Cathepsin B-like and cell death in the unicellular human pathogen Leishmania

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H₂O₂), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.     

Gentamicin-Attenuated Leishmania infantum Vaccine: Protection of Dogs against Canine Visceral Leishmaniosis in Endemic Area of Southeast of Iran

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A vaccine trial was conducted using 103 naive dogs from a leishmaniosis non-endemic area (55 vaccinated and 48 unvaccinated) brought into an endemic area of southeast Iran. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination. The efficacy of the vaccine was evaluated after 24 months (4 sandfly transmission seasons) by serological, parasitological analyses and clinical examination. In western blot analysis of antibodies to L. infantum antigens, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs which were seropositive at >100, recognized the 21 kDa antigen of L. infantum wild-type (WT). Nine out of 31 (29%) unvaccinated dogs, but none of vaccinated dogs, were positive for the presence of Leishmania DNA. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. These results suggest that gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniosis in the endemic area.     

The Escherichia coli btuE gene, encodes a glutathione peroxidase that is induced under oxidative stress conditions

Most aerobic organisms are exposed to oxidative stress. Looking for enzyme activities involved in the bacterial response to this kind of stress, we focused on the btuE-encoded Escherichia coli BtuE, an enzyme that shares homology with the glutathione peroxidase (GPX) family. This work deals with the purification and characterization of the btuE gene product.Purified BtuE decomposes in vitro hydrogen peroxide in a glutathione-dependent manner. BtuE also utilizes preferentially thioredoxin A to decompose hydrogen peroxide as well as cumene-, tert-butyl-, and linoleic acid hydroperoxides, confirming that its active site confers non-specific peroxidase activity. These data suggest that the enzyme may have one or more organic hydroperoxide as its physiological substrate.The btuE gene was induced when cells were exposed to oxidative stress elicitors that included potassium tellurite, menadione and hydrogen peroxide, among others, suggesting that BtuE could participate in the E. coli response to reactive oxygen species. To our knowledge, this is the first report describing a glutathione peroxidase in E. coli.     

Decreased memory T-cell response and function in human immunodeficiency virus-infected patients with tegumentary leishmaniasis

The effects of human immunodeficiency virus (HIV) on the immune response in patients with cutaneous leishmaniasis have not yet been fully delineated. This study quantified and evaluated the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and effector memory (EM) CD4+T-cells in response to SLA were quantified using flow cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected patients in response to SLA were significantly lower than those in patients with Leishmania monoinfection (p < 0.05). The proportions of CM and EM CD4+T-cells in response to SLA were similar between the coinfected patients and patients with Leishmania monoinfection. However, the median CM and EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05). The reduction in the lymphoproliferative response to Leishmania antigens coincides with the decrease in the absolute numbers of both EM and CM CD4+T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania.     

The overexpression of a new ABC transporter in Leishmania is related to phospholipid trafficking and reduced infectivity

This paper reports the characterization of a new ABC transporter (LtrABC1.1), related to the human ABCA subfamily, in the protozoan parasite Leishmania tropica. LtrABC1.1 is a tandem duplicated gene flanked by inverted repeats. LtrABC1.1 is expressed mainly in the flagellar pocket of the parasite. Drug resistance studies in Leishmania overexpressing LtrABC1.1 showed the transporter not to confer resistance to a range of unrelated drugs. LtrABC1.1 appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. The activity of this protein may also affect membrane movement processes since secreted acid phosphatase (SAP) activity was significantly lower in promastigotes overexpressing LtrABC1.1. In vitro infection experiments with macrophages indicated LtrABC1.1-transfected parasites to be significantly less infective. Together, these results suggest that this new ABC transporter could play a role in lipid movements across the plasma membrane, and that its activity might influence vesicle trafficking. This is the first ABCA-like transporter described in unicellular eukaryotes.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Kolaviron and selenium reduce hydrogen peroxide-induced alterations of the inflammatory response

