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Author(s): Marie Gottar [1]; Vanessa Gobert [1]; Tatiana Michel [1]; Marcia Belvin [2]; Geoffrey Duyk [2]; Jules A. Hoffmann [1]; Dominique Ferrandon (corresponding author) [1, 3]; Julien Royet (corresponding author) [...]

Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. We find that Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. We report that mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Serratia marcescens is an opportunistic bacterium that infects a wide range of hosts including humans. It is a potent pathogen in a septic injury model of Drosophila melanogaster since a few bacteria directly injected in the body cavity kill the insect within a day. In contrast, flies do not succumb to ingested bacteria for days even though some bacteria cross the intestinal barrier into the hemolymph within hours. The mechanisms by which S. marcescens attacks enterocytes and damages the intestinal epithelium remain uncharacterized. To better understand intestinal infections, we performed a genetic screen for loss of virulence of ingested S. marcescens and identified FliR, a structural component of the flagellum, as a virulence factor. Next, we compared the virulence of two flagellum mutants fliR and flhD in two distinct S. marcescens strains. Both genes are required for S. marcescens to escape the gut lumen into the hemocoel, indicating that the flagellum plays an important role for the passage of bacteria through the intestinal barrier. Unexpectedly, fliR but not flhD is involved in S. marcescens-mediated damages of the intestinal epithelium that ultimately contribute to the demise of the host. Our results therefore suggest a flagellum-independent role for fliR in bacterial virulence., (© 2022. The Author(s).)

The detailed pathogenesis of eosinophilic bronchitis (EB) remains unclear. Transglutaminase 2 (TG2) has been implicated in many respiratory diseases including asthma. Herein, we aim to assess preliminarily the relationship of TG2 with EB in the context of the development of an appropriate EB model through ovalbumin (OVA) sensitization and challenge in the C57BL/6 mouse strain. Our data lead us to propose a 50 μg dose of OVA challenge as appropriate to establish an EB model in C57BL/6 mice, whereas a challenge with a 400 μg dose of OVA significantly induced asthma. Compared to controls, TG2 is up-regulated in the airway epithelium of EB mice and EB patients. When TG2 activity was inhibited by cystamine treatment, there were no effects on airway responsiveness; in contrast, the lung pathology score and eosinophil counts in bronchoalveolar lavage fluid were significantly increased whereas the cough frequency was significantly decreased. The expression levels of interleukin (IL)-4, IL-13, IL-6, mast cell protease7 and the transient receptor potential (TRP) ankyrin 1 (TRPA1), TRP vanilloid 1 (TRPV1) were significantly decreased. These data open the possibility of an involvement of TG2 in mediating the increased cough frequency in EB through the regulation of TRPA1 and TRPV1 expression. The establishment of an EB model in C57BL/6 mice opens the way for a genetic investigation of the involvement of TG2 and other molecules in this disease using KO mice, which are often generated in the C57BL/6 genetic background.

Microsporidia are located at the base of the fungal evolutionary tree. They are obligate intracellular parasites and harness host metabolism to fuel their growth and proliferation. However, how the infestation of cells affects the whole organism and how the organism contributes to parasite proliferation remain poorly understood. Here, we have developed a Tubulinosema ratisbonensis systemic infection model in the genetically amenable Drosophila melanogaster host, in which parasite spores obtained in a mammalian cell culture infection system are injected into adult flies. The parasites proliferate within flies and ultimately kill their hosts. As commonly observed for microsporidia infecting insects, T. ratisbonensis preferentially grows in the fat body and ultimately depletes the host metabolic stores. We find that supplementing the fly diet with yeast does not benefit the host but the parasite, which increases its proliferation. Unexpectedly, fatty acids and not carbohydrates or amino acids are the critical components responsible for this phenomenon. Our genetic dissection of host lipid metabolism identifies a crucial compound hijacked by T. ratisbonensis: phosphatidic acid. We propose that phosphatidic acid is a limiting precursor for the synthesis of the parasite membranes and, hence, of its proliferation.

When Drosophila melanogaster feeds on Pseudomonas aeruginosa , some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4- mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense., (© 2018 The Authors.)

Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism.

The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin production. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity.