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

Reviewer #1 (Public review):

Summary:

The work of Bechara Rahme and colleagues provides an explanation as to how bacterially infected flies eventually die. While widespread tissue and multiorgan damage are to be expected in the latest stages of a systemic infection, the mechanisms leading to the host's death remain unresolved. To this end, this work illustrates the role of PrtA, a metalloproteinase found within Outer Membrane Vesicles (OMVs) secreted by Serratia marcescens, in inducing neuronal apoptosis and paralysis before death. Another interesting aspect of the work is the compromise of blood blood-brain barrier (BBB) by OMVs. BBB is different between mammals and flies; however, it merits scientific attention.

Strengths:

The strength of evidence lies in a wealth of experiments involving disparate innate immune mechanisms that either contribute (Imd, PPO1/2, Nox, Duox, SOD2) or oppose (hemocytes and Hayan protease) host defense. Moreover, the role of neuronal JNK and apoptic signaling is shown to contribute to host death.

Genetics is supported by experiments using chemical treatments (Vitamin C and mito-TEMPO) as host-protecting antioxidants, and the biochemical purification and quantification of OMVs and the PrtA protease.

Weaknesses:

However, the reliance on non-isogenised flies to provide quantitative data is unsafe, and at this point, the strength of the evidenceis apparently incomplete. The mutant flies used for the genes Key, Myd88, Hayan, and Nos are doubtfully comparable to the control fly strains used in terms of the general genetic background. The latter is of utmost importance in assessing quantitative traits.

The general background difference between control and test flies is also an issue when using tissue-specific expression via GAL4/UAS, because the UAS lines used are only apparently but not truly isogenic to the w flies used as controls.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate the mechanisms underlying the virulence of OMVs using a Drosophila model. They reveal a complex interplay between host defenses and OMV pathogenicity. Although the study enhances our understanding of Drosophila innate immunity, additional evidence is needed to strengthen the conclusions.

Strengths:

(1) In Figure 1, Toll pathway mutants infected with OMVs displayed three distinct phenotypic outcomes: mildly enhanced resistance to OMV infection, a response similar to that of the control, or increased susceptibility. Therefore, in addition to Imd and Kenny mutants from the Imd pathway, further mutants, such as Relish and PGRP-LC, should be examined to assess whether the Imd pathway is involved in host defense against OMVs.

(2) Plasmatocytes clear particles via phagocytosis or endocytosis. However, flies lacking all hemocytes showed increased resistance to OMV challenge, raising the question of whether hemocytes actually aid the pathogen. To explore this hypothesis, the uptake of fluorescently tagged OMVs should be examined.

(3) Hayan cleaves PPO into active PO. However, Hayan and PPO mutants exhibit opposite phenotypes upon OMV injection, raising the question of whether OMV-induced pathogenesis is linked to melanization.

(4) Puckered mRNA levels were used as a read-out for JNK pathway activity. A transient induction of the JNK pathway was observed in head and thorax tissues. It would be beneficial if the authors could directly examine JNK activation in neuronal cells using immunostaining for pJNK.

(5) In Figure 4B, the kayak was knocked down using the pan-neuronal driver elav-Gal4. To confirm the specificity and validity of this observation, the experiment should be repeated using another neural-specific driver.

Weaknesses:

It is unclear how many Serratia marcescens cells a 69 nL injection of 0.1 ng/nL OMVs corresponds to.

Reviewer #3 (Public review):

Summary:

The authors investigate deficiencies in various immune responses, and also the prtA toxin's role in OMV toxicity. Some key interpretations are that the Imd pathway contributes to preventing OMV toxicity, but not Toll, and that Hayan and Eater somehow mediate OMV or PrtA toxicity. This descriptive effort is a solid set of experiments, although some experimental results may require further validation.

Strengths:

The breadth of experiments tests multiple immune parameters, providing a systematic effort that ensures a number of potentially relevant interactions can be recovered. Certain findings, such as the PrtA toxicity to flies, appear solid, and some interesting findings regarding Hayan and eater will be of interest to the fly immunity field.

Weaknesses:

It appears almost all results rely on the use of a single mutant representing the deletion of the gene. It's not clear if the mutations are always in the same genetic background, but this can be clarified. There are a couple of results that are confusing and may be internally contradicting, and should be additionally validated and clarified.

Author response:

We thank the reviewers and editors for the careful evaluation of our manuscript. Below, we provide a first refutation of some of the concerns expressed by reviewers.

