<Emphasis Type="Italic">Leishmania major</Emphasis> protein disulfide isomerase as a drug target

Leishmaniasis is a major health problem worldwide and tools available for their control are limited. Effective vaccines are still lacking, drugs are toxic and expensive, and parasites develop resistance to chemotherapy. In this context, new antimicrobials are urgently needed to control the disease in both human and animal. Here, we report the enzymatic and functional characterization of a Leishmania virulence factor, Leishmania major Protein disulfide isomerase (LmPDI) that could constitute a potential drug target. LmPDI possesses domain structure organization similar to other PDI family members (a, a′, b, b′ and c domains), and it displays the three enzymatic and functional activities specific of PDI family members: isomerase, reductase and chaperone. These results suggest that LmPDI plays a key role in assisting Leishmania protein folding via its capacity to catalyze formation, breakage, and rearrangement of disulfide bonds in nascent polypeptides. Moreover, Bacitracin, a reductase activity inhibitor, and Ribostamycin, a chaperone activity inhibitor, were tested in LmPDI enzymatic assays and versus Leishmania promastigote in vitro cultures and Leishmania amastigote multiplication inside infected THP-1-derived macrophages. Bacitracin inhibited both isomerase and reductase activities, while Ribostamycin had no effect on the chaperone activity. Interestingly, Bacitracin blocked in vitro promastigote growth as well as amastigote multiplication inside macrophages with EC₅₀ values of 39 μM. These results suggest that LmPDI may constitute an interesting target for the development of new anti-Leishmania drugs.     

In vitro antileishmanial activity of resveratrol and its hydroxylated analogues against <Emphasis Type="Italic">Leishmania major</Emphasis> promastigotes and amastigotes

Dermatitis caused by penetrating bird schistosome cercariae is an emerging global public health problem. Infections may be prevented by the use of topical formulations that inhibit cercarial skin penetration. We evaluated nine water resistant formulations by exposing treated arms of volunteers to Trichobilharzia szidati cercariae. Six formulations protected from cercarial invasion. However, after immersion of the treated skin in water (2 × 20 min), only two formulations offered full protection: (1) Safe Sea™, a cream protecting against jelly fish, (2) niclosamide in water resistant sun protecting cream formulations at concentrations as low as 0.05%. In an in vitro system Safe Sea™ and a 0.1% niclosamide formulation caused a high damage rate in T. szidati (92% and 99% after 5 min; only niclosamide with lethal effect) but not in Schistosoma mansoni (1% and 72%; both formulations with lethal effect). However, a 1% niclosamide formulation damaged S. mansoni sufficiently (100% after 5 min) and might offer full penetration protection.     

<Emphasis Type="Italic">PTR1</Emphasis>-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite <Emphasis Type="Italic">Leishmania major</Emphasis>

Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major, which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1 ⁻ null mutants were 18-fold more sensitive to H₂O₂ than PTR1-overproducing lines, and significant three- to fivefold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H₂O₂ susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1- but not dihydrofolate reductase-dependent manner, implicating H₄B metabolism specifically. Neither H₂O₂ consumption nor the level of intracellular oxidative stress was affected by PTR1 levels. Coupled with the fact that reduced pteridines are at least 100-fold less abundant than cellular thiols, these data argue strongly that reduced pteridines act through a mechanism other than scavenging. The ability of unconjugated pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes.     

Functional analysis of <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> carboxylic acids permeases: heterologous expression of <Emphasis Type="Italic">KlJEN1</Emphasis> and <Emphasis Type="Italic">KlJEN2</Emphasis> genes

The present work describes a detailed physiological and molecular characterization of the mechanisms of transport of carboxylic acids in Kluyveromyces lactis. This yeast species presents two homologue genes to JEN1 of Saccharomyces cerevisiae: KlJEN1 encodes a monocarboxylate permease and KlJEN2 encodes a dicarboxylic acid permease. In the strain K. lactis GG1888, expression of these genes does not require an inducer and activity for both transport systems was observed in glucose-grown cells. To confirm their key role for carboxylic acids transport in K. lactis, null mutants were analyzed. Heterologous expression in S. cerevisiae has been performed and chimeric fusions with GFP showed their proper localization in the plasma membrane. S. cerevisiae jen1Δ cells transformed with KlJEN1 recovered the capacity to use lactic acid, as well as to transport labeled lactic acid by a mediated mechanism. When KlJEN2 was heterologously expressed, S. cerevisiae transformants gained the ability to transport labeled succinic and malic acids by a mediated mechanism, exhibiting, however, a poor growth in malic acid containing media. The results confirmed the role of KlJen1p and KlJen2p as mono and dicarboxylic acids permeases, respectively, not subjected to glucose repression, being fully functional in S. cerevisiae. O. Queirós and L. Pereira contributed equally to this work.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

The importance of <Emphasis Type="Italic">Helicobacter pylori tnpA</Emphasis>, <Emphasis Type="Italic">tnpB</Emphasis>, and <Emphasis Type="Italic">cagA</Emphasis> genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.     

