Direct evidence for cyanide-insensitive quinol oxidase (alternative oxidase) in apicomplexan parasite Cryptosporidium parvum: phylogenetic and therapeutic implications

Cryptosporidium parvum is a parasitic protozoan that causes the diarrheal disease cryptosporidiosis, for which no satisfactory chemotherapy is currently available. Although the presence of mitochondria in this parasite has been suggested, its respiratory system is poorly understood due to difficulties in performing biochemical analyses. In order to better understand the respiratory chain of C. parvum, we surveyed its genomic DNA database in GenBank and identified a partial sequence encoding cyanide-insensitive alternative oxidase (AOX). Based on this sequence, we cloned C. parvum AOX (CpAOX) cDNA from the phylum apicomplexa for the first time. The deduced amino acid sequence (335 a.a.) of CpAOX contains diiron coordination motifs (-E-, -EXXH-) that are conserved among AOXs. Phylogenetic analysis suggested that CpAOX is a mitochondrial-type AOX, possibly derived from mitochondrial endosymbiont gene transfer. The recombinant enzyme expressed in Escherichia coli showed quinol oxidase activity. This activity was insensitive to cyanide and highly sensitive to ascofuranone, a specific inhibitor of trypanosome AOX.     

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp.: Its mitochondrial respiratory chain lacks a plant-like alternative oxidase

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, α-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.     

Cytological evaluation of the effect of azoxystrobin and alternative oxidase inhibitors in Botrytis cinerea

Azoxystrobin (AZ), a strobilurin-derived fungicide, is known to inhibit mitochondrial respiration in fungi by blocking the electron transport chain in the inner mitochondrial membrane. Germination was strongly inhibited when Botrytis cinerea spore suspension was treated with AZ and the alternative oxidase (AOX) inhibitors, salicylhydroxamic acid (SHAM) and n-propyl gallate. However, chemical death indicators trypan blue and propidium iodide showed that those spores were still alive. When the spore suspension in the AZ and SHAM solution was replaced with distilled water, the germination rate almost recovered, at least during the first 2 days of incubation with AZ and SHAM solution. No morphological alteration was detected in the cells treated with AZ and SHAM, especially in mitochondria, using transmission electron microscopy. Therefore, simultaneous application of AZ and AOX inhibitors has a fungistatic, rather than a fungicidal, action.     

A simple protocol to obtain highly pure Wolbachia endosymbiont DNA for genome sequencing

Most genome sequencing projects using intracellular bacteria face difficulties in obtaining sufficient bacterial DNA free of host contamination. We have developed a simple and rapid protocol to isolate endosymbiont DNA virtually free from fly and mosquito host DNA. We purified DNA from six Wolbachia strains in preparation for genome sequencing using this method, and achieved up to 97% pure Wolbachia sequence, even after using frozen insects. This is a significant improvement for future Wolbachia and other endosymbiont genome projects.     

Cloning and functional expression of the mitochondrial alternative oxidase gene (aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

Role of the putative structural protein Sed1p in mitochondrial genome maintenance

The nuclear gene MIP1 encodes the mitochondrial DNA polymerase responsible for replicating the mitochondrial genome in Saccharomyces cerevisiae. A number of other factors involved in replicating and segregating the mitochondrial genome are yet to be identified. Here, we report that a bacterial two-hybrid screen using the mitochondrial polymerase, Mip1p, as bait identified the yeast protein Sed1p. Sed1p is a cell surface protein highly expressed in the stationary phase. We find that several modified forms of Sed1p are expressed and the largest of these forms interacts with the mitochondrial polymerase in vitro. Deletion of SED1 causes a 3.5-fold increase in the rate of mitochondrial DNA point mutations as well as a 4.3-fold increase in the rate of loss of respiration. In contrast, we see no change in the rate of nuclear point mutations indicating the specific role of Sed1p function in mitochondrial genome stability. Indirect immunofluorescence analysis of Sed1p localization shows that Sed1p is targeted to the mitochondria. Moreover, Sed1p is detected in purified mitochondrial fractions and the localization to the mitochondria of the largest modified form is insensitive to the action of proteinase K. Deletion of the sed1 gene results in a reduction in the quantity of Mip1p and also affects the levels of a mitochondrially-expressed protein, Cox3p. Our results point towards a role for Sed1p in mitochondrial genome maintenance.     

Alternative oxidases (AOX1a and AOX2) can functionally substitute for plastid terminal oxidase in Arabidopsis chloroplasts

The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing the activity of PTOX in the desaturation steps of carotenogenesis. Similar results were obtained when AOX1a was reengineered to target the plastid. Chloroplast-localized AOX2 formed monomers and dimers, reminiscent of AOX regulation in mitochondria. Both AOX2 and AOX1a were present in higher molecular weight complexes in plastid membranes. The presence of these proteins did not generally affect steady state photosynthesis, aside from causing enhanced nonphotochemical quenching in both lines. Because AOX2 was imported into chloroplasts using its own transpeptide, we propose that AOX2 is able to function in chloroplasts to supplement PTOX activity during early events in chloroplast biogenesis. We conclude that the ability of AOX1a and AOX2 to substitute for PTOX in the correct physiological and developmental contexts is a striking example of the capacity of a mitochondrial protein to replace the function of a chloroplast protein and illustrates the plasticity of the photosynthetic apparatus.     