Both reviewer 1 &3 underscore the importance of controlling for genetic backgrounds. This is actually an issue only for a limited part of the study and this criticism should not apply to major findings of this study, with some exceptions, as detailed below.

It is important to note that we have identified ourselves several of the mutant lines we have been using. For instance, key and MyD88 mutant alleles have been identified in the Exelixis transposon insertion collection that we have screened in collaboration with this firm (e.g., [3, 4, 5]). This resource has been generated in a isogenized w [A5001] strain[6], which we are using as matched control for these mutants (Figs 1B,D). Of note, while they share a common genetic background, the phenotypes of key and MyD88 are opposite in terms of sensitivity to OMV challenge. The imd^shadok null allele had been identified during our chemical mutagenesis screen with EMS in a yw cn bw background [5, 7, 8, 9], which was used as a control (FigS1A).

With respect to Hayan (Fig. 2C, Fig. S2C) and eater (Fig. S2A-B) mutants[10, 11, 12], we find a similarly strong phenotype with two independent mutants in distinct genetic backgrounds (actually three for Hayan, as we have not included in our original manuscript the Hayan^SK3allele generated in the Lemaitre laboratory in which OMVs displayed also impaired virulence). We have shown that the Hayan mutants do display the expected phenotype in terms of PPO cleavage (Fig. S2D). Please, also note that in Fig. S2C the two mutant alleles are tested in the same experiment: even though there is some variation between the w¹¹¹⁸ and the w[A5001] strains, the two mutants behave in a remarkably similar manner. As regards the role of the cellular response, we note that we obtained results similar to those obtained with eater mutants using genetic ablation of hemocytes (Fig. 2A) or by saturating the phagocytosis apparatus (Fig. 2B), a confirmation by two totally-independent approaches.

Of note, the observed eater and Hayan phenotypes are strong and not relatively small and thus unlikely to be due to the genetic background.

The PPO mutants have been isogenized in the w¹¹¹⁸ by the lab of Bruno Lemaitre[13, 14] and are also validated biochemically in Fig. S2D. These mutants have been extensively tested in the Lemaitre laboratory[13, 14, 15].

With respect to RNAi silencing driven ubiquitously or in specific tissues using the UAS-Gal4 system, we have mostly used transgenes from the Trip collection and have used as a control the mCherry RNAi provided by this resource[16]. As the RNAi transgenes have been generated in the same genetic background, it follows that independently of the driver used, the genetic background used in mCherry and genes-of-interest (Duox, Nox, Jafrac2) silenced flies is controlled for (Fig. 3D,E).

For UAS-Gal4-mediated overexpression of fly superoxide dismutase genes, we have used SOD1 and SOD2 transgenes that have both been generated by the same laboratory (Phillips laboratory, University of Guelph) presumably in the same genetic background. Using two distinct drivers we find a strongly enhanced susceptibility phenotype when using UAS-SOD2 but not UAS-SOD1 transgenes (Fig. 3F, Fig. 4E). Importantly, the former is associated with mitochondria whereas the other is expressed in the endoplasmic reticulum: we independently confirm this phenotype using the mitoTempo mitochondrial ROS inhibitor.

We shall thus address the criticism with NOS mutants, where genetic background control is indeed critical and for the UAS-kay RNAi line using a Trip line and its associated mCherry RNAi control transgene.

With respect to the Toll pathway mutants, we agree that some of the variability of the phenotypes may be due to the genetic background, especially as regards tube and pelle. The SPE and grass mutants have been retrieved in a screen performed by the group of Jean-Marc Reichhart in our Research Unit. They thus have been generated in the same genetic background, yet grass displays a mildly decreased virulence of injected OMVs whereas SPE mutants display an opposite phenotype (compare Fig. S1E to S1I; the survival experiment shave been performed in the same set of experiments and have been separated for clarity). We do not intend to analyze further the mutants of the Toll pathway as our data suggest that the canonical Toll pathway, likely activated through psh (Fig. S1F) appears to be activated to detectable levels too late by comparison with the time course of OMV pathogenicity. In our opinion, the contribution of the Toll pathway in the host defense against OMV pathogenicity is minor, albeit we acknowledge that some of the findings, especially with SPE are puzzling.

With respect to the IMD pathway, we shall test also PGRP-LC and Relish mutants, as suggested by reviewers 2&3.

