A leishmaniose, doença parasitária vetoriada causada por protozoários do gênero Leishmania, é uma das principais doenças tropicais negligenciadas do mundo. Os medicamentos atualmente disponíveis para o tratamento das leishmanioses são insatisfatórios, principalmente devido à baixa efetividade dos mesmos, surgimento de resistência do parasito ou reações adversas graves apresentadas pelos pacientes. Nas últimas décadas, tem havido um interesse renovado em produtos naturais derivados de micro-organismos como fonte para a concepção de novas drogas. As bactérias entomopatogênicas Xenorhabdus nematophila e Photorhabdus luminescens produzem grande número de metabólitos secundários, muitos deles têm efeitos tóxicos específicos sobre as células eucarióticas. O objetivo deste trabalho foi avaliar a atividade leishmanicida de sobrenadantes de culturas destas bactérias. Os testes in vitro foram realizados sobre formas promastigotas e amastigotas de Leishmania amazonensis e incluíram o efeito citotóxico dos sobrenadantes sobre macrófagos. Ambos os sobrenadantes de culturas de P. luminescens e X. nematophila mostraram atividade leishmanicida significativa contra as formas promastigotas de L. amazonensis (valores de CI50 de 7,5 % e 0,63 % (v/v), respectivamente). O sobrenadante de cultura de X. nematophila foi o mais efetivo e o mais estável ao calor. Além disso, ambos os sobrenadantes de culturas continham pequenas moléculas que estimularam a atividade leishmanicida de macrófagos por um mecanismo independente de óxido nítrico. Estes resultados revelaram que estas bactérias entomopatogênicas são fontes potenciais para a concepção de novos medicamentos contra a leishmaniose. / Leishmaniasis, a vector-borne parasitic disease caused by protozoa of the genus Leishmania, is one of the main neglected tropical diseases in the world. The drugs currently available for the treatment are unsatisfactory, mainly due to their low effectiveness, parasite resistance emergence or serious adverse reactions presented by the patients. In recent decades, there has been a renewed interest in natural products derived from microorganisms as a source for the design of new drugs. The Entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus luminescens produce a large number of secondary metabolites, many of them have specific toxic effects on eukaryotic cells. The objective of this study was to evaluate the leishmanicidal activity of these bacteria culture supernatants. In vitro tests were performed on promastigote and amastigote forms of L. amazonensis and included the cytotoxic effect of the supernatants on macrophages. Both supernatants from P. luminescens and X. nematophila cultures showed significant leishmanicidal activity against promastigotes forms of L. amazonensis (IC50 values of 7.5% and 0.63 % (v/v), respectively). The supernatant from X. nematophila was the most effective and more heat-stable. Furthermore, both culture supernatants contained small molecules that stimulated the leishmanicidal activity of macrophages by a mechanism independent of nitric oxide. These results revealed that these entomopathogenic bacteria are potential sources for the development of new drugs against leishmaniasis.

Photorhabdus are motile Gram-negative bacteria that have a mutualistic association with entomopathogenic Heterorhabditis nematodes. Nematodes vector the bacteria from one insect host to another, while the bacterial symbiont produces toxins and secondary metabolites that kill that the insect host. In this study, we characterize the bacterial symbiont of Heterorhabditis sonorensis, recently discovered in the Sonoran desert. Biochemical and molecular methods including sequence data from five genes: 16s rDNA, gyrB, recA, gltX, dnaN were considered. Evolutionary relationships of this new Photorhabdus subsp. were inferred considering maximum parsimony and Bayesian analyses. We also surveyed for secondary metabolites (SM) produced by this microorganism, considering HPLC and mass spectrometry analyses. SM crude extracts showed activity against the corn ear worm Helicoverpa zea, the root-knot nematode (Meloidogyne incognita), the bacterium Pseudomonas syringae, and the fungus Fusarium oxysporum; and were more toxic that those produced by related species. Results from these studies showed that Photorhabdus l. sonorensis' secondary metabolites have potent antagonistic activity against these plant pathogens.

Photorhabdus luminescens MN7 é uma enterobactéria associada a seus simbionte, nematoides da espécie Heterorhabditis baujardi LPP7, coletada em Monte Negro (RO), Brasil. As bactérias do gênero Algumas linhagens de P. luminescens possuem no seu citoplasma dois cristais proteicos compostos das CIPs (Crystal Inclusion Proteins A e B). Na fase estacionária de crescimento esses cristais são até 40% do total de proteínas na célula. Fizemos uma estratégia para clonar e expressar a ORF completa dos genes que codificam CipA, CipB e CipB2 de MN7. Os três genes foram amplificados e subclonados, mas apenas CipA foi expressa em Escherichia coli. A proteína recombinante foi purificada em coluna de Níquel N2+ e utilizada para inóculo em camundongos Balb/c. CipB foi clonada no plasmídeo de expressão, mas não foi expressa quando induzida. CipB2 foi amplificada, mas não foi clonada no plasmídeo de expressão. Também foi amplificado e subclonado o fragmento de miniSOG (mini Singlet Oxygen Generator), para ser ligado aos genes das CIPs que foram clonados para ensaios de fluorescência. / Photorhabdus luminescens MN7 is an enterobacterium associated with its symbiont, nematodes of the Heterorhabditis baujardi LPP7 species, collected in Monte Negro (RO), Brazil. Bacteria of the genus of P. luminescens lineages do not have their cytoplasm two protein crystals composed of CIPs (Crystal Inclusion Proteins A and B). In the stationary phase of growth of the crystals are up to 40% of the total proteins in the cell. We have devised a strategy to clone and express a complete ORF of the genes encoding MN7 CipA, CipB and CipB2. All three genes were amplified and subcloned, but only CipA was expressed in Escherichia coli. The recombinant protein was purified on a Nickel N2+ column and used for inoculum in Balb / c mice. CipB was cloned without expression plasmid, but was not expressed when induced. CipB2 was amplified, but was not cloned without expression plasmid. The miniSOG (mini Singel Oxygen Generator) fragment was also amplified and subcloned to be linked to the genes of the CIPs that were so cloned for fluorescence assays.

Entomopathogenic nematodes (EPNs) (genera Heterorhabditis and Steinernema) kill their invertebrate hosts with the aid of a mutualistic bacterium. The bacteria (Xenorhabdus spp. for steinernematids and Photorhabdus spp. for heterorhabditids) are primarily responsible for killing the host and providing the nematodes with nutrition and defense against secondary invaders. Photorhabdus is a Gram-negative bacterium in the Enterobacteriaceae family with high virulence towards their insect hosts. To achieve high mortality rates Photorhabdus produces a variety of virulence factors such as toxins, lipases, proteases, secretion systems, and fimbriae. EPNs are amenable to laboratory rearing and mass production for biocontrol applications against insects using in vivo or in vitro methods; however, in vitro liquid culture is considered to be the most efficient. In this method the symbiotic bacteria are cultured prior to the addition of their partner EPN. This can leave the bacteria susceptible to a number of problems such as genetic drift and inadvertent selection. Regardless of the culture method the symbiotic bacteria exhibit trait deterioration or changes due to laboratory rearing. This project had three primary aims: 1) investigate the role of nutrition in trait deterioration, 2) examine virulence evolution using a phylogenetic context, and 3) identify genes that are necessary for survival and virulence inside the insect host. Prior to studying these objectives we first determined the optimal conditions for growing and counting viable cells of Photorhabdus. We discovered that growth is enhanced by the addition of pyruvate to growth media. To determine the role of nutrition in trait deterioration we repeatedly sub-cultured Photorhabdus in three different media types. Throughout this study we found that, in contrast to previous studies, trait deterioration does not always happen and the environment influences trait deterioration. Furthermore, based on our phylogenetic studies we found that Photorhabdus spp. are evolving to an increase in insect virulence. Lastly, using Tn-seq we determined a list of 84 genes that are needed for efficient virulence inside the insect host and provide suggestions for ongoing research efforts.

