Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus.
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.
Bowen
David J
DJ
Department of Entomology, University of Wisconsin-Madison, Madison, USA.
Rocheleau
Thomas A
TA
Department of Entomology, University of Wisconsin-Madison, Madison, USA.
Grutzmacher
Cathy K
CK
Department of Entomology, University of Wisconsin-Madison, Madison, USA.
Meslet
Laurence
L
Institut de Génétique et Microbiologie, CNRS UMR 8621, Bâtiment 360, Université Paris XI, 91405 Orsay Cedex, France.
Valens
Michelle
M
Institut de Génétique et Microbiologie, CNRS UMR 8621, Bâtiment 360, Université Paris XI, 91405 Orsay Cedex, France.
Marble
Daniel
D
Department of Entomology, University of Wisconsin-Madison, Madison, USA.
Dowling
Andrea
A
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
Ffrench-Constant
Richard
R
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
Blight
Mark A
MA
Institut de Génétique et Microbiologie, CNRS UMR 8621, Bâtiment 360, Université Paris XI, 91405 Orsay Cedex, France.
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006790:Host-Parasite Interactions; D007313:Insecta / Q000469:parasitology; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009348:Nematoda / Q000382:microbiology; D009876:Operon; D020637:Photorhabdus / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C079508:PrtA metalloprotease