Reviewer 2 query: “It is unclear how many Serratia marcescens cells a 69 nL injection of 0.1 ng/nL OMVs corresponds to.”

OMVs were purified from 600 mL of SmDb11 cultures grown to an average OD₆₀₀ of 2.0. Based on a cell density of 0.8 × 10⁸ cells/mL per OD unit, this corresponds to approximately 9.6 × 10¹⁰ total bacterial cells.

Each OMV preparation was concentrated into a final volume of 400 µL, resulting in a concentration factor of ~1500× relative to the original culture. Therefore, an injection dose of 69 nL of OMVs is equivalent to 0.1 mL of the starting bacterial culture, which corresponds to:

0.2 OD units

Approximately 1.6 × 10⁷ bacterial cells

It is likely that such high concentrations occur only toward the end of the infection, if OMVs are produced at the same rate in the host and in vitro.

With respect to other Reviewer 2 queries, we shall give a try at labeling OMVs with the FM4-64 lipophilic dye and examining whether they are taken up by hemocytes. However, an issue may arise with potentially high background, which has been encountered in cell culture. Of note, OMVs are known to attack cultured human THP1 cells, a monocyte cell line [17].Of note, determining whether OMVs are taken up by hemocytes may only be a starting point to understand how they promote the pathogenicity of OMVs. This question constitutes the topic of a full study that we are currently unable to undertake.

We shall also test whether we can document phospho-JNK expression in neural tissues.

Finally, we shall also confirm the data obtained with two elav-Gal4 drivers (including an inducible one) with the nsyb-Gal4 driver line.

References

(1) Xu R, et al. The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins. EMBO Rep 24, e56036 (2023).

(2) Huang J, et al. A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium. Proc Natl Acad Sci U S A 120, e2205140120 (2023).

(3) Gobert V, et al. Dual Activation of the Drosophila Toll Pathway by Two Pattern Recognition Receptors. Science 302, 2126-2130 (2003).

(4) Gottar M, et al. Dual Detection of Fungal Infections in Drosophila via Recognition of Glucans and Sensing of Virulence Factors. Cell 127, 1425-1437 (2006).

(5) Gottar M, et al. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416, 640-644 (2002).

(6) Thibault ST, et al. A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat Genet 36, 283-287 (2004).

(7) Rutschmann S, Jung AC, Hetru C, Reichhart J-M, Hoffmann JA, Ferrandon D. The Rel protein DIF mediates the antifungal, but not the antibacterial, response in Drosophila. Immunity 12, 569-580 (2000).

(8) Rutschmann S, Jung AC, Rui Z, Silverman N, Hoffmann JA, Ferrandon D. Role of Drosophila IKKg in a Toll-independent antibacterial immune response. Nat Immunology 1, 342-347 (2000).

(9) Jung A, Criqui M-C, Rutschmann S, Hoffmann J-A, Ferrandon D. A microfluorometer assay to measure the expression of ß-galactosidase and GFP reporter genes in single Drosophila flies. Biotechniques 30, 594- 601 (2001).

(10) Nam HJ, Jang IH, You H, Lee KA, Lee WJ. Genetic evidence of a redox-dependent systemic wound response via Hayan protease-phenoloxidase system in Drosophila. Embo J 31, 1253-1265 (2012).

(11) Kocks C, et al. Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila. Cell 123, 335-346 (2005).

(12) Bretscher AJ, et al. The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster. Biology open 4, 355-363 (2015).

(13) Binggeli O, Neyen C, Poidevin M, Lemaitre B. Prophenoloxidase activation is required for survival to microbial infections in Drosophila. PLoS Pathog 10, e1004067 (2014).

(14) Dudzic JP, Kondo S, Ueda R, Bergman CM, Lemaitre B. Drosophila innate immunity: regional and functional specialization of prophenoloxidases. BMC Biol 13, 81 (2015).

(15) Dudzic JP, Hanson MA, Iatsenko I, Kondo S, Lemaitre B. More Than Black or White: Melanization and Toll Share Regulatory Serine Proteases in Drosophila. Cell reports 27, 1050-1061 e1053 (2019).

(16) Perkins LA, et al. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics 201, 843-852 (2015).

(17) Goman A, et al. Uncovering a new family of conserved virulence factors that promote the production of host-damaging outer membrane vesicles in gram-negative bacteria. J Extracell Vesicles 14, e270032 (2025).