RESUMO
To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru.
Assuntos
Citocromos b/genética , Leishmania/classificação , Leishmania/genética , Leishmaniose Cutânea/epidemiologia , Manose-6-Fosfato Isomerase/genética , Filogeografia , Proteínas de Protozoários/genética , Humanos , Leishmania/isolamento & purificação , Peru/epidemiologiaRESUMO
The FTA card (Whatman) was assessed for its utility as a molecular epidemiological tool in collecting samples from patients with leishmaniasis in Peru because the card has a variety of merits; it is less invasive for patients and easy to handle for both physicians and other medical personnel for sample collection or diagnosis, in addition to its simplicity and easy countrywide and/or intercountry transportation for analysis. Samples were collected from 132 patients suspected of having leishmaniasis, and Leishmania species were successfully identified in samples from 81 patients in 15 departments of Peru by cytochrome b and mannose phosphate isomerase gene analyses. Of these, 61.7% were identified as Leishmania (Viannia) peruviana, 22.2% as L. (V.) braziliensis, 12.3% as L. (V.) guyanensis, 2.5% as L. (V.) shawi, and 1.2% as L. (V.) lainsoni. The three predominant species, L. (V.) peruviana, L. (V.) braziliensis, and L. (V.) guyanensis, were mainly found in the Andean highlands, in the tropical rainforest, and in northern and central rainforest regions, respectively. This is the first time L. (V.) shawi has been identified outside Brazil. The present study showed that the FTA card will be a useful tool for the ecological study of different forms of leishmaniasis. Furthermore, collecting samples directly from patients' lesions by using the FTA card eliminates (i) the possibility of contamination of Leishmania isolates during short- and/or long-term passages of culture in vitro in each laboratory and (ii) pain and suffering of patients from taking samples by skin biopsy.
Assuntos
Leishmania/classificação , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/patologia , Parasitologia/métodos , Manejo de Espécimes/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Citocromos b/genética , Feminino , Humanos , Lactente , Recém-Nascido , Leishmania/genética , Masculino , Manose-6-Fosfato Isomerase/genética , Pessoa de Meia-Idade , Peru , Proteínas de Protozoários/genética , Adulto JovemRESUMO
Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.
Assuntos
Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Genes Bacterianos , Polissacarídeos Bacterianos/biossíntese , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/genética , Hidroliases/genética , Hidroliases/metabolismo , Lipopolissacarídeos/biossíntese , Manose-6-Fosfato Isomerase/genética , Manose-6-Fosfato Isomerase/metabolismo , Mutação , Plasmídeos/genética , Estresse FisiológicoRESUMO
All New World Leishmania species can cause cutaneous lesions, while only Leishmania (Viannia) braziliensis has been associated with mucosal metastases. Multilocus enzyme electrophoresis (MLEE) is the optimal standard for species identification but is slow and costly. New methods for species identification are needed to ensure proper identification and therapy. The coding regions of four metabolic enzyme markers in the MLEE typing method: mannose phosphate isomerase (MPI), malate dehydrogenase (MDH), glucose-6-phosphate isomerase (GPI), and 6-phosphogluconate dehydrogenase (6PGD), were analysed from seven species of New World Leishmania isolated from patients with either cutaneous or mucosal lesions to identify specific genetic polymorphisms responsible for the phenotypic variations observed in the MLEE typing scheme. We identified species-specific polymorphisms and determined that a combination of sequencing of the mpi and 6pgd genes was sufficient to differentiate among seven closely related species of New World Leishmania and among isolates of L. braziliensis shown previously to have atypical MLEE patterns. When DNA isolated from 10 cutaneous lesion biopsies were evaluated, the sequence typing method was 100% concordant with the published MLEE/monoclonal antibody identification methods. The identification of species-specific polymorphisms can be used to design a DNA-based test with greater discriminatory power that requires shorter identification times. When the causative agent of the disease is L. braziliensis, this method ensures correct species identification, even when the agent is a genetic variant. Proper identification could facilitate adequate treatment, preventing the onset of the disfiguring mucosal form of the disease.
Assuntos
Loci Gênicos , Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Animais , América Central , DNA de Protozoário/análise , DNA de Protozoário/genética , Variação Genética , Genoma de Protozoário , Glucose-6-Fosfato Isomerase/análise , Glucose-6-Fosfato Isomerase/genética , Humanos , Leishmania/enzimologia , Leishmaniose Cutânea/enzimologia , Leishmaniose Cutânea/parasitologia , Malato Desidrogenase/análise , Malato Desidrogenase/genética , Manose-6-Fosfato Isomerase/análise , Manose-6-Fosfato Isomerase/genética , Dados de Sequência Molecular , Fosfogluconato Desidrogenase/análise , Fosfogluconato Desidrogenase/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , América do Sul , Especificidade da EspécieRESUMO
A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.
Assuntos
Citrus sinensis/genética , Manose-6-Fosfato Isomerase/genética , Manose/farmacologia , Plantas Geneticamente Modificadas/genética , Citrus sinensis/efeitos dos fármacos , Citrus sinensis/crescimento & desenvolvimento , Técnicas de Cultura , Frutose/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos/genética , Manose-6-Fosfato Isomerase/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sacarose/farmacologiaRESUMO
Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates. Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa. To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype. Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose. The mutants are unable to synthesize phosphoglycan repeats [-6-Gal beta 1-4Man alpha 1-PO(4)-] and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose. The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development. L. mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function.
Assuntos
Glicoconjugados/biossíntese , Leishmania mexicana/metabolismo , Manose-6-Fosfato Isomerase/fisiologia , Animais , Sequência de Bases , Cromatografia em Camada Fina , Leishmania mexicana/patogenicidade , Macrófagos/parasitologia , Manose/metabolismo , Manose/farmacologia , Manose-6-Fosfato Isomerase/química , Manose-6-Fosfato Isomerase/genética , Dados de Sequência Molecular , Polissacarídeos/biossíntese , Swainsonina/farmacologia , VirulênciaRESUMO
Azotobacter vinelandii forms desiccation-resistant cysts which contain a high proportion of the exopolysaccharide alginate in their envelope. We have previously shown that the A. vinelandii alginate biosynthetic genes algA and algL are transcribed from a promoter located somewhere upstream of algL. In this study we sequenced the A. vinelandii algX, algL, algV, algI and algF genes located between algG and algA. We carried out primer extension analysis of the algG, algX and algL genes and detected transcription start sites upstream algG but not upstream algX or algL, implying that algG and algX form part of the previously identified algL-A operon. A promoter upstream algA was also detected; however, transcription of algA exclusively from this promoter is not sufficient for the AlgA levels required for alginate production. An algF mutant (AJ34) was constructed by insertion of the Omega-tetracycline cassette in the non-polar orientation. As expected, AJ34 produced unacetylated alginate. Viability of 35day old cysts formed by strain AJ34, but not of those formed by the wild type, was reduced, indicating that acetylation of alginate plays a role in cyst resistance to desiccation.