like disulphide reductase from the ancient protozoan parasite, Giardia duodenalis. This dimeric flavoprotein contains 1 mol FAD per subunit and had an apparent subunit molecular mass of 35 kDa. The purified enzyme catalysed the NADPH-dependent ( K m = 8 μM) reduction of 5,5′-dithio-bis(2-nitrobenzoic acid) to thionitrobenzoate and was unable to utilise NADH as an electron donor. The sulphydryl-active compounds, N-ethylmaleimide, sodium arsenite and Zn 2+ ions, strongly inhibited the enzyme suggesting that a thiol component forms part of the active site. Purified enzyme was able to utilise a variety of substrates, including cystine and oxidised glutathione, which suggests that it is a broad-range disulphide reductase, probably accounting for the majority of thiol cycling activity in this organism. While the G. duodenalis enzyme does not require an intermediate electron transport protein, analogous to thioredoxin, for activity, we have identified a candidate carrier protein which enhances DTNB turnover six fold, therefore implying that Giardia contains a thioredoxin-like system. Physical, enzymatic and spectral properties of the G. duodenalis disulphide reductase are also consistent with it being a member of the thioredoxin reductase-class of disulphide reductases. Furthermore, the internal amino acid sequence of a tryptic peptide generated from the purified protein was highly homologous with thioredoxin reductases from other sources. This is the first report of a disulphide reductase to be purified from the anaerobic protozoa and explains the so called ‘glutathione-induced thiol-reductase activity’ previously observed in G. duodenalis.

β-Tubulin genes of Trichomonas vaginalis

Microtubules, formed by polymerization of α and β-tubulins, are major structural components of the mitotic spindle, cytoskeleton, and flagella, and are also an important target for the antiparasitic benzimidazole drugs. Trichomonas vaginalis, a flagellated protozoan responsible for urogenital tract infections in humans, is highly sensitive to certain benzimidazoles in vitro. As a first step towards defining the roles of microtubules in this organism, the regulation of their expression, and the basis for their benzimidazole sensitivity, we have characterized the genes encoding T. vaginalis β-tubulin. A combination of genomic DNA cloning using bacteriophage lambda and PCR amplification using conserved β-tubulin gene primers was employed . Southern blots of DNA from two different T. vaginalis strains suggest there are 6–7 β-tubulin gene copies. Sequencing identified three distinct genes: btub1, btub2, and btub3. Amplification of cDNA with gene-specific primers indicated that the relative expression of RNA transcripts was btub1 > ⪢ btub3. The promoter region from btub1 includes a 15-bp repeat also found (with 1-bp difference) upstream of the T. vaginalis ferred oxin gene. Primer extension suggests the 5′ leader of the mRNA transcribed from tub1 is only 10 nucleotides long, similar to the lengths found in other anaerobic protozoa. In 152 residues examined by PCR, btub2 and btub3 differed by 1 and 12 amino acids, respectively, from btub1. All three sequences, however, have diverged considerably (20–24%) from β-tubulins of other protozoa. T. vaginalis β-tubulins include residues Tyr167 and Phe200, previously implicated in resistance and sensitivity, respectively, to the benzimidazole derivative benomyl.

Benthic trophic dynamics in California coastal basin and continental slope communities inferred using inverse analysis

The isolation of the deep-sea benthos makes it difficult to measure more than a few elemental transformation rates in a given ecosystem. The measurements usually made include the transfer rates of such biologically important materials as oxygen, particulate carbon, and ammonia between water and sediment. We have developed inverse analysis techniques that produce descriptions which include material flows between trophic groups within the sediments. The techniques use a limited set of process rate and biomass measurements and known physiological and chemical information. Estimated material flows include rates of consun~ption, production, respiration, excretion, and egestion of the major trophic groups in benthic food webs. We analyzed benthic communities in a lowoxygen environment (Santa Monica Basin) and in a higher-oxygen environment on the conhnental slope (Patton Escarpment), both bordering southern Cahfornia (USA). The inverse analyses suggested that the low oxygen community was dominated by anaerobic protozoa and bactena and supported only a small populations of grazers. The dominance of bacteria with high growth rates caused sedimentary detrital carbon and nitrogen there to decompose in a few days. The Patton Escarpment community, overlain with oxygen-rich water, had a more complex food web dominated by the higher trophic level protozoa, meiofauna, and macrofauna. We suggest that carbon and nitrogen were retained in the biomass of these larger grazers for months. The analyses showed that grazers and microbial organisms speciahze in using resources of different nutritional quality. Specialization of this type may have led to a more complete oxidation of sedimenting detritus at Patton Escarpment than at Santa Monica Basin. Organisms in both food webs had low gross production efficiencies that averaged 10 % at the Patton Escarpment and 7 % at the Santa Monica Basin sites. These results suggest that inverse analysis can be a powerful tool to analyze benthic communities.

