DNA analysis of cattle parasitic protozoan Tritrichomonas foetus after photodynamic therapy.

Photodynamic therapy (PDT) is a modality of therapy that involves the activation of photosensitive substances and the generation of cytotoxic oxygen species and free radicals to promote the selective destruction of target tissues. This study analyzed the application of PDT to Tritrichomonas foetus, a scourged and etiological agent of bovine trichomoniasis, a sexually transmitted infectious disease. As it is an amitochondrial and aerotolerant protozoan, it produces energy under low O2 tension via hydrogenosome. T. foetus from an axenic culture was incubated with photosensitizer tetrasulfonated aluminium phthalocyanine and then irradiated with a laser source (InGaAIP) at a density of 4.5Jcm-2. The DNA integrity of the control and treated group parasites was analyzed by conventional gel electrophoresis and comet assay techniques. In previous results, morphological changes characterized by apoptotic cell death were observed after T. foetus was submitted to PDT treatment. In the treated groups, T. foetus DNA showed a higher concentration of small fragments, about 200pb, in gel electrophoresis after PDT. In the comet assay, the DNA tail percentage was significantly higher in the treated groups. These results demonstrate that PDT leads to DNA fragmentation with changes in nuclear morphology and apoptotic features.

Photo-induced Leishmania DNA degradation by silver-doped zinc oxide nanoparticle: an in-vitro approach.

Recently, the authors reported newly synthesised polyethylene glycol (PEG)ylated silver (9%)-doped zinc oxide nanoparticle (doped semiconductor nanoparticle (DSN)) which has high potency for killing Leishmania tropica by producing reactive oxygen species on exposure to sunlight. The current report is focused on Leishmania DNA interaction and damage caused by the DSN. Here, we showed that the damage to Leishmania DNA was indirect, as the DSN was unable to interact with the DNA in intact Leishmania cell, indicating the incapability of PEGylated DSN to cross the nucleus barrier. The DNA damage was the result of high production of singlet oxygen on exposure to sunlight. The DNA damage was successfully prevented by singlet oxygen scavenger (sodium azide) confirming involvement of the highly energetic singlet oxygen in the DNA degradation process.

Sunlight inhibits growth and induces markers of programmed cell death in Plasmodium falciparum in vitro.

BACKGROUND: Plasmodium falciparum is responsible for the majority of global malaria deaths. During the pathogenic blood stages of infection, a rapid increase in parasitaemia threatens the survival of the host before transmission of slow-maturing sexual parasites to the mosquito vector to continue the life cycle. Programmed cell death (PCD) may provide the parasite with the means to control its burden on the host and thereby ensure its own survival. Various environmental stress factors encountered during malaria may induce PCD in P. falciparum. This study is the first to characterize parasite cell death in response to natural sunlight. METHODS: The 3D7 strain of P. falciparum was cultured in vitro in donor erythrocytes. Synchronized and mixed-stage parasitized cultures were exposed to sunlight for 1 h and compared to cultures maintained in the dark, 24 h later. Mixed-stage parasites were also subjected to a second one-hour exposure at 24 h and assessed at 48 h. Parasitaemia was measured daily by flow cytometry. Biochemical markers of cell death were assessed, including DNA fragmentation, mitochondrial membrane polarization and phosphatidylserine externalization. RESULTS: Sunlight inhibited P. falciparum growth in vitro. Late-stage parasites were more severely affected than early stages. However, some late-stage parasites survived exposure to sunlight to form new rings 24 h later, as would be expected during PCD whereby only a portion of the population dies. DNA fragmentation was observed at 24 and 48 h and preceded mitochondrial hyperpolarization in mixed-stage parasites at 48 h. Mitochondrial hyperpolarization likely resulted from increased oxidative stress. Although data suggested increased phosphatidylserine externalization in mixed-stage parasites, results were not statistically significant. CONCLUSION: The combination of biochemical markers and the survival of some parasites, despite exposure to a lethal stimulus, support the occurrence of PCD in P. falciparum.

