Intracellular Density of Wolbachia Is Mediated by Host Autophagy and the Bacterial Cytoplasmic Incompatibility Gene cifB in a Cell Type-Dependent Manner in Drosophila melanogaster

Abstract

Autophagy is an intracellular degradation pathway involved in innate immunity. Pathogenic bacteria have evolved several mechanisms to escape degradation or exploit autophagy to acquire host nutrients. In the case of endosymbionts, which often have commensal or mutualistic interactions with the host, autophagy is not well characterized. We utilized tissue-specific autophagy mutants to determine if Wolbachia, a vertically transmitted obligate endosymbiont of Drosophila melanogaster, is regulated by autophagy in somatic and germ line cell types. Our analysis revealed core autophagy proteins Atg1 and Atg8 and a selective autophagy-specific protein Ref(2)p negatively regulate Wolbachia in the hub, a male gonad somatic cell type. Furthermore, we determined that the Wolbachia effector protein, CifB, modulates autophagy-Wolbachia interactions, identifying a new host-related pathway which these bacterial proteins interact with. In the female germ line, the cell type necessary for inheritance of Wolbachia through vertical transmission, we discovered that bulk autophagy mediated by Atg1 and Atg8 positively regulates Wolbachia density, whereas Ref(2)p had no effect. Global metabolomics of fly ovaries deficient in germ line autophagy revealed reduced lipid and carbon metabolism, implicating metabolites from these pathways as positive regulators of Wolbachia. Our work provides further understanding of how autophagy affects bacteria in a cell type-dependent manner. IMPORTANCE Autophagy is a eukaryotic intracellular degradation pathway which can act as an innate immune response to eliminate pathogens. Conversely, pathogens can evolve proteins which modulate the autophagy pathway to subvert degradation and establish an infection. Wolbachia, a vertically transmitted obligate endosymbiont which infects up to 40% of insect species, is negatively regulated by autophagy in whole animals, but the specific molecular mechanism and tissue which govern this interaction remain unknown. Our studies use cell type-specific autophagy mutants to reveal that Wolbachia is negatively regulated by selective autophagy in the soma, while nonselective autophagy positively regulates Wolbachia in the female germ line. These data provide evidence that cell type can drive different basal autophagy programs which modulate intracellular microbes differently. Additionally, we identified that the Wolbachia effector CifB acts in the selective autophagy pathway to aid in intracellular bacterial survival, providing a new function for CifB beyond its previously identified role in reproductive manipulation.

Restriction ofWolbachiabacteria in early embryogenesis of neotropicalDrosophilaspecies via ER-mediated autophagy

“…In contrast to wWil, wPau and wStv Wolbachia, might have evolved a mechanism to remove the ubiquitination mark but still be cleared with autophagy through a different pathway. It was recently demonstrated that the wMelCS strain, but not the closely related wMel, might have developed a trick to subvert the autophagy machinery by actively avoiding the ubiquitination in D. melanogaster hub cells (Deehan et al, 2021). These data show that even closely related Wolbachia strains exhibit stark differences in interaction with the host machinery, which might also be the case in our study.…”

“…This defence strategy of the symbiont also coincides with the downregulation of autophagy genes as observed in ovaries of the wasp Asobara tabida and the woodlouse Armadillidium vulgare(Kremer et al, 2012;Chevalier et al, 2012). Additionally, a recent study demonstrated that wMelCS strain of Wolbachia evolved a mechanism to subvert host autophagy in order to survive in hub cells and both wMel and wMelCS are able to avoid elimination in the developing egg(Deehan et al, 2021). On the other hand, the PGCs themselves might lack extensive autophagic activity and thereby provide a safe environment for the Wolbachia to survive, replicate and being successfully transmitted via oocytes.…”

“…keeping the cost-benefit equilibrium in balance. Also for facultative symbiotic associations it was shown that Wolbachia density is regulated by autophagy(Voronin et al, 2012;Le Clec'h et al, 2012;Deehan et al, 2021). In our study, we observed Wolbachia accumulation mostly in PGCs during embryogenesis, whereas the rest of infection in the somatic part is being massively eliminated and subsequently restricted to certain isolated areas.…”

“…Autophagy was considered a potential mechanism for bacterial clearance because it has previously been demonstrated as a key cellular strategy for controlling Wolbachia density and tropism in Brugia malayi nematodes and D. melanogaster flies in vivo, as well as in vitro in cell lines of D. melanogaster and Aedes albopictus (Voronin et al, 2012). Moreover, it was recently shown that the density of Wolbachia in D. melanogaster is mediated by host autophagy in a cell typedependent manner (Deehan et al, 2021). To test our hypothesis, we conducted sequential FISH and immunofluorescent analysis using an anti-GABARAP antibody, which is diagnostic for maturing autophagosomes in a cell.…”

DiversewMel variants ofWolbachia pipientisdifferentially rescue fertility and cytological defects of thebag of marblespartial loss of function mutation inDrosophila melanogaster

“…Through these methods, the W. pipientis genes cifA and cifB (orthologs in wMel and wPip) that cause CI have been identified and functionally validated in D. melanogaster (Beckmann et al 2017;Le Page et al 2017). These transgenic tools have been used to further define the CI phenotype (Shropshire et al 2018), investigate the consequences of genetic variation at cifA and cifB on CI (Shropshire et al 2021), and identify other host phenotypes affected by cifA and cifB (Deehan et al 2021). Another group has identified the W. pipientis TomO locus which is one of likely multiple W. pipientis loci that interact with Sex lethal, a germline stem cell (GCS) gene that controls GSC maintenance and sex determination (Ote et al 2016).…”