Male-killing symbiont damages host's dosage-compensated sex chromosome to induce embryonic apoptosis

Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner.

In Vitro Culture of the Insect Endosymbiont Spiroplasma poulsonii Highlights Bacterial Genes Involved in Host-Symbiont Interaction

Mario Gonzalo Garcia Arraez, Bruno Lemaitre, Florent François Masson, Fanny Schüpfer

Endosymbiotic bacteria associated with eukaryotic hosts are omnipresent in nature, particularly in insects. Studying the bacterial side of host-symbiont interactions is, however, often limited by the unculturability and genetic intractability of the symbionts. Spiroplasma poulsonii is a maternally transmitted bacterial endosymbiont that is naturally associated with several Drosophila species. S. poulsonii strongly affects its host's physiology, for example by causing male killing or by protecting it against various parasites. Despite intense work on this model since the 1950s, attempts to cultivate endosymbiotic Spiroplasma in vitro have failed so far. Here, we developed a method to sustain the in vitro culture of S. poulsonii by optimizing a commercially accessible medium. We also provide a complete genome assembly, including the first sequence of a natural plasmid of an endosymbiotic Spiroplasma species. Last, by comparing the transcriptome of the in vitro culture to the transcriptome of bacteria extracted from the host, we identified genes putatively involved in host-symbiont interactions. This work provides new opportunities to study the physiology of endosymbiotic Spiroplasma and paves the way to dissect insect-endosymbiont interactions with two genetically tractable partners.IMPORTANCE The discovery of insect bacterial endosymbionts (maternally transmitted bacteria) has revolutionized the study of insects, suggesting novel strategies for their control. Most endosymbionts are strongly dependent on their host to survive, making them uncultivable in artificial systems and genetically intractable. Spiroplasma poulsonii is an endosymbiont of Drosophila that affects host metabolism, reproduction, and defense against parasites. By providing the first reliable culture medium that allows a long-lasting in vitro culture of Spiroplasma and by elucidating its complete genome, this work lays the foundation for the development of genetic engineering tools to dissect endosymbiosis with two partners amenable to molecular study. Furthermore, the optimization method that we describe can be used on other yet uncultivable symbionts, opening new technical opportunities in the field of host-microbes interactions

2018

Role of Nimrod receptors in the Drosophila melanogaster cellular immune response

Claudia Melcarne

The use of modern molecular biology tools and simple but powerful tractable model organisms such as Drosophila has contributed significantly to our recent advances in the innate immunity field, notably phagocytosis. Since 2001, many phagocytic transmembrane receptors, potential opsonins, and in some cases their ligands, have been discovered. Among them, the Nimrod family, which contains key phagocytic receptors and potential opsonins, deserves special interest as accumulating genetic and biochemical evidence points to their critical role in phagocytosis of both bacteria and apoptotic cells. In this PhD thesis we provided a deeper characterization of two Nimrod transmembrane receptors, Eater and NimC1. In particular, by using single or combined mutations in eater and NimC1, we aimed to further elucidate i) the involvement of each receptor in bacterial phagocytosis and ii) the role of Eater in hemocytes sessility and adhesion. Both receptors have been shown to be specifically expressed in hemocytes, the insect blood cells, and to mediate Gram-positive but not Gram-negative bacteria phagocytosis. In this work we generated a novel NimC1 mutant and demonstrated that the single NimC1 deletion does not affect phagocytosis of bacteria. However, by using the compound NimC1;eater mutant we were able to show that these receptors contribute in a synergistic way to microbes engulfment, but that Eater can bypass the requirement for NimC1. Moreover, we discovered that NimC1, but not Eater, is required for phagocytosis of non-immunogenic particles, namely latex beads and yeast zymosan. Therefore, this work provides evidences of Eater and NimC1 being the main receptors for bacteria phagocytosis, and suggests that those proteins likely play distinct roles in microbial uptake, as tethering and docking receptors. The second part of this thesis re-addresses the role of Eater in hemocyte sessility and adhesion. A previous study showed that this phagocytic receptor is essential to mediate blood cell sessility to the animal integument during larval stages. However, a series of questions about Eater-mediated sessility remains still open. For example, is Eater mediating hemocyte adhesion also in adult flies? And yet, does Eater mediate hemocyte sessility by regulating the expression of cell adhesion genes, therefore functioning as a signalling receptor? By using eater1 mutant adult flies, we showed that, as in larval stages, Eater is required in adult hemocytes to confer adhesion and sessility. Interestingly, we uncovered a potential role of sessility in hemocyte survival during adulthood, since eater1 flies have a decreased number of blood cells. Preliminary transcriptomics analysis led us to hypothesise that this receptor might not work as a signalling molecule, but rather as an adhesion protein providing the initial hemocyte contact to its target surface. In line with Eaterâs role as an adhesion molecule, its over-expression in hemocytes leads to the formation of enlarged cells compared to control. To conclude, this thesis extended our knowledge on the Drosophila cellular immune response, by providing new insights on two Nimrod phagocytic receptors.

EPFL2020

Vertical transmission of a Drosophila endosymbiont via cooption of the yolk transport and internalization machinery

Jeremy Keith Herren, Bruno Lemaitre, Juan Camilo Paredes Escobar, Fanny Schüpfer

Spiroplasma is a diverse bacterial clade that includes many vertically transmitted insect endosymbionts, including Spiroplasma poulsonii, a natural endosymbiont of Drosophila melanogaster. These bacteria persist in the hemolymph of their adult host and exhibit efficient vertical transmission from mother to offspring. In this study, we analyzed the mechanism that underlies their vertical transmission, and here we provide strong evidence that these bacteria use the yolk uptake machinery to colonize the germ line. We show that Spiroplasma reaches the oocyte by passing through the intercellular space surrounding the ovarian follicle cells and is then endocytosed into oocytes within yolk granules during the vitellogenic stages of oogenesis. Mutations that disrupt yolk uptake by oocytes inhibit vertical Spiroplasma transmission and lead to an accumulation of these bacteria outside the oocyte. Impairment of yolk secretion by the fat body results in Spiroplasma not reaching the oocyte and a severe reduction of vertical transmission. We propose a model in which Spiroplasma first interacts with yolk in the hemolymph to gain access to the oocyte and then uses the yolk receptor, Yolkless, to be endocytosed into the oocyte. Cooption of the yolk uptake machinery is a powerful strategy for endosymbionts to target the germ line and achieve vertical transmission. This mechanism may apply to other endosymbionts and provides a possible explanation for endosymbiont host specificity. IMPORTANCE: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. Studies have shown that many facultative endosymbionts, including Spiroplasma, confer protection against different classes of parasites on their hosts and therefore are attractive tools for the control of vector-borne diseases. The ability to be efficiently transmitted from females to their offspring is the key feature shaping associations between insects and their inherited endosymbionts, but to date, little is known about the mechanisms involved. In oviparous animals, yolk accumulates in developing eggs and serves to meet the nutritional demands of embryonic development. Here we show that Spiroplasma coopts the yolk transport and uptake machinery to colonize the germ line and ensure efficient vertical transmission. The uptake of yolk is a female germ line-specific feature and therefore an attractive target for cooption by endosymbionts that need to maintain high-fidelity maternal transmission.

American Society for Microbiology2013