Blind killing of both male and female Drosophila embryos by a natural variant of the endosymbiotic bacterium Spiroplasma poulsonii

Spiroplasma poulsonii is a vertically transmitted endosymbiont of Drosophila melanogaster that causes male-killing, that is the death of infected male embryos during embryogenesis. Here, we report a natural variant of S. poulsonii that is efficiently vertically transmitted yet does not selectively kill males, but kills rather a subset of all embryos regardless of their sex, a phenotype we call 'blind-killing'. We show that the natural plasmid of S. poulsonii has an altered structure: Spaid, the gene coding for the male-killing toxin, is deleted in the blind-killing strain, confirming its function as a male-killing factor. Then we further investigate several hypotheses that could explain the sex-independent toxicity of this new strain on host embryos. As the second non-male-killing variant isolated from a male-killing original population, this new strain raises questions on how male-killing is maintained or lost in fly populations. As a natural knock-out of Spaid, which is unachievable yet by genetic engineering approaches, this variant also represents a valuable tool for further investigations on the male-killing mechanism.

The double-edged toxins

Mario Gonzalo Garcia Arraez

Many insect species are associated with endosymbiotic bacteria which have the particularity of living within host tissues. Endosymbionts benefit from this stable and nutritious environment, while providing ecological advantages to their host, such as protection against parasites or thermal tolerance. Spiroplasma poulsonii is an endosymbiotic bacterium that infects natural populations of Drosophila melanogaster. Spiroplasma poulsonii lacks a cell wall, a fact that renders it invisible to the host immune system and allows it to thrive in the host hemolymph. It invades the female germline by co-opting the hostâs yolk transport and uptake machinery, which ensures its vertical transmission. Its efficient transmission is associated with a phenotype called male-killing, whereby infected male embryos die during their early development while infected females survive. The mechanisms that ensure the stability of Drosophila-Spiroplasma symbiosis are increasingly well understood, but the bacterial genes involved remain poorly known because of the intractability of Spiroplasma. This project aims at better characterizing the Drosophila-Spiroplasma interaction, with particular focus on the bacterial side. In the first part, I developed a method to cultivate Spiroplasma poulsonii in vitro by optimizing a commercially available medium. This culture method allowed comparing the transcriptome of in vitro grown versus host-grown Spiroplasma, enabling us to identify putative genes involved in the interaction with the host. Interestingly, inside its insect host, S. poulsonii up-regulates genes coding for toxins of the Ribosomal Inactivating Protein (RIP) family. RIPs were previously known for their role in host protection against macro-parasites, such as wasps and nematodes. Their up-regulation in unparasitized hosts compared to culture was thus peculiar, raising the question what effect these RIPs have on host biology. Thus, in the second part, I studied the function of S. poulsonii RIPs in the absence of parasites. I showed that two of them are constantly expressed within the host and provide evidence that these toxins shorten host life span and increase embryonic mortality. Interestingly, the expression of RIPs was more toxic to male embryos than females, suggesting that RIPs contribute to S. poulsonii-induced male-killing. Last, I studied the D. melanogaster response to RIPs, and how the host mitigates the deleterious effects of these toxins by up-regulating the cytosolic chaperone Heat-Shock-Protein 70B (HSP70B). This protein carries out essential functions in protein homeostasis under normal and stressful conditions such as folding, refolding, or increasing the half-life of proteins. Interestingly, up-regulation of Hsp70B in the presence of RIPs results in an increased lifespan and in a better tolerance to heat stress, which may be ecologically advantageous. Altogether, this work illustrates how Spiroplasma-derived RIP toxins can differentially affect Drosophila depending on the ecological context, ranging from beneficial upon parasite infection or heat shock to detrimental in the absence of such environmental pressures.

EPFL2018

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

Toshiyuki Harumoto, Bruno Lemaitre

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.

Nature Publishing Group2016

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