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. Its mitochondrial respiratory chain lacks a plant-like alternative oxidase

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, alpha-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.     

Functional expression of plant alternative oxidase decreases antimycin A-induced reactive oxygen species production in human cells

Alternative oxidase (AOX) plays a pivotal role in cyanide-resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria-targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin-A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.     

Mitochondrial alternative oxidase is determinant for growth and sporulation in the early diverging fungus Blastocladiella emersonii

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.     

Yeast cytochrome c is a sequence-specific DNA-binding protein

A protein binding to the alcohol oxidase 2 upstream activation sequence (AOX2UAS) of the methylotropic yeast, Pichia pastoris, has been purified and identified as cytochrome c (cyt c). Cyt c purified from P. pastoris or Saccharomyces cerevisiae binds to AOX2UAS. Specific point mutations in AOX2UAS abolish cyt c binding. We conclude that yeast cyt c is a sequence-specific DNA-binding protein and may have a regulatory role in the nucleus.     

工业真菌中的交替氧化酶

真菌是现代微生物发酵产业的主力军之一。交替呼吸途径(Alternative respiration pathway,ARP),以其非磷酸化电子传递途径,起到了能量溢流(Energy overflow)的作用,调节细胞能量代谢,平衡碳代谢和电子传递,有利于代谢产物的积累。此外,交替呼吸对真菌的抗逆反应和条件致病菌的生理作用也都具有非常重要的影响。交替氧化酶(Alternative oxidase,AOX)是线粒体中交替呼吸途径的末端氧化酶,广泛存在于高等植物及部分真菌和藻类中。由于交替氧化酶对水杨氧肟酸(Salicylhydroxamic acid,SHAM)敏感而对细胞色素呼吸抑制剂氰化物不敏感,交替氧化酶AOX介导的交替呼吸途径又被称为抗氰呼吸途径(Cyanide-resistant respiration,CRR)。近年来,研究交替呼吸途径和交替氧化酶已成为细胞呼吸代谢领域的热门课题。本文主要对真菌交替呼吸途径和交替氧化酶的结构与其在工业真菌体内功能的最新研究进展作一简要的综述。     

Heterogeneous expression and functional analysis of two distinct replication protein A large subunits from Cryptosporidium parvum

Replication protein A is a single stranded DNA-binding protein that has multiple roles in eukaryotic DNA metabolism. Typically, eukaryotic replication protein A is a stable heterotrimeric complex with three subunits of 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3). We have previously cloned and characterised an RPA1 subunit from Cryptosporidium parvum, which shares high homology with other eukaryotic replication protein A 1 proteins, but lacks an N-terminal domain. Here, we have identified a second replication protein A 1 (termed CpRPA1B) from the ongoing C. parvum genome-sequencing project. The deduced protein sequence to CpRPA1B shows only 16% sequence identity with CpRPA1, indicating that two different types of RPA1 subunits are present in C. parvum. The CpRPA1B gene predicts a 75.5 kDa peptide similar in size to those of higher eukaryotes, but in contrast to the 53.9 kDa N-terminal short-type CpRPA1 protein. However, western blot analysis suggested that, although the entire CpRPA1B open reading frame might be translated, the protein may be cleaved by posttranslational modification, similar to that observed with the replication protein A 1 gene product in Plasmodium falciparum. Indirect immunofluorescence studies indicated a diffused pattern for both proteins in sporozoites. However, differential localisation was observed with CpRPA1 to the anterior region that contains the apical-complex and CpRPA1B to the central region in/or around the nuclei of the sporozoites. Both CpRPA1 and CpRPA1B full-length open reading frames were expressed for functionality assays. The CpRPA1 and CpRPA1B recombinant proteins were expressed in bacterial Escherichia coli as maltose-binding protein fusion proteins and the entire fusion proteins were assayed for their DNA-binding properties. Studies indicate that CpRPA1B binds ssDNA of >or=5 nucleotides (dT), while CpRPA1 only binds ssDNA >or=20 nucleotides (dT). This study indicates that C. parvum possesses two different types of replication protein A large subunits (replication protein A 1), both differing significantly from their hosts.     

Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco)

The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aox1. Sense and antisense DNA constructs of Nicotiana tabacum Aox1 were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified. Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture. Transgenic cells with increased AOX protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration. Thus, genetic alteration of the level of AOX protein was sufficient to alter the capacity for electron transport through the alternative pathway. Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type. However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment. This suggests that a critical function of AOX may be to support respiration when the cytochrome pathway is impaired. The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.