An anaerobic protozoon, with symbiotic methanogens, living in municipal landfill material

We have established that anaerobic protozoa do live in municipal landfill material although they probably spend much of the time encysted, especially in the drier (< 40% water) site. At least eight species were observed; they were readily isolated by adding anoxic water to dry landfill samples. The ciliate Metopus palaeformis was frequently i isolated; it appears to be ubiquitous in anaerobic landfills. It has a polymorphic life cycle, it is positive for hydrogenase, each ciliate contains about 500 bromoethanesulfonate-sensitive methanogen symbionts (probably Methanobacterium formicicum), and maximum cell densities in culture exceed 3000 per ml. The methanogens are not attached to the hydrogenosomes, neither do they undergo morphological transformation; the ciliate receives no measurable energetic advantage from its symbionts. The ciliate encysts in response to a shortage of food or water, and the methanogens remain viable within the cysts. When the protozoon excysts, the methanogens resume growth and cell division within the trophic form of the ciliate. Unlike free-living methanogens, the M. palaeformis-methanogen consortium is not particularly sensitive to oxygen; the symbiotic methanogens remain viable following exposure of the consortium to atmospheric oxygen for several days. Dispersal of methanogen-bearing protozoan cysts through oxygenated environments is a potential mechanism of transfer between landfill sites and other anaerobic environments. Anaerobic protozoan consortia are theoretically capable of making a significant contribution to methane generation from wet landfill sites.

Novel biochemical pathways in parasitic protozoa.

Throughout evolution, enzymes and their metabolites have been highly conserved. Parasites are no exception to this and differ most markedly by the absence of metabolic pathways that are present in the mammalian host. In general, parasites are metabolically lazy and rely on the metabolism of the host both for a supply of prefabricated components such as purines, fatty acids, sterols and amino acids and for the removal of end-products. Nonetheless, parasites are metabolically highly sophisticated in that (1) they retain the genetic capacity to induce many pathways, when needed, and (2) they have developed complex mechanisms for their survival in the host. Certain unique features of the metabolism of trypanosomes, leishmania, malaria and anaerobic protozoa will be discussed. This will include (1) glycolysis and electron transport with reference to the unique organelles: the glycosome and the hydrogenosome, (2) purine salvage, pyrimidine biosynthesis and folic acid metabolism and (3) polyamine and thiol metabolism with special reference to the role of the unique metabolite of trypanosomes and leishmanias, trypanothione.

Hydrogenosomes in the rumen fungus Neocallimastix patriciarum.

Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.

Nutrition and Growth Characteristics of Trichomitopsis termopsidis , a Cellulolytic Protozoan from Termites

Putatively axenic cultures of Trichomitopsis termopsidis 6057, isolated by M. A. Yamin (J. Protozool. 25:535-538, 1978) from the hindgut of Zootermopsis termites, apparently contained methanogenic bacteria, inasmuch as small amounts of CH(4) were produced during growth. However, T. termopsidis could be "cured" of methanogenic activity by incubation in the presence of bromoethanesulfonate. Both the cured derivative (6057C) and the parent strain (6057) required NaHCO(3) and fetal bovine serum for good growth; the presence of yeast extract in media was stimulatory. Growth of both strains was markedly improved by substituting heat-killed cells of Bacteroides sp. strain JW20 (a termite gut isolate) for heat-killed rumen bacteria in media as a source of bacterial cell material. Heat-killed Bacteroides sp. strain JW20 was the best of a number of bacteria tested, and under these conditions H(2) was a major protozoan fermentation product. Growth of T. termopsidis strains was further improved by co-cultivation in the presence of Methanospirillum hungatii. M. hungatii was the best of a number of H(2)-consuming bacteria tested, and under these conditions CH(4), but not H(2), was produced, indicating interspecies transfer of H(2) between the protozoa and M. hungatii. Both strains of T. termopsidis used powdered, particulate forms of cellulose (e.g., pure cellulose, corncob, cereal leaves) as fermentable energy sources, although powdered wood, chitin, or xylan supported little or no growth. Cells of the cellulose-forming coccus Sarcina ventriculi also served as a fermentable energy source, but these were used poorly as a source of bacterial cell material. The only substantial difference between T. termopsidis 6057 and 6057C was that the latter grew poorly or not at all with rumen bacteria as a source of bacterial cell material. The improved growth of T. termopsidis in vitro should facilitate further studies on the cell biology and biochemistry of these symbiotic, anaerobic protozoa.

Hydrogenosomes in a mixed isolate of Isotricha prostoma and Isotricha intestinalis from ovine rumen contents

1. 1. Both Isotricha intestinalis and I. prostoma possess microbody-like organelles, with a highly granular appearance. 2. 2. These organelles, which are sedimentable at 10 5 g-min, bear no morphological similarity to mitochondria, but are enzymatically similar to organelles possessed by certain other anaerobic protozoa and termed hydrogenosomes. 3. 3. The hydrogenosomes isolated from a preparation of mixed isotrichs bear a closer similarity to those isolated from the other rumen holotrich, Dasytricha ruminantium, than those recently identified in a mixed entodiniomorph preparation, or the trichomonads, in that the enzyme malate dehydrogenase (decarboxylating) is non-sedimentable and phosphoacetyl transferase together with acetate kinase are involved in the transformation of acetyl-CoA to acetate. 4. 4. The results enable a scheme of acetate, CO 2 and H 2 formation from carbohydrates to be proposed and extends the number of protozoa known to possess this organelle.

Mode of action of metronidazole on anaerobic bacteria and protozoa.

Metronidazole and related 5-nitroimidazoles are relatively nontoxic. Reduction of their nitro group, however, leads to the production of short-lived cytotoxic intermediates, which finally decompose into nontoxic end products. The toxicity of the intermediates is due to their interaction with deoxyribonucleic acid and possibly with other macromolecules. Metabolic pathways of low redox potential that are linked to ferredoxin or flavodoxin-like electron transport components reduce nitroimidazoles with great efficacy. Such pathways are characteristic of susceptible anaerobic protozoa and bacteria and are absent in nonsusceptible aerobic cells of the host.