Quorum-sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization.

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.

Fluorescent-fipronil: Design and synthesis of a stable conjugate.

Fipronil is a phenyl pyrazole molecule widely used across the world as both insecticide and veterinary drug. The main goal of this work was to synthesize a fluorescently labeled fipronil derivative for cellular imaging without affecting its intrinsic properties. We selected fluorescein as fluorescent probe and we investigated different strategies for stable chemical ligation between both entities, such as thiourea and direct peptide bond. While thiourea bond displayed low stability, direct peptide bond was difficult to achieve due to problems of steric hindrance. The best result was obtained by conjugation using click chemistry, which allowed to obtain fipronil stably labeled with the fluorescent probe.

Phagocytosis Is the Sole Arm of Drosophila melanogaster Known Host Defenses That Provides Some Protection Against Microsporidia Infection.

Microsporidia are obligate intracellular parasites able to infest specifically a large range of species, including insects. The knowledge about the biology of microsporidial infections remains confined to mostly descriptive studies, including molecular approaches such as transcriptomics or proteomics. Thus, functional data to understand insect host defenses are currently lacking. Here, we have undertaken a genetic analysis of known host defenses of the Drosophila melanogaster using an infection model whereby Tubulinosema ratisbonensis spores are directly injected in this insect. We find that phagocytosis does confer some protection in this infection model. In contrast, the systemic immune response, extracellular reactive oxygen species, thioester proteins, xenophagy, and intracellular antiviral response pathways do not appear to be involved in the resistance against this parasite. Unexpectedly, several genes such as PGRP-LE seem to promote this infection. The prophenol oxidases that mediate melanization have different functions; PPO1 presents a phenotype similar to that of PGRP-LE whereas that of PPO2 suggests a function in the resilience to infection. Similarly, eiger and Unpaired3, which encode two cytokines secreted by hemocytes display a resilience phenotype with a strong susceptibility to T. ratisbonensis.

Phosphatidic acid as a limiting host metabolite for the proliferation of the microsporidium Tubulinosema ratisbonensis in Drosophila flies.

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.

Draft Genome Sequence of Tubulinosema ratisbonensis, a Microsporidian Species Infecting the Model Organism Drosophila melanogaster.

We present the draft genome sequence of Tubulinosema ratisbonensis, a microsporidium species infecting Drosophila melanogaster A total of 3,013 protein-encoding genes and an array of transposable elements were identified. This work represents a necessary step to develop a novel model of host-parasite relationships using the highly tractable genetic model D. melanogaster.