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.

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.

The Gram-negative bacterium Photorhabdus luminescens is a pathogen of insects. It is able to secrete a variety of toxins and effectors against its host in order to escape its immune defences. The model insect Manduca sexta is able to mount a variety of humoral and cellular responses against pathogen attack. Ultimately these prove ineffective against P. luminescens. The pre-treatment of M. sexta with Escherichia coli provides protection against the pathogenesis of P. luminescens. Here, I use RNA interference and Fluorescence-assisted cell sorting techniques to investigate interactions between pathogen and host to further elucidate the roles of various host factors in mounting the immune response. I also investigate the nutrient requirements of the bacteria for pathogenesis. I show data that peptidoglycan recognition protein (PGRP) is essential for the up-regulation of antimicrobial peptides, an important immune defence. I also show that P. luminescens has a requirement for two types of iron during pathogenesis of M. sexta. And lastly I show that P. luminescens is able to avoid phagocytosis, another important immune defence.