UV irradiation responses in Giardia intestinalis.

The response to ultraviolet light (UV) radiation, a natural stressor to the intestinal protozoan parasite Giardia intestinalis, was studied to deepen the understanding of how the surrounding environment affects the parasite during transmission. UV radiation at 10 mJ/cm(2) kills Giardia cysts effectively whereas trophozoites and encysting parasites can recover from UV treatment at 100 mJ/cm(2) and 50 mJ/cm(2) respectively. Staining for phosphorylated histone H2A showed that UV treatment induces double-stranded DNA breaks and flow cytometry analyses revealed that UV treatment of trophozoites induces DNA replication arrest. Active DNA replication coupled to DNA repair could be an explanation to why UV light does not kill trophozoites and encysting cells as efficiently as the non-replicating cysts. We also examined UV-induced gene expression responses in both trophozoites and cysts using RNA sequencing (RNA seq). UV radiation induces small overall changes in gene expression in Giardia but cysts show a stronger response than trophozoites. Heat shock proteins, kinesins and Nek kinases are up-regulated, whereas alpha-giardins and histones are down-regulated in UV treated trophozoites. Expression of variable surface proteins (VSPs) is changed in both trophozoites and cysts. Our data show that Giardia cysts have limited ability to repair UV-induced damage and this may have implications for drinking- and waste-water treatment when setting criteria for the use of UV disinfection to ensure safe water.

Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica.

Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan.

The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human breast cancer cells.

MicroRNAs (miRNAs) are important regulators of cell fate determination and homeostasis. Expression of these small RNA genes is tightly regulated during development and in normal tissues, but they are often misregulated in cancer. MiRNA expression is also affected by DNA damaging agents, such as radiation. In particular, mammalian miR-34 is upregulated by p53 in response to radiation, but little is known about the role of this miRNA in vivo. Here we show that Caenorhabditis elegans with loss-of-function mutations in the mir-34 gene have an abnormal cellular survival response to radiation; these animals are highly radiosensitive in the soma and radioresistant in the germline. These findings show a role for mir-34 in both apoptotic and non-apoptotic cell death in vivo, much like that of cep-1, the C. elegans p53 homolog. These results have been additionally validated in vitro in breast cancer cells, wherein exogenous addition of miR-34 alters cell survival post-radiation. These observations confirm that mir-34 is required for a normal cellular response to DNA damage in vivo resulting in altered cellular survival post-irradiation, and point to a potential therapeutic use for anti-miR-34 as a radiosensitizing agent in p53-mutant breast cancer.

Effects of DNA damage induced by UV irradiation on gene expression in the protozoan parasite Entamoeba histolytica.

Previously, we provided evidence for the role of E. histolytica RAD52 epistasis group genes and the EhRAD51 recombinase in DNA damage response. To identify other genes participating in DNA repair in this protozoan parasite, here we analyzed the transcriptional response to genetic damage induced by ultraviolet light (UV) using cDNA microarrays. We found that 11.6% (350 ORFs) and 17.2% (522 ORFs) of genes were modulated at 5 min and 3h after UV irradiation, respectively. Most genes were less than 2-fold changed evidencing a weak transcriptional activation. The genes encoding so-called "classical" DNA repair proteins were slightly regulated in trophozoites submitted to UV irradiation. We also observed the over-expression of genes encoding for Fe-S clusters-containing proteins, potentially involved in the stress adaptation in response to DNA damage. Several genes encoding cytoskeleton proteins were repressed suggesting that actin dynamics was impaired after UV irradiation. Our analysis highlights novel genes potentially involved in DNA damage response, and these data will contribute to further elucidation of mechanisms regulating genome integrity in this early branch protozoan.

Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair.

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.

UV-induced cell damage is species-specific among aquatic phagotrophic protists.