Photorhabdus é um gênero de enterobactérias simbiontes de Heterorhabditis, um gênero de nematoides entomopatogênicos. Dentre as enzimas secretadas por P. luminescens TTO1, destaca-se PrtA, uma metaloprotease que pertence a subfamília das serralisinas. PrtS, uma protease capaz de induzir uma forte resposta de melanização no inseto, foi identificada em P. temperata Az29 mas não em P. luminescens TTO1. Neste trabalho foram detectadas e caracterizadas bioquimicamente algumas proteases secretadas pelo isolado MN7 de P. luminescens luminescens. Foram detectadas duas atividades mais proeminentes: uma de 50 kDa, e outra de 38 kDa. Com o sequenciamento do genoma desta bactéria, pudemos confirmar a presença no genoma de genes codificando proteínas de alta identidade com as descritas PrtA e PrtS, de massas similares às detectadas nas nossas zimografias. As proteases são inibidas por inibidores específicos de metaloproteases. P. luminescens MN7 apresenta secreção, portanto, de uma protease já descrita algumas vezes, e de outra presente apenas em P. temperata Az29. / Photorhabdus is a genus of Enterobacteriaceae, symbionts of Heterorhabditis, a genus of entomopathogenic nematodes. Among the enzymes secreted by P. luminescens TTO1 stands out PrtA, a metalloprotease that belongs to the subfamily of serralysins. PrtS, a protease capable of inducing a strong melanotic response from the insect, was identified in P. temperata Az29 but not in P. luminescens TTO1. In this work were detected and characterized biochemically few isolated proteases secreted by P. luminescens luminescens MN7. Two most prominent activities were detected: one with 50 kDa and one with 38 kDa. With the sequencing of the genome of MN7, we could confirm the presence in the genome of genes encoding proteins with high identity to PrtA and PrtS already described, with similar masses to those detected in our zimographies. These proteases are inhibited by specific inhibitors of metalloproteases. P. luminescens MN7 secretes a protease already described a few times, and other present only in P. temperata Az29.

Photorhabdus é um gênero de enterobactérias simbiontes de Heterorhabditis, um gênero de nematoides entomopatogênicos. Dentre as enzimas secretadas por P. luminescens TTO1, destaca-se PrtA, uma metaloprotease que pertence a subfamília das serralisinas. PrtS, uma protease capaz de induzir uma forte resposta de melanização no inseto, foi identificada em P. temperata Az29 mas não em P. luminescens TTO1. Neste trabalho foram detectadas e caracterizadas bioquimicamente algumas proteases secretadas pelo isolado MN7 de P. luminescens luminescens. Foram detectadas duas atividades mais proeminentes: uma de 50 kDa, e outra de 38 kDa. Com o sequenciamento do genoma desta bactéria, pudemos confirmar a presença no genoma de genes codificando proteínas de alta identidade com as descritas PrtA e PrtS, de massas similares às detectadas nas nossas zimografias. As proteases são inibidas por inibidores específicos de metaloproteases. P. luminescens MN7 apresenta secreção, portanto, de uma protease já descrita algumas vezes, e de outra presente apenas em P. temperata Az29. / Photorhabdus is a genus of Enterobacteriaceae, symbionts of Heterorhabditis, a genus of entomopathogenic nematodes. Among the enzymes secreted by P. luminescens TTO1 stands out PrtA, a metalloprotease that belongs to the subfamily of serralysins. PrtS, a protease capable of inducing a strong melanotic response from the insect, was identified in P. temperata Az29 but not in P. luminescens TTO1. In this work were detected and characterized biochemically few isolated proteases secreted by P. luminescens luminescens MN7. Two most prominent activities were detected: one with 50 kDa and one with 38 kDa. With the sequencing of the genome of MN7, we could confirm the presence in the genome of genes encoding proteins with high identity to PrtA and PrtS already described, with similar masses to those detected in our zimographies. These proteases are inhibited by specific inhibitors of metalloproteases. P. luminescens MN7 secretes a protease already described a few times, and other present only in P. temperata Az29.

In this study, we assessed pirAB toxin transcription in Photorhabdus luminescens laumondii (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: Galleria mellonella and Manduca sexta. Three portals of entry were utilized for the forced infection assays: (a) integument; (b) the digestive route (via mouth and anus); and (c) the tracheal route (via spiracles). We also assessed plu4093-2 transcription during the course of a natural infection; this is when the bacteria are delivered by Heterorhabditis bacteriophora nematodes. Transcript abundance in G. mellonella was higher than in M. sexta at two of the observed time points: 15 and 18 h. Expression of pirAB plu4093-2 reached above endogenous control levels at 22 h in G. mellonella but not in M. sexta. Overall, pirAB plu4093-2 transcripts were not as highly expressed in M. sexta as in G. mellonella, from 15 to 22 h. This is the first study to directly compare pirAB plu4093-2 toxin transcript production considering different portals of entry.

Os fármacos atualmente disponíveis para o tratamento da Doença de Chagas e leishmaniose possuem eficácia insatisfatória, principalmente devido à resistência parasitária e reações adversas severas. Duas entomobactérias, Photorhabdus luminescens e Xenorhabdus nematophila, produzem uma variedade de metabólicos secundários tóxicos a células eucarióticas. Diante disto, testou-se a toxicidade de metabólitos secretados por P. luminescens e X. nematophila sobre Leishmania amazonensis e Trypanosoma cruzi, in vitro. Os meios condicionados de ambas bactérias mostraram significativo efeito parasiticida de forma concentração e tempo-dependente (L. amazonensis: IC50 P. luminescens = 21,80 μg/mL e X. nematophila = 0,33 mg/mL; T. cruzi: IC50 P. luminescens = 1,0 mg/mL e IC50 X. nematophila = 0,34 mg/mL) e apresentaram alta seletividade ao parasito (L. amazonensis: SIP. luminescens = 3.92 e SIX. nematophila = 19,85; T. cruzi: SIP. luminescens = 7,23 e SIX. nematophila = 14.17 para promastigotas e tripomastigotas, respectivamente). Além disso, os metabolitos estimulam a atividade de macrófagos contra amastigotas por um mecanismo independente de óxido nítrico. Com relação à caracterização dos compostos antiparasitários, sugere-se que moléculas com diferentes características atuem sobre cada parasito. P. luminescens secreta uma molécula leishmanicida de natureza peptídica menor que 3 kDa e uma molécula tripanocida de natureza não proteica, resistente a aquecimento a 100 ºC. X. nematophila produz uma molécula leishmanicida de polaridade inferior à tripanocida, uma vez que a atividade antiparasitária ficou em fases diferentes na extração com metanol. O mecanismo de ação de ambas bactérias sobre promastigotas parece estar relacionado à lesão mitocondrial, uma vez que ambas levaram à despolarização da membrana mitocondrial. X. nematophila, além disso, estimula a produção de ROS pelas formas promastigotas. A seletividade pelo parasito aliada a baixa citotoxicidade tornam estas bactérias promissoras fontes de compostos com potencial terapêutico contra leishmanioses e doença de Chagas. / Drugs currently available for Chagas disease and leishmaniasis have unsatisfactory efficacy, mainly due to parasitic resistance and severe adverse reactions. Two entomobacteria, Photorhabdus luminescens and Xenorhabdus nematophila, produce a variety of secondary metabolites toxic to eukaryotic cells. So, the toxicity of the metabolites secreted by Photorhabdus luminescens and Xenorhabdus nematophila were tested against Trypanosoma cruzi and Leishmania amazonensis. The mean values of both bacteria showed a significant concentration-dependent and time-dependent effect 14.17 (L. amazonensis: IC50P. luminescens = 21.80 μg / mL and IC50X. nematophila = 0.33 mg / mL, T. cruzi: IC50P. luminescens = 1,0 mg/mL and IC50X. nematophila = 0 , 34 mg / mL), and showed a high selectivity to the parasite (L. amazonensis: SIP. luminescens = 3,92 and SIX.nematophila = 19.85, T. cruzi: SIP. luminescens = 7.23 and SIX.nematophila = 14.17 for promastigotes and trypomastigotes, respectively). In addition, cultures stimulate the activity of macrophages against amastigotes by an independent mechanism of nitric oxide. Regarding the characterization of antiparasitic compounds, it is suggested that molecules with different characteristics act on each parasite. P. luminescens secretes a leishmanicidal peptide molecule lesser than 3 kDa and a trypanocidal molecule of non-protein nature, resistant to heating at 100 °C. X. nematophila produces a leishmanicidal molecule of lower polarity than trypanocidal, since antiparasitic activity was at different phases in methanol extraction. The mechanism of action of both bacteria on promastigotes seems to be related to the mitochondrial injury, since both led to the depolarization of the mitochondrial membrane. X. nematophila, furthermore, stimulates the production of ROS by the promastigote. Selectivity by the parasite coupled with low cytotoxicity makes these bacteria promising sources of compounds with therapeutic potential against leishmaniasis or Chagas' disease.

