The Photorhabdus asymbiotica virulence cassettes deliver protein effectors directly into target eukaryotic cells
- University Hospital of Wales, United Kingdom
- Warwick University, United Kingdom
- Beijing Friendship Hospital, Capital Medical University, China
Figures
Figure 1 with 4 supplements
Genetic organisation of PVC operons.
(A) Similarity between PVCs and two diverse protein secretion systems, (i) the P. luteoviolacea mac gene cluster and (ii) The type-VI secretion system (T6SS) from Burkholderia mallei. Homologous …
Figure 1—figure supplement 1
PVC operons in relation to putative regions of horizontal gene transfer as identified by Alien Hunter.
(A) The PVCpnf and (B) the PVCcif operons are highlighted by the blue rectangle. Alien Hunter regions of HGT are designated by the features in tones of red. In red are the regions with the highest …
Figure 1—figure supplement 2
Boxplots of the mean GC content across 16 different pvc operons of Photorhabdus.
The GC was calculated for each of the 16 structural loci (clustered by annotated/predicted function and syntenic position in operon consistent with the nomenclature devised in this paper). GC …
Figure 1—figure supplement 3
Box plots of amino acid similarity across homologous protein sequences for these same 16 operons.
Amino acid sequences were clustered together as in (A), by annotation and syntenic position. Global Multiple Sequence Alignments were created with CLUSTAL Omega, using the default parameters (Gonnet …
Figure 1—figure supplement 4
A map of the model class I PaATCC43949 PVCpnf operon showing two effector genes in the payload region in red and a table, both colour matched to the description of subunit-structure relationship described in the recent CryoEM structure paper (Jiang et al., 2019).
Payload effectors are in red and ‘NR’ means subunit not resolved in the structure.
Figure 2 with 4 supplements
Examination of PVC operon expression using gfp reporter constructs in Photorhabdus.
(A) Confocal micrographs of showing in vitro (A1–A3) and in vivo (B1–B3) expression in PlTT01 of gfp transcription-translation reporter constructs of PaATCC43949 PVCpnf operon gene promoters. These …
Figure 2—figure supplement 1
A representative selection of images for four time points, for P. asymbiotica PB68.1 (PaPB68) harbouring.
(A) the PaPB68 PVCpnf pvc1 promoter fusion construct or (B) pAGAG negative control reporter plasmid with no promoter. For (A) quadruplicate images are displayed vertically as representative of the …
Figure 2—figure supplement 2
Quantification of population heterogeneity of PVC expression using image analysis of gfp expression.
(A) Time dependant swarm-plots of GFP intensity of individual cells in the population of P. asymbiotica. PB68.1 harbouring the PaPB68 PVCpnf pvc1 promoter fusion construct compared to the pAGAG …
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Figure 2—figure supplement 2—source data 1
Cell intensity counts used in image analysis quantification shown in Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.46259.011
Figure 2—figure supplement 3
A representative selection of images for four growth time points, for P. asymbiotica.
PB68.1 harbouring four different pvc-operon reporter constructs and the pAGAG negative control reporter plasmid containing no promoter. Wide field (left) and a magnified subsection (right) panels …
Figure 2—figure supplement 4
A representative selection of images for four growth time points, for P. luminescens.
TT01 harbouring four different pvc-operon reporter constructs and the pAGAG negative control reporter plasmid containing no promoter. Wide field (left) and a magnified subsection (right) panels are …
Figure 3
Confirmation of the PVCpnf-operon transcription in vitro and in vivo using RT-PCR.
(A) RT-PCR analysis of gene transcription of various genes of the PaATCC43949 PVCpnf operon over time in vitro at insect (28°C) and human (37°C) relevant temperatures and in vivo during Manduca sexta…
Figure 4
Pnf protein can only be detected in discharged or disrupted needle complexes using antibody based analysis.
(A) Representative images of immuno-gold stained transmission electron microscopy grids confirming the Pnf-payload toxin is associated with the needle complex. PVCpnf needle complexes (PVC-NC) were …
Figure 5
Visualisation of PVC needle complexes on the cell surface.
Cryo-SEM analysis of ex vivo hemocytes from 5th instar Manduca sexta that had been injected with (A) native or (B) heat inactivated enriched preparations of PaATCC43949 PVCpnf needle complexes …
Figure 6 with 1 supplement
Pnf needs to gain access to the host cell cytoplasm to induce F-actin formation and multi-nucleation in cultured HeLa cells.
Wild-type and inactive toxoid mutant Pnf protein was delivered topically using BioPORTER. Cell cytoskeleton is stained with TRITC-Phalloidin and the cell nuclei with DAPI. This gave rise to …
Figure 6—figure supplement 1
Ab initio structural prediction of the Pnf toxin and toxoid derivative.
Top. Predicted structural models of Pnf wild-type protein (Pa_Pnf) and the toxoid (Pa_Pnd_C190A) produced using Phyre2 with a 100% confidence score by comparison to an E. coli CNF1/2 family toxin …
Figure 7
Pnf transglutaminates and deamidates purified mammalian RhoGTPAses at Gln63 (RhoA) and Gln 61 (Rac1).
(A) For transglutamination assays a 2:1 molar ratio of small Rho GTPase to purified Pnf was incubated in transglutamination buffer in the presence of ethylediamine for 1 hr at 37°C. Note …
Additional files
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Supplementary file 1
Table of PCR primers.
Oligonuclotides used for the construction of pAGAG based reporter plasmids used in Figure 2—figure supplement 2, 3 and 4.
- https://doi.org/10.7554/eLife.46259.020
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Transparent reporting form
- https://doi.org/10.7554/eLife.46259.021
