Serratia marcescens Outer Membrane Vesicles rapidly paralyze Drosophila melanogaster through triggering apoptosis in the nervous system

Bechara Sina Rahme
Roberto E Bruna
Marion Draheim
Chuping Cai
Maria Victoria Molino
Yaotang Wu
Miriam Wennida Yamba
Gisela Di Venanzio
Matthieu Lestradet
Eleonora García Véscovi author has email address
Dominique Ferrandon author has email address

Université de Strasbourg, CNRS, M3I UPR 9022 du CNRS, Strasbourg, France
Instituto de Biología Molecular y Cellular de Rosario, Consejo Nacional de Investigaciones Cientificas y Tecnológicas, and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
Sino-French Hoffmann Institute, Guangzhou Medical University, Guangzhou, China
Department of Molecular Microbiology, Washington University School of Medicine, St Louis, United States

https://doi.org/10.7554/eLife.108709.1

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Figures and data

The IMD, but not the Toll pathway, is implicated in the host defense against S. marcescens OMVs.
(A) Survival test of w [A5001] flies after the injection of different doses of purified S. marcescens OMVs. (B, D) Survival test of key^-/- (B) and MyD88^-/- (D) mutant flies to the injection of either 0.1 ng/μL of OMVs or HEPES buffer in comparison to wild-type flies (w [A5001]). Each graph represents one out of three independent experiments that yielded similar results. Error bars represent the standard error of the mean. Statistical tests were performed using the Logrank test. (C, E) RT-qPCR analysis of the Diptericin (C) and Drosomycin (E) expression in w [A5001] flies in response to OMV or HEPES buffer injection. Each dot represents the relative expression of the gene to Rp49 (RpL32) of five adult females. Each graph represents the pooled data from three independent experiments. The middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Statistical tests were performed using the Mann-Whitney test.

Phagocytes and Hayan promote the pathogenicity of S. marcescens OMVs, while PPOs play a protective role against OMVs.
(A) Survival test of “hemoless” flies (hmlG4>UAS-rpr, UAS-hid) to the injection of either 0.1 ng/μL of OMVs or HEPES buffer in comparison to control flies (hmlG4>yw). (B) Survival test of phagocytosis-impaired flies injected with latex beads (Lxb) 24h prior to the injection of either 0.1 ng/μL of OMVs or HEPES buffer in comparison to flies that had been previously injected with PBS as a control. (C-D) Survival test of Hayan¹ (C) and PPO1, PPO2 and PPO1-PPO2 double mutants (PPO12) (D) flies to the injection of either 0.1 ng/μL of OMVs or HEPES buffer in comparison to wild-type flies (w¹¹¹⁸). (A-D) Each graph represents one out of three independent experiments that yielded similar results. Error bars represent the standard error of the mean. Statistical tests were performed using Logrank.

Mitochondrial ROS play a detrimental role in the stress response against S. marcescens OMVs.
(A) Relative H₂O₂ measurements on single whole flies injected with 0.1 ng/μL of OMVs in comparison to HEPES injection. (B) Survival test of flies co-injected with 20 mM of Vitamin C and either 0.1 ng/μL of OMVs or HEPES in comparison to OMV only-injected flies. The yellow curve corresponds to OMVs that have been exposed to vitamin C for a limited period before its removal by filtration, prior to subsequent injection. (C) Survival test of NOS-deficient flies to OMV injection in comparison to cantonS control flies. (D) Survival test of DuOx- and Nox-impaired flies (ubiG4G80>UAS-DuOx^RNAi and ubiG4G80>UAS-NOX^RNAi) to the injection of OMVs in comparison to control flies (ubiG4G80>mcherry^RNAi). (E) Survival test of Jafrac-deficient flies (ubiG4>UAS-jaf2^RNAi) to the injection of 0.1 ng/μL of OMVs in comparison to control flies (ubiG4>mcherry^RNAi). (F) Survival test of SOD-overexpressing flies (ubiG4>UAS-SOD1 and ubiG4>UAS-SOD2) to the injection of 0.1 ng/μL of OMVs in comparison to control flies (ubiG4>UAS-GFP). (G) Survival test of flies co-injected with 20 μM of mitoTEMPO and either 0.1 ng/μL of OMVs or HEPES buffer in comparison to HEPES- or OMV-injected flies. The yellow curve corresponds to OMVs that have been exposed to mitoTEMPO for a limited period before its removal by filtration, prior to subsequent injection. (H) Relative H₂O₂ measurements on single whole fly co-injected with 20 μM of mitoTEMPO or vitaminC and either 0.1 ng/μL of OMVs or HEPES buffer in comparison to HEPES and OMV injection. (A-H) Each graph represents one out of three independent experiments that yielded similar results. (B-G) Statistical tests were performed using Logrank, and error bars represent the standard error of the mean. (A, H) The middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Statistical tests were performed using Mann-Whitney.

