Dataset Information

Trypanothione S-transferase activity in a trypanosomatid ribosomal elongation factor 1B.

ABSTRACT: Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the alpha, beta, and gamma subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the gamma subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bgamma genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1Bgamma had no S-transferase activity, suggesting that eEF1Bgamma is unstable in the absence of the other subunits.

SUBMITTER: Vickers TJ

PROVIDER: S-EPMC3428924 | biostudies-literature | 2004 Jun

REPOSITORIES: biostudies-literature

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Trypanothione S-transferase activity in a trypanosomatid ribosomal elongation factor 1B.

Vickers Tim J TJ Fairlamb Alan H AH

The Journal of biological chemistry 20040408 26

Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transfer ...[more]

PMID: 15073172

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Project description:UnlabelledRibosomal elongation factor 4 (EF4) is highly conserved among bacteria, mitochondria, and chloroplasts. However, the EF4-encoding gene, lepA, is nonessential and its deficiency shows no growth or fitness defect. In purified systems, EF4 back-translocates stalled, posttranslational ribosomes for efficient protein synthesis; consequently, EF4 has a protective role during moderate stress. We were surprised to find that EF4 also has a detrimental role during severe stress: deletion of lepA increased Escherichia coli survival following treatment with several antimicrobials. EF4 contributed to stress-mediated lethality through reactive oxygen species (ROS) because (i) the protective effect of a ?lepA mutation against lethal antimicrobials was eliminated by anaerobic growth or by agents that block hydroxyl radical accumulation and (ii) the ?lepA mutation decreased ROS levels stimulated by antimicrobial stress. Epistasis experiments showed that EF4 functions in the same genetic pathway as the MazF toxin, a stress response factor implicated in ROS-mediated cell death. The detrimental action of EF4 required transfer-messenger RNA (tmRNA, which tags truncated proteins for degradation and is known to be inhibited by EF4) and the ClpP protease. Inhibition of a protective, tmRNA/ClpP-mediated degradative activity would allow truncated proteins to indirectly perturb the respiratory chain and thereby provide a potential link between EF4 and ROS. The connection among EF4, MazF, tmRNA, and ROS expands a pathway leading from harsh stress to bacterial self-destruction. The destructive aspect of EF4 plus the protective properties described previously make EF4 a bifunctional factor in a stress response that promotes survival or death, depending on the severity of stress.ImportanceTranslation elongation factor 4 (EF4) is one of the most conserved proteins in nature, but it is dispensable. Lack of strong phenotypes for its genetic knockout has made EF4 an enigma. Recent biochemical work has demonstrated that mild stress may stall ribosomes and that EF4 can reposition stalled ribosomes to resume proper translation. Thus, EF4 protects cells from moderate stress. Here we report that EF4 is paradoxically harmful during severe stress, such as that caused by antimicrobial treatment. EF4 acts in a pathway that leads to excessive accumulation of reactive oxygen species (ROS), thereby participating in a bacterial self-destruction that occurs when cells cannot effectively repair stress-mediated damage. Thus, EF4 has two opposing functions-at low-to-moderate levels of stress, the protein is protective by allowing stress-paused translation to resume; at high-levels of stress, EF4 helps bacteria self-destruct. These data support the existence of a bacterial live-or-die response to stress.

Project description:BackgroundBats are a highly successful, globally dispersed order of mammals that occupy a wide array of ecological niches. They are also intensely parasitized and implicated in multiple viral, bacterial and parasitic zoonoses. Trypanosomes are thought to be especially abundant and diverse in bats. In this study, we used 18S ribosomal RNA metabarcoding to probe bat trypanosome diversity in unprecedented detail.Methodology/principal findingsTotal DNA was extracted from the blood of 90 bat individuals (17 species) captured along Atlantic Forest fragments of Espírito Santo state, southeast Brazil. 18S ribosomal RNA was amplified by standard and/or nested PCR, then deep sequenced to recover and identify Operational Taxonomic Units (OTUs) for phylogenetic analysis. Blood samples from 34 bat individuals (13 species) tested positive for infection by 18S rRNA amplification. Amplicon sequences clustered to 14 OTUs, of which five were identified as Trypanosoma cruzi I, T. cruzi III/V, Trypanosoma cruzi marinkellei, Trypanosoma rangeli, and Trypanosoma dionisii, and seven were identified as novel genotypes monophyletic to basal T. cruzi clade types of the New World. Another OTU was identified as a trypanosome like those found in reptiles. Surprisingly, the remaining OTU was identified as Bodo saltans-closest non-parasitic relative of the trypanosomatid order. While three blood samples featured just one OTU (T. dionisii), all others resolved as mixed infections of up to eight OTUs.Conclusions/significanceThis study demonstrates the utility of next-generation barcoding methods to screen parasite diversity in mammalian reservoir hosts. We exposed high rates of local bat parasitism by multiple trypanosome species, some known to cause fatal human disease, others non-pathogenic, novel or yet little understood. Our results highlight bats as a long-standing nexus among host-parasite interactions of multiple niches, sustained in part by opportunistic and incidental infections of consequence to evolutionary theory as much as to public health.

Dataset Information

Trypanothione S-transferase activity in a trypanosomatid ribosomal elongation factor 1B.

Publications

Trypanothione S-transferase activity in a trypanosomatid ribosomal elongation factor 1B.

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