The sensitivity to ultraviolet radiation (UVR, 280-400 nm) of ten species of freshwater and marine phagotrophic protists was assessed in short-term (4 h) laboratory experiments. Changes in the motility and morphology of the cells, as well as direct quantification of DNA damage, were evaluated. The net amount of cyclobutane pyrimidine dimers formed after exposure of the organisms to a weighted dose (Setlow DNA normalized at 300 nm) of 1.7 kJ m(-2) was quantified by an immunoassay using a monoclonal specific antibody directed against thymine dimers (T<>Ts). This is the first application of this method to aquatic protists. The results indicated that marine and freshwater heterotrophic nanoflagellates, representatives from the order Kinetoplastida (Bodo caudatus and Bodo saltans, respectively) accumulate significantly higher DNA damage than protists representatives of the orders Chrysomonadida, Cryptomonadida or Scuticociliatida. The high proportion of A:T bases in the unique kinetoplast DNA, may explain the higher accumulation of T<>Ts found in bodonids. Experiments made with B. saltans to study the dynamics of DNA damage accumulation in the presence of UVR and photorepairing light, indicated that the mechanisms of DNA repair in this species are very inefficient. Furthermore, the dramatic changes observed in the cell morphology of B. saltans probably compromise its recovery. Our results show that sensitivity to UVR among aquatic phagotrophic protists is species-specific and that different cell targets are affected differently among species. While DNA damage in B. saltans was accompanied by motility reduction, altered morphology, and finally mortality, this was not observed in other bodonids as well as in the other species tested.

A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II-null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II-null cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

Characterization by pulse-field electrophoresis of a new region of DNA amplification containing the M2 subunit of ribonucleotide reductase in hydroxyurea-resistant Leishmania.

An extrachromosomal circular DNA of of approximately 50-kb size was amplified in the hydroxyurea-resistant variant of Leishmania mexicana amazonensis. The amplicon carried the M2 gene of ribonuleotide reductase as part of the gene encoding resistance to hydroxyurea. The amplicon was unstable. It disappeared rapidly as shown in pulse-field gradient electrophoresis gels after reversion of the cells for 20-80 days. This loss of amplified DNA was accompanied by a rapid loss of resistance to hydroxyurea during the same period. The amplicon was not hybridized to specific probes from any of the four regions of DNA amplification previously reported for Leishmania. This region of amplification thus appears to be a new region of DNA amplification in Leishmania.

Evidence for UV-B-induced DNA degradation in Euglena gracilis mediated by activation of metal-dependent nucleases.

It is demonstrated that in vivo irradiation with artificial UV-B for several hours significantly reduces the amount of large DNA extractable from immobilized Euglena in comparison with non-irradiated controls. This UV-B effect can be eliminated by a drastic reduction of the divalent ion concentration in the extracellular medium, i.e. the substitution of the culture medium by Tris-buffered agarose. Moreover, in vitro degradation of large DNA is demonstrated for crude protein extracts isolated from non-irradiated or UV-B-irradiated Euglena. The nuclease activity is shown for both crude protein extracts and purified nucleases; in both cases, two protein bands possessing nuclease activity are obtained with apparent molecular masses of 26 and 40 kDa and their activity is inhibited by specific nuclease inhibitors, i.e. aurintricarboxylic acid and ATP, applied at a concentration as low as 10(-8) M. Moreover, in vitro, nuclease activity clearly depends on the pH, with an optimum around pH 4.5, and on the ion composition of the extracellular medium. A strong stimulating effect is shown for Ca2+ with an optimum around 10(-4) M; this effect is potentiated by Zn2+ and Mn2+, but strongly counteracted by Mg2+ and the calmodulin inhibitors trifluoperazine and N- (6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W5). These results favour the concept which explains the lethal UV-B effect on Euglena as arising from a change in the general metabolic state of the cell and an activation of a DNA-degrading system, i.e. activation of metal-dependent nucleases (U.K. Tirlapur, D.-P. Häder and R. Scheuerlein, UV-B mediated damage in the photosynthetic flagellate, Euglena gracilis, studied by image analysis, Beitr. Biol. Pflanzen, 67 (1992) 305-317).