The abilities of kolaviron and selenium (either separately or in combination) to prevent hydrogen peroxide-induced alterations in cell viability and activation were investigated. The cell line U937 was incubated with the antioxidants (i.e. kolaviron or selenium) for 24?h before exposure to hydrogen peroxide and cell viability was assessed via trypan blue dye exclusion assay. The U937 cells were also transformed to the macrophage form, incubated with the antioxidants before exposure to hydrogen peroxide. Subsequently, production of nitric oxide and pro-inflammatory cytokines were assessed as indices of macrophage activation. The myoblast cell line H9c2 was also incubated with Se and kolaviron for 24?h before exposure to hydrogen peroxide. Cell viability and generation of reactive oxygen species (ROS) were assessed via MTT and DCHF assays. The results revealed that hydrogen peroxide significantly reduced (p?<?0.05) the viability of U937 cells which was ameliorated by kolaviron and selenium. Kolaviron and selenium also reduced hydrogen peroxide-induced secretion of nitric oxide, TNF-α, IL-1 and IL-6 by transformed U937 cells. Hydrogen peroxide also significantly reduced (p?<?0.05) the viability of H9c2 cells which was significantly restored by kolaviron. Though selenium had no effect on the proliferation of H9c2 cells, co-treatment with kolaviron significantly reduced hydrogen peroxide-induced alterations. Both kolaviron and selenium also reduced hydrogen peroxide-mediated ROS production by H9c2 cells. In all cases, the combined action of kolaviron and selenium offered greater amelioration of the hydrogen peroxide-induced alterations than their separate effects (p?<?0.05) but may not be synergistic or additive.     

Peroxide metabolism in the cestodes Hymenolepis diminuta and Moniezia expansa

The enzymes of hydrogen peroxide metabolism have been investigated in the cestodes H. diminuta and M. expansa. Neither catalase, lipoxygenase, glutathione peroxidase, NADH peroxidase nor NADPH peroxidase could be detected in homogenates of either species. However, both H. diminuta and M. expansa possessed a peroxidase which had a high affinity for reduced cytochrome c. The peroxidase was characterized by substrate and inhibitor studies and cell fractionation showed the enzyme to be located in the mitochondrial membrane fraction. The peroxidase could act as a substitute for catalase, by destroying metabolic hydrogen peroxide. Appreciable superoxide dismutase activity was found in M. expansa and H. diminuta and it is possible that this enzyme is the source of helminth hydrogen peroxide.     

灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。     

Effects of HIV aspartyl-proteinase inhibitors on Leishmania sp.

In this work, we have found an antiproliferative effect on Leishmania sp. promastigotes and axenic amastigotes by the human immunodeficiency virus (HIV) aspartyl-proteinase inhibitors, Ac-Leu-Val-Phenylalaninal, Saquinavir mesylate and Nelfinavir, the latter two being used as part of antiretroviral therapy. This effect appears to be the result of cell division blockage. In addition, these drugs induced in culture a decrease in the percentage of co-infected HIV/Leishmania monocytes and amastigotes of Leishmania per macrophage. The finding of a dose-dependent inhibition of Leishmania promastigotes aspartyl-proteinase activity by these drugs allows us to propose this activity as the drug parasite target. A direct action of these HIV aspartyl-proteinase inhibitors on the parasite, would be correlated with the effect that highly active antiretroviral therapy have had in the decrease of HIV/Leishmania coinfection, opening an interesting perspective for new drugs research development based on this novel parasite proteinase family.     

Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management

Reactive oxygen and nitrogen species (ROS and RNS) produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS) plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL). There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS^-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed environments encountered during infection and can be targeted for chemotherapeutic purpose to treat visceral leishmaniasis.     

Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia

The first autochthonous Leishmania infection in Australia was reported by Rose et al. (2004) and the parasite was characterised as a unique species. The host was the red kangaroo (Macropus rufus) but the transmitting vector was unknown. To incriminate the biological vector, insect trapping by a variety of methods was undertaken at two field sites of known Leishmania transmission. Collected sand flies were identified to species level and were screened for Leishmania DNA using a semi-quantitative real-time PCR. Collections revealed four species of sand fly, with a predominance of the reptile biter Sergentomyia queenslandi (Hill). However, no Leishmania-positive flies were detected. Therefore, alternative vectors were investigated for infection, giving startling results. Screening revealed that an undescribed species of day-feeding midge, subgenus Forcipomyia (Lasiohelea) Kieffer, had a prevalence of up to 15% for Leishmania DNA, with high parasitemia in some individuals. Manual gut dissections confirmed the presence of promastigotes and in some midges material similar to promastigote secretory gel, including parasites with metacyclic-like morphology. Parasites were cultured from infected midges and sequence analysis of the Leishmania RNA polymerase subunit II gene confirmed infections were identical to the original isolated Leishmania sp. Phylogenetic analysis revealed the closest known species to be Leishmania enriettii, with this and the Australian species confirmed as members of Leishmania sensu stricto. Collectively the results strongly suggest that the day-feeding midge (F. (Lasiohelea) sp. 1) is a potential biological vector of Leishmania in northern Australia, which is to our knowledge the first evidence of a vector other than a phlebotomine sand fly anywhere in the world. These findings have considerable implications in the understanding of the Leishmania life cycle worldwide.     

