Purification and Characterization of an Extracellular Protease from
            <i>Xenorhabdus nematophila</i>
            Involved in Insect Immunosuppression

Caldas, Cristina; Cherqui, Anas; Pereira, Aluisio José; Simões, Nélson

doi:10.1128/aem.68.3.1297-1304.2002

Cited by 99 publications

(72 citation statements)

References 42 publications

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“…luminescens secretes many enzymes that contribute to insect death and result in bioconversion of the insect cadaver 29 . In X. nematophila a zinc metalloprotease, PrtA, is involved in the immunosuppression of the insect 30 . In P. luminescens, the protein is encoded by the operon prtA-inh-prtBCD and is secreted by a type I secretory system 31 .…”

Section: Secreted Proteinsmentioning

confidence: 99%

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens

et al. 2003

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Photorhabdus luminescens is an enterobacterium that is symbiotic with soil entomopathogenic nematodes and pathogenic to a wide range of insects. P. luminescens promotes its own transmission among susceptible insect populations using its nematode host as vector 1 . Its life cycle comprises a symbiotic stage in the nematode's gut and a virulent stage in the insect larvae, which it kills through toxemia and septicemia. After the nematode attacks a prey insect and P. luminescens is released, the bacterium produces a wide variety of virulence factors ensuring rapid insect killing. Bioconversion of the insect cadaver by exoenzymes produced by the bacteria allows the bacteria to multiply and the nematode to reproduce. During this process P. luminescens produces antibiotics to prevent invasion of the insect cadaver by bacterial or fungal competitors. Finally, elimination of competitors allows P. luminescens and the nematode to reassociate specifically before leaving the insect cadaver 2,3 .To better understand this complex life style, we determined the genome sequence of P. luminescens subspecies laumondii strain TT01 4 , a symbiont of the nematode Heterorhabditis bacteriophora isolated on Trinidad and Tobago. RESULTS General featuresStrain TT01 possesses a single circular chromosome of 5,688,987 bp with an average GC content of 42.8%. No plasmid replicon was found.A total of 4,839 protein-coding genes, including 157 pseudogenes, seven complete sets (23S, 5S and 16S) of ribosomal RNA operons and 85 tRNA genes, were predicted ( Fig. 1; Supplementary Table 1 online). Toxins against insectsMore toxin genes were predicted in the P. luminescens genome than in any other bacterial genome sequenced yet. A large number of these toxins may be involved in the killing of a wide variety of insects. Some may act synergistically or use redundancy for 'overkill' 5 , ensuring a quick death of the host. In addition, some may kill insects by interfering with their development. In the TT01 genome, two paralogs, plu4092 and plu4436, encode proteins similar to juvenile hormone esterases (JHEs) of the insect Leptinotarsa decemlineata 6 . Juvenile hormone maintains the insect in a larval state. Its inactivation by JHE allows metamorphosis to proceed. JHEs may be used to trigger the insect endocrine machinery at an inappropriate time and thus represents a promising approach for insect control 7 . These genes are located downstream of highly related orphan genes (plu4093 and plu4437), suggesting a locus duplication.The toxicity of the proteins encoded by these two loci was verified experimentally. Two Escherichia coli clones, containing the recombinant BAC1A02 and BAC8C11, were shown to be toxic toward insects. BAC1A02, which contains the locus plu4093-plu4092, exhibited substantial oral toxicity toward three mosquito species, Aedes aegypti,

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Section: Secreted Proteinsmentioning

confidence: 99%

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens

et al. 2003

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“…The optimum pH of KB76 was 7.4, which is closely similar to that of Xenorhabdus nematophila protease (Caldas et al 2002). The significant decrease in the enzyme activity at lower pHs may be attributed to the pI of the protein (5.5) where enzyme precipitation occurs.…”

Section: Discussionmentioning

confidence: 57%

Purification of toxic protease from Brevibacterium otitidis KB76 with both metal and hydrosulfuryl at the active site

Kotb¹,

Abouel-Hawa²,

Tohamy³

et al. 2013

Biologia

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An extracellular toxic protease, KB76 from Brevibacterium otitidis was successfully purified to 31.3-fold by anion-exchange and gel filtration chromatography. The molecular mass was determined to be 47 kDa using SDS-PAGE. The optimum temperature and pH of the protease were 7.4 and 40• C, respectively. Ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride inhibited the activity of the enzyme but soybean trypsin inhibitor and aprotinin had no obvious inhibition, which suggested the presence of both metal and hydrosulfuryl at or near the active site. Additionally, the isoelectric point of this protein was 5.5 ± 0.2. Its apparent Km and Vmax for the synthetic substrate N-succinyl-Lphenylalanine p-nitroanilide were 2.41 mM and 21.74 µM/min, respectively. Further, studying the lethality of the protease on mice by intraperitoneal injection, it exhibited 48-h LD50 value of 9.6 mg/kg body weight. Gross and electron microscopic study in mice revealed that purified protease was capable of eliciting a variety of tissue responses resulted in liver necrosis. In conclusion, this protease produced by B. otitidis represents a potential toxic agent.

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“…4). An interesting difference in enzyme activity compared to the Protease II secreted by Xenorhabdus nematophila (Caldas et al, 2002), also an RTX zinc metalloprotease, is that the range of artificial substrates employed by the latter authors were not cleaved by PrtA. To date, we have not identified a cleavable colorimetric artificial substrate permitting a Michaelis-Menten analysis of protease activity.…”

Section: Protease Purification and Activitymentioning

confidence: 88%

“…The apparent molecular mass and zinc dependence of PrtA suggest that this enzyme is of the same family of metzincin metalloendopeptidases belonging to the RTX family. Interestingly, K122 PrtA was inhibited by EDTA with a 50 % inhibition of approximately 70 mM, being considerably more sensitive than the X. nematophilus protease II, with 43 % inhibition at 20 mM EDTA (Caldas et al, 2002), despite similar quantities of protease being tested.…”

Section: Inhibition Of the Prta Proteasementioning

confidence: 99%

Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus

et al. 2003

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Proteases play a key role in the interaction between pathogens and their hosts. The bacterial entomopathogen Photorhabdus lives in symbiosis with nematodes that invade insects. Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect. Here they secrete factors which kill the host and also convert the host tissues into food for the replicating bacteria and nematodes. One of the secreted proteins is PrtA, which is shown here to be a repeats-in-toxin (RTX) alkaline zinc metalloprotease. PrtA has high affinity for artificial substrates such as casein and gelatin and can be inhibited by zinc metalloprotease inhibitors. The metalloprotease also shows a calcium-and temperature-dependent autolysis. The prtA gene carries the characteristic RTX repeated motifs and predicts high similarity to proteases from Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. The prtA gene resides in a locus encoding both the protease ABC transporter (prtBCD) and an intervening ORF encoding a protease inhibitor (inh). PrtA activity is detectable 24 h after artificial bacterial infection of an insect, suggesting that the protease may play a key role in degrading insect tissues rather than in overcoming the insect immune system. Purified PrtA also shows cytotoxicity to mammalian cell cultures, supporting its proposed role in bioconversion of the insect cadaver into food for bacterial and nematode development.

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Purification and Characterization of an Extracellular Protease from Xenorhabdus nematophila Involved in Insect Immunosuppression

Cited by 99 publications

References 42 publications

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens

Purification of toxic protease from Brevibacterium otitidis KB76 with both metal and hydrosulfuryl at the active site

Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus

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