The neuronal JNK pathway promotes the pathogenicity of S. marcescens OMVs (A, C-D) RT-qPCR analysis of puckered.
(A), hid (C) and reaper (D) expression in w [A5001] flies in response to the OMV or HEPES buffer injection or in response to the co-injection of 20 mM of Vitamin C with either OMV or HEPES at 0.5, 1, 1.5 and 2 h after injection. Each dot represents the relative expression of the gene to Rp49 (RpL32) of 5 adult females. The expression of each gene for each condition is relative to its expression in the HEPES-injected flies. Each graph represents the pooled data from two independent experiments. The middle bar of the box plots represents the median, and the upper and lower limits of boxes indicate the first and third quartiles, respectively; the whiskers define the minima and maxima. Statistical tests were performed using Mann-Whitney. (B) Survival test of flies in which JNK expression has been silenced by RNAi specifically in neurons (elavG4>UAS-kayak^RNAi) to the injection of either 0.1 ng/μL of OMVs or HEPES buffer in comparison to control flies (elav-G4 and UAS-kayak^RNAi). (E) Survival test of flies in which SOD genes are overexpressed in neurons (elavG4>UAS-SOD1 and elavG4>UAS-SOD2) to the injection of either OMVs or HEPES buffer in comparison to control flies (elavG4>UAS-GFP). (F) Survival test of flies in which the baculovirus p35 gene is overexpressed in neurons (elavG4>UAS-p35) to the injection of OMVs in comparison to control flies (elavG4>UAS-GFP). (B, E & F) Each graph represents one out of three independent experiments that yielded similar results. Statistical tests were performed using Logrank, and error bars represent the standard error of the mean.

The virulence of S. marcescens OMVs is mediated by the PrtA metalloprotease.
(A) Survival test of w [A5001] flies after the injection of 0.1 ng/μL of OMVs purified from either WT (WT OMVs) or prtA mutant (prtA^- OMVs) S. marcescens. (B) Survival test of w [A5001] flies after the injection of different doses of OMVs purified from prtA mutant S. marcescens. (C) Survival test of w [A5001] flies after the injection of 0.1 ng/μL of OMVs or 0.025 ng/μL of purified PrtA protease from WT S. marcescens. (D) Survival test of w [A5001] flies after the co-injection of 0.1 ng/μL of prtA mutant OMVs and 0.025 ng/μL of purified PrtA (prtA^- OMVs complementation) in comparison to flies injected with 0.1 ng/μL of WT OMVs. (E) Survival test of kenny, eater and Hayan mutants to the injection of 0.27 ng/μL of purified PrtA in comparison to HEPES injection and w [A5001] control flies. (F) Survival test of w [A5001] flies after the injection of prtA mutant S. marcescens bacteria in comparison to the injection of WT RM66262 bacteria. (A-F) Each graph represents one out of three independent experiments that yielded similar results. Statistical tests were performed using Logrank, and error bars represent the standard error.

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