A metalloprotease secreted by the insect pathogen Photorhabdus luminescens induces melanization

Abstract

Photorhabdus luminescens is a gram-negative insect pathogen that enters the hemocoel of infected hosts and produces a number of secreted proteins that promote colonization and subsequent death of the insect. In initial studies to determine the exact role of individual secreted proteins in insect pathogenesis, concentrated culture supernatants from various P. luminescens strains were injected into the tobacco hornworm Manduca sexta. Culture supernatants from P. luminescens TT01, the genome-sequenced strain, stimulated a rapid melanization reaction in M. sexta. Comparison of the profiles of secreted proteins from the various Photorhabdus strains revealed a single protein of approximately 37 kDa that was significantly overrepresented in the TT01 culture supernatant. This protein was purified by DEAE ion-exchange and Superdex 75 gel filtration chromatography and identified by matrix-assisted laser desorption ionization-time of flight analysis as the product of the TT01 gene plu1382 (NCBI accession number NC_005126); we refer to it here as PrtS. PrtS is a member of the M4 metalloprotease family. Injection of PrtS into larvae of M. sexta and Galleria mellonella and into adult Drosophila melanogaster and D. melanogaster melanization mutants (Bc) confirmed that the purified protein induced the melanization reaction. The prtS gene was transcribed by P. luminescens injected into M. sexta before death of the insect, suggesting that the protein was produced during infection. The exact function of this protease during infection is not clear. The bacteria might survive inside the insect despite the melanization process, or it might be that the bacterium is specifically activating melanization in an attempt to circumvent this innate immune response.

FIG. 1.

PrtS is found in abundance in Photorhabdus luminescens strain TT01 culture supernatant. Photorhabdus cultures were grown for 72 h at 30°C, and supernatant proteins were separated by 12% SDS-PAGE and stained with Coomassie blue. Lanes: A, P. asymbiotica (ATCC 43950); B, P. luminescens TT01; C, P. luminescens Hb; D, P. temperata C1; E, P. luminescens Hm. The asterisk indicates the position of PrtS.

FIG. 2.

PrtS stimulates melanization in M. sexta. (A) Coomassie blue-stained 12% SDS-polyacrylamide gel of purified PrtS (lane 1) and BSA (lane 2). (B) Photograph of M. sexta fifth-instar larvae at 71 h post-injection of 4.4 μg of PrtS (panel 1) and BSA (panel 2). The black pigment on the larva in panel 1 is an indication of melanization response.

FIG. 3.

Amino acid sequence of PrtS from Photorhabdus luminescens strain TT01 plu1382. The protein shown in Fig. 2 was sent for MALDI-TOF analysis. Peptides obtained from that analysis are underlined. Residues in bold are the characteristic HEXXH motif of M4 metalloproteases. The arrow denotes the last residue of the cleaved leader sequence.

FIG. 4.

rPrtS is active and can cleave rPrtS-E168A. rPrtS and rPrtS-E168A were incubated at room temperature in the presence (+) and absence (−) of the metalloprotease inhibitor 1,10-phenanthroline (8 mM) for 0 h (lanes A to E), 2 h (lanes F to J), and 18 h (lanes K to M). Lanes A, F, and K are 2.2 μg rPrtS-E168A; lanes B, D, G, and I are 2.2 μg rPrtS-E168A with 0.01 μg rPrtS; lanes C, E, H, J, L, and M are 2.2 μg rPrtS-E168A with 0.02 μg rPrtS; and lane N is rPrtS used as a size control.

FIG. 5.

rPrtS but not rPrtS-E168A induces melanization in Drosophila. (A) Wild-type fly pricked with 2.6 μg μl⁻¹ rPrtS-E168A. (B) Wild-type fly pricked with 2.6 μg μl⁻¹ rPrtS; the arrow points to the site of injection. (C) Untreated ebony fly as color control.

FIG. 6.

The prtS gene is transcribed during infection of M. sexta. (A) Growth of P. luminescens TT01 in M. sexta. M. sexta hemocoel was injected with either 10 CFU P. luminescens TT01 or 10,000 CFU E. coli VCS257. At indicated time points postinjection, 100 μl of hemolymph was collected from the horn of the insect and dilutions were made in PBS and plated to obtain colony counts. At 48 h postinjection with P. luminescens, the M. sexta died. (B) Detection of prtS transcript using RT-PCR. Hemolymph samples from M. sexta were collected as described above, total RNA was isolated, and RT-PCR was performed as described in Materials and Methods. Samples from three M. sexta insects per time point were examined. Hours postinjection are indicated above the horizontal lines. “0” represents hemolymph sample immediately after injection; − represents samples from uninfected controls.