Changes in oxidative stress parameters and acid phosphatase activity in the pre-regressing and regressing tail of Indian jumping frog Polypedates maculatus (Anura,Rhacophoridae)

Activities of acid phosphatase (normal and Co²⁺-sensitive), superoxide dismutase and catalase and levels of lipid peroxidation, hydrogen peroxide were compared in the tails of tadpoles of stage III, XVIII, XXI and XXIII, respectively, of the Indian Jumping frog Polypedates maculatus. It is noticed that acid phosphatase activity (normal and Co²⁺-sensitive), and levels of lipid peroxidation and hydrogen peroxide increased during tail regression. There is also an increase in the level of superoxide dismutase and catalase in the regressing tail. A positive correlation between activity of acid phosphatase and lipid peroxidation, hydrogen peroxide and lipid peroxidation, acid phosphatase and hydrogen peroxide was noticed in the tail of tadpoles during different developmental stages, suggesting a critical interaction between reactive oxygen species and lysosomal activity during metamorphosis.     

Effect of water activity control on the catalytic performance of surfactant–Arthromyces ramosus peroxidase complex in toluene

Arthromyces ramosus peroxidase (ARP) was successfully modified with a synthetic surfactant for one-electron oxidation reaction of a hydrophobic substrate in toluene. Although UV–visible absorption spectrum of surfactant–ARP complex in toluene showed slight red shift of Soret band compared to that in water, the complex can catalyze oxidation reaction of o-phenylenediamine (o-PDA) with hydrogen peroxide. It appeared that thermodynamic water activity in the reaction system has dominant effect on either the catalytic activity or the stability in the catalytic cycle. Steady-state kinetics under the optimal condition revealed that the specific constant (k_cat/K_m) of ARP complex for o-PDA was 2 orders of magnitude lower than that in aqueous media, while only 13-fold lower for hydrogen peroxide. The reduction of catalytic activity caused by altering the reaction media from water to toluene was found to be mainly due to the low specific constant of ARP complex for o-PDA rather than hydrogen peroxide.     

Esteblishment and characterization of the line of Fagopyrum tataricum morphogenic callus tolerant to aminotriazole

It is shown that the inhibitor of catalase 3-amino-1,2,4-triazole (AT) at the concentration of 2 mM affected differently growth of tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) callus lines differing in the morphogenecity. In some cases, AT induced the death of a great fraction of non-morphogenic callus cells; in other cases, it inhibited growth and reduced viability of morphogenic callus. The death of non-morphogenic callus cells may be related to the accumulation of hydrogen peroxide and the development of oxidative stress. After morphogenic callus treatment with AT, we obtained a modified line 1?C8 AT tolerant to AT and differing from the original line in morphology, cell sizes, proliferative activity, and some biochemical characteristics. In the 1?C8 AT line, catalase was sensitive to this inhibitor action. In this case, catalase inactivation with AT did not increase the content of hydrogen peroxide in the cells, which may indicate the compensatory functioning of another/others mechanism(s) destroying hydrogen peroxide.     

Nox4 modulates collagen production stimulated by transforming growth factor β1 in vivo and in vitro

The synthesis of extracellular matrix including collagen during wound healing responses involves signaling via reactive oxygen species (ROS). We hypothesized that NADPH oxidase isoform Nox4 facilitates the stimulatory effects of the profibrotic cytokine transforming growth factor (TGF) β₁ on collagen production in vitro and in vivo. TGFβ₁ stimulated collagen synthesis and hydrogen peroxide generation in mouse cardiac fibroblasts, and both responses were attenuated by a scavenger of superoxide and hydrogen peroxide (EUK-134). Furthermore, by expressing a dominant negative form of Nox4 (Adv-Nox4^ΔNADPH) in fibroblasts, TGFβ₁-induced hydrogen peroxide production and collagen production were abrogated, suggesting that Nox4-dependent ROS are important for TGFβ₁ signaling in collagen production. This was confirmed by the inhibitory effect of an adenovirus carrying siRNA targeting Nox4 (Adv-Nox4i) on TGFβ₁-induced collagen synthesis and expression of activated myofibroblasts marker smooth muscle alpha actin. Finally we used a mouse model of subcutaneous sponge implant to examine the role of Nox4 in the local stimulatory effects of TGFβ₁ on collagen accumulation in vivo. TGFβ₁-induced collagen accumulation was significantly reduced when the sponges were instilled with Adv-Nox4^ΔNADPH. In conclusion, Nox4 acts as an intermediary in the signaling of TGFβ₁ to facilitate collagen synthesis.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.