How heterologously expressed <Emphasis Type="Italic">Escherichia coli</Emphasis> genes contribute to understanding DNA repair processes in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

DNA-damaging agents constantly challenge cellular DNA; and efficient DNA repair is therefore essential to maintain genome stability and cell viability. Several DNA repair mechanisms have evolved and these have been shown to be highly conserved from bacteria to man. DNA repair studies were originally initiated in very simple organisms such as Escherichia coli and Saccharomyces cerevisiae, bacteria being the best understood organism to date. As a consequence, bacterial DNA repair genes encoding proteins with well characterized functions have been transferred into higher organisms in order to increase repair capacity, or to complement repair defects, in heterologous cells. While indicating the contribution of these repair functions to protection against the genotoxic effects of DNA-damaging agents, heterologous expression studies also highlighted the role of the DNA lesions that are substrates for such processes. In addition, bacterial DNA repair-like functions could be identified in higher organisms using this approach. We heterologously expressed three well characterized E. coli repair genes in S. cerevisiae cells of different genetic backgrounds: (1) the ada gene encoding O⁶-methylguanine DNA-methyltransferase, a protein involved in the repair of alkylation damage to DNA, (2) the recA gene encoding the main recombinase in E. coli and (3) the nth gene, the product of which (endonuclease III) is responsible for the repair of oxidative base damage. Here, we summarize our results and indicate the possible implications they have for a better understanding of particular DNA repair processes in S. cerevisiae.     

Identification and characterization of a novel <Emphasis Type="Italic">Tritimovirus</Emphasis> species isolated from wild <Emphasis Type="Italic">Trisetum flavescens</Emphasis> L., family <Emphasis Type="Italic">Poaceae</Emphasis>

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3′ end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).     

<Emphasis Type="Italic">Leishmania major</Emphasis> infection in a patient with visceral leishmaniasis: treatment with Amphotericin B

We have reported an acute form of visceral leishmaniasis, which was caused by Leishmania major, in Southern Iran. The parasite was isolated from a 30-year-old man living in Bushehr Province, Southern Iran, and an endemic region of kala-azar in south of Iran. The patient had typical clinical signs of Mediterranean kala-azar, and besides parasitological, biochemical, and immunological findings confirming diagnosis of visceral leishmaniasis, the agent was identified as L. major using Nested polymerase chain reaction. The patient was treated successfully with Amphotericin B during a 1-month period.     

Prevalence of the <Emphasis Type="Italic">ica</Emphasis> operon and insertion sequence IS<Emphasis Type="Italic">256</Emphasis> among <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> prosthetic joint infection isolates

Joint replacement surgery has improved the quality of life for hundreds of thousands of patients. However, the infection of a joint implant is an important and serious complication, though the prevalence is low. Staphylococcus epidermidis is the most important pathogen involved in foreign-body infections. S. epidermidis is also a commensal that comprises a substantial part of the normal skin flora of humans. The possibility to demonstrate potential specific virulence markers may facilitate the interpretation of the bacteriological findings, as well as the clinical decision. The prevalence of the ica locus and insertion sequence IS256 by using polymerase chain reaction (PCR) among 32 clinical S. epidermidis isolates from prosthetic joint infections (PJIs) and 24 commensal isolates from nares and skin was investigated. Sixteen (50%) of the 32 PJI isolates harbored the ica operon compared with one-third of the commensal isolates obtained from the samples of the skin and nares of healthy individuals. The IS256 was demonstrated in 26 (81%) out of 32 PJI isolates. By contrast, IS256 was found in one of 24 commensal isolates. In conclusion, IS256 may be superior to the ica operon as a marker of the invasive capacity of S. epidermidis, since it was found in most of the PJI isolates, but rarely among commensals.     

Structure of nuclear-localized <Emphasis Type="Italic">cox3</Emphasis> genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and in its colorless close relative <Emphasis Type="Italic">Polytomella</Emphasis> sp.

Several chlorophyte algae do not have the cox3 gene, encoding subunit III of cytochrome c oxidase, in their mitochondrial genomes. The cox3 gene is nuclear-encoded in the photosynthetic alga Chlamydomonas reinhardtii and in the colorless alga Polytomella sp. In this work, the genomic sequences of the cox3 genes of these two closely related algae are reported. The cox3 genes of both C. reinhardtii and Polytomella sp. contain four introns in the region encoding the putative mitochondrial-targeting sequences. These four introns show low sequence identities, but their locations are conserved between these species. The cox3 gene of C. reinhardtii has five additional introns in the region encoding the mature subunit III of cytochrome c oxidase. Sequence analysis of intron 6 of the cox3 gene of C. reinhardtii revealed similarity with two sequence elements present in introns of several other nuclear genes from this green alga. In the majority of the genes, these conserved sequences are located either near the 3' end or near the 5' end of the introns. Based on these data, we propose that the colorless genus Polytomella separated from C. reinhardtii after the cox3 gene was transferred to the nucleus. The data also support the evolutionary hypothesis of a recent acquisition of introns in C. reinhardtii.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

<Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">aeruginosa</Emphasis> Biofilms in CF Infection

Pseudomonas aeruginosa is an opportunistic pathogen of immunocompromised hosts. In cystic fibrosis (CF), P. aeruginosa causes acute and chronic lung infections that result in significant morbidity and mortality. P. aeruginosa possesses several traits that contribute to its ability to colonize and persist in acute and chronic infections. These include high resistance to antimicrobials, ability to form biofilms, plethora of virulence products, and metabolic versatility. In P. aeruginosa, a cell-to-cell communication process termed quorum sensing (QS) regulates many of these factors that contribute to its pathogenesis. Recent evidence suggests that the CF lung environment presents a specialized niche for P. aeruginosa. The relationship of P. aeruginosa QS, biofilm formation, and the CF lung environment is discussed.     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.