Os fármacos atualmente disponíveis para o tratamento da Doença de Chagas e leishmaniose possuem eficácia insatisfatória, principalmente devido à resistência parasitária e reações adversas severas. Duas entomobactérias, Photorhabdus luminescens e Xenorhabdus nematophila, produzem uma variedade de metabólicos secundários tóxicos a células eucarióticas. Diante disto, testou-se a toxicidade de metabólitos secretados por P. luminescens e X. nematophila sobre Leishmania amazonensis e Trypanosoma cruzi, in vitro. Os meios condicionados de ambas bactérias mostraram significativo efeito parasiticida de forma concentração e tempo-dependente (L. amazonensis: IC50 P. luminescens = 21,80 μg/mL e X. nematophila = 0,33 mg/mL; T. cruzi: IC50 P. luminescens = 1,0 mg/mL e IC50 X. nematophila = 0,34 mg/mL) e apresentaram alta seletividade ao parasito (L. amazonensis: SIP. luminescens = 3.92 e SIX. nematophila = 19,85; T. cruzi: SIP. luminescens = 7,23 e SIX. nematophila = 14.17 para promastigotas e tripomastigotas, respectivamente). Além disso, os metabolitos estimulam a atividade de macrófagos contra amastigotas por um mecanismo independente de óxido nítrico. Com relação à caracterização dos compostos antiparasitários, sugere-se que moléculas com diferentes características atuem sobre cada parasito. P. luminescens secreta uma molécula leishmanicida de natureza peptídica menor que 3 kDa e uma molécula tripanocida de natureza não proteica, resistente a aquecimento a 100 ºC. X. nematophila produz uma molécula leishmanicida de polaridade inferior à tripanocida, uma vez que a atividade antiparasitária ficou em fases diferentes na extração com metanol. O mecanismo de ação de ambas bactérias sobre promastigotas parece estar relacionado à lesão mitocondrial, uma vez que ambas levaram à despolarização da membrana mitocondrial. X. nematophila, além disso, estimula a produção de ROS pelas formas promastigotas. A seletividade pelo parasito aliada a baixa citotoxicidade tornam estas bactérias promissoras fontes de compostos com potencial terapêutico contra leishmanioses e doença de Chagas. / Drugs currently available for Chagas disease and leishmaniasis have unsatisfactory efficacy, mainly due to parasitic resistance and severe adverse reactions. Two entomobacteria, Photorhabdus luminescens and Xenorhabdus nematophila, produce a variety of secondary metabolites toxic to eukaryotic cells. So, the toxicity of the metabolites secreted by Photorhabdus luminescens and Xenorhabdus nematophila were tested against Trypanosoma cruzi and Leishmania amazonensis. The mean values of both bacteria showed a significant concentration-dependent and time-dependent effect 14.17 (L. amazonensis: IC50P. luminescens = 21.80 μg / mL and IC50X. nematophila = 0.33 mg / mL, T. cruzi: IC50P. luminescens = 1,0 mg/mL and IC50X. nematophila = 0 , 34 mg / mL), and showed a high selectivity to the parasite (L. amazonensis: SIP. luminescens = 3,92 and SIX.nematophila = 19.85, T. cruzi: SIP. luminescens = 7.23 and SIX.nematophila = 14.17 for promastigotes and trypomastigotes, respectively). In addition, cultures stimulate the activity of macrophages against amastigotes by an independent mechanism of nitric oxide. Regarding the characterization of antiparasitic compounds, it is suggested that molecules with different characteristics act on each parasite. P. luminescens secretes a leishmanicidal peptide molecule lesser than 3 kDa and a trypanocidal molecule of non-protein nature, resistant to heating at 100 °C. X. nematophila produces a leishmanicidal molecule of lower polarity than trypanocidal, since antiparasitic activity was at different phases in methanol extraction. The mechanism of action of both bacteria on promastigotes seems to be related to the mitochondrial injury, since both led to the depolarization of the mitochondrial membrane. X. nematophila, furthermore, stimulates the production of ROS by the promastigote. Selectivity by the parasite coupled with low cytotoxicity makes these bacteria promising sources of compounds with therapeutic potential against leishmaniasis or Chagas' disease.

Os fármacos atualmente disponíveis para o tratamento da Doença de Chagas e leishmaniose possuem eficácia insatisfatória, principalmente devido à resistência parasitária e reações adversas severas. Duas entomobactérias, Photorhabdus luminescens e Xenorhabdus nematophila, produzem uma variedade de metabólicos secundários tóxicos a células eucarióticas. Diante disto, testou-se a toxicidade de metabólitos secretados por P. luminescens e X. nematophila sobre Leishmania amazonensis e Trypanosoma cruzi, in vitro. Os meios condicionados de ambas bactérias mostraram significativo efeito parasiticida de forma concentração e tempo-dependente (L. amazonensis: IC50 P. luminescens = 21,80 μg/mL e X. nematophila = 0,33 mg/mL; T. cruzi: IC50 P. luminescens = 1,0 mg/mL e IC50 X. nematophila = 0,34 mg/mL) e apresentaram alta seletividade ao parasito (L. amazonensis: SIP. luminescens = 3.92 e SIX. nematophila = 19,85; T. cruzi: SIP. luminescens = 7,23 e SIX. nematophila = 14.17 para promastigotas e tripomastigotas, respectivamente). Além disso, os metabolitos estimulam a atividade de macrófagos contra amastigotas por um mecanismo independente de óxido nítrico. Com relação à caracterização dos compostos antiparasitários, sugere-se que moléculas com diferentes características atuem sobre cada parasito. P. luminescens secreta uma molécula leishmanicida de natureza peptídica menor que 3 kDa e uma molécula tripanocida de natureza não proteica, resistente a aquecimento a 100 ºC. X. nematophila produz uma molécula leishmanicida de polaridade inferior à tripanocida, uma vez que a atividade antiparasitária ficou em fases diferentes na extração com metanol. O mecanismo de ação de ambas bactérias sobre promastigotas parece estar relacionado à lesão mitocondrial, uma vez que ambas levaram à despolarização da membrana mitocondrial. X. nematophila, além disso, estimula a produção de ROS pelas formas promastigotas. A seletividade pelo parasito aliada a baixa citotoxicidade tornam estas bactérias promissoras fontes de compostos com potencial terapêutico contra leishmanioses e doença de Chagas. / Drugs currently available for Chagas disease and leishmaniasis have unsatisfactory efficacy, mainly due to parasitic resistance and severe adverse reactions. Two entomobacteria, Photorhabdus luminescens and Xenorhabdus nematophila, produce a variety of secondary metabolites toxic to eukaryotic cells. So, the toxicity of the metabolites secreted by Photorhabdus luminescens and Xenorhabdus nematophila were tested against Trypanosoma cruzi and Leishmania amazonensis. The mean values of both bacteria showed a significant concentration-dependent and time-dependent effect 14.17 (L. amazonensis: IC50P. luminescens = 21.80 μg / mL and IC50X. nematophila = 0.33 mg / mL, T. cruzi: IC50P. luminescens = 1,0 mg/mL and IC50X. nematophila = 0 , 34 mg / mL), and showed a high selectivity to the parasite (L. amazonensis: SIP. luminescens = 3,92 and SIX.nematophila = 19.85, T. cruzi: SIP. luminescens = 7.23 and SIX.nematophila = 14.17 for promastigotes and trypomastigotes, respectively). In addition, cultures stimulate the activity of macrophages against amastigotes by an independent mechanism of nitric oxide. Regarding the characterization of antiparasitic compounds, it is suggested that molecules with different characteristics act on each parasite. P. luminescens secretes a leishmanicidal peptide molecule lesser than 3 kDa and a trypanocidal molecule of non-protein nature, resistant to heating at 100 °C. X. nematophila produces a leishmanicidal molecule of lower polarity than trypanocidal, since antiparasitic activity was at different phases in methanol extraction. The mechanism of action of both bacteria on promastigotes seems to be related to the mitochondrial injury, since both led to the depolarization of the mitochondrial membrane. X. nematophila, furthermore, stimulates the production of ROS by the promastigote. Selectivity by the parasite coupled with low cytotoxicity makes these bacteria promising sources of compounds with therapeutic potential against leishmaniasis or Chagas' disease.