Hrdina, A. | Search bioRxiv

bioRxiv2023

The endosymbiont Spiroplasma poulsonii increases Drosophila melanogaster resistance to pathogens by enhancing iron-sequestration and melanization

Facultative endosymbiotic bacteria, such as Wolbachia and Spiroplasma species, are commonly found in association with insects and can dramatically alter their host physiology. Many endosymbionts are defensive and protect their hosts against parasites or pathogens. Despite the widespread nature of defensive insect symbioses and their importance for the ecology and evolution of insects, the mechanisms of symbiont-mediated host protection remain poorly characterized. Here, we utilized the fruit fly Drosophila melanogaster and its facultative endosymbiont Spiroplasma poulsonii to characterize the mechanisms underlying symbiont-mediated host protection against bacterial and fungal pathogens. Our results indicate a variable effect of S. poulsonii on infection outcome, with endosymbiont-harbouring flies being more resistant to Rhyzopus oryzae, Staphylococcus aureus, and Providencia alcalifaciens, but more sensitive or as sensitive as endosymbiont-free flies to the infections with Pseudomonas species. Further focusing on the protective effect, we identified Transferrin-mediated iron sequestration induced by Spiroplasma as being crucial for the defense against R. oryzae and P. alcalifaciens. In case of S. aureus, enhanced melanization in Spiroplasma-harbouring flies plays a major role in the protection. Both iron sequestration and melanization induced by Spiroplasma require the host immune sensor protease Persephone, suggesting a role of proteases secreted by the symbiont in the activation of host defense reactions. Hence, our work reveals a broader defensive range of Spiroplasma than previously appreciated and adds nutritional immunity and melanization to the defensive arsenal of symbionts. ImportanceDefensive endosymbiotic bacteria conferring protection to their hosts against parasites and pathogens are widespread in insect populations. However, the mechanisms by which most symbionts confer protection are not fully understood. Here, we studied the mechanisms of protection against bacterial and fungal pathogens mediated by the Drosophila melanogaster endosymbiont Spiroplasma poulsonii. We demonstrate that besides previously described protection against wasps and nematodes, Spiroplasma also confers increased resistance to pathogenic bacteria and fungi. We identified Spiroplasma-induced iron sequestration and melanisation as key defense mechanisms. Our work broadens the known defense spectrum of Spiroplasma and reveals a previously unappreciated role of melanization and iron sequestration in endosymbiont-mediated host protection. We propose that the mechanisms we have identified here may be of broader significance and could apply to other endosymbionts, particularly to Wolbachia, and potentially explain their protective properties.

microbiology↗

bioRxiv2022

Lipopolysaccharide-mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster

Bacteria from the genus Providencia are ubiquitous Gram-negative opportunistic pathogens, causing "travelers diarrhea", urinary tract, and other nosocomial infections in humans. Some Providencia strains have also been isolated as natural pathogens of Drosophila melanogaster. Despite clinical relevance and extensive use in Drosophila immunity research, little is known about Providencia virulence mechanisms and the corresponding insect host defenses. To close this knowledge gap, we investigated the virulence factors of a representative Providencia species - P. alcalifaciens which is highly virulent to fruit flies and amenable to genetic manipulations. We generated a P. alcalifaciens transposon mutant library and performed an unbiased forward genetics screen in vivo for attenuated mutants. Our screen uncovered 23 mutants with reduced virulence. The vast majority of them had disrupted genes linked to lipopolysaccharide (LPS) synthesis or modifications. These LPS mutants were sensitive to cationic antimicrobial peptides (AMPs) in vitro and their virulence was restored in Drosophila mutants lacking most AMPs. Thus, LPS-mediated resistance to host AMPs is one of the virulence strategies of P. alcalifaciens. Another subset of P. alcalifaciens attenuated mutants exhibited increased susceptibility to reactive oxygen species (ROS) in vitro and their virulence was rescued by chemical scavenging of ROS in flies prior to infection. Using genetic analysis, we found that the enzyme Duox specifically in hemocytes is the source of bactericidal ROS targeting P. alcalifaciens. Consistently, the virulence of ROS-sensitive P. alcalifaciens mutants was rescued in flies with Duox knockdown in hemocytes. Therefore, these genes function as virulence factors by helping bacteria to counteract the ROS immune response. Our reciprocal analysis of host-pathogen interactions between D. melanogaster and P. alcalifaciens identified that AMPs and hemocyte-derived ROS are the major defense mechanisms against P. alcalifaciens, while the ability of the pathogen to resist these host immune responses is its major virulence mechanism. Thus, our work revealed a host-pathogen conflict mediated by ROS and AMPs. Author summaryPathogens express special molecules or structures called virulence factors to successfully infect a host. By identifying these factors, we can learn how hosts fight and how pathogens cause infections. Here, we identified virulence factors of the human and fruit fly pathogen Providencia alcalifaciens, by infecting flies with a series of mutants of this pathogen. In this way, we detected 23 mutants that were less virulent. Some of these less virulent mutants were hypersensitive to fruit fly immune defense molecules called antimicrobial peptides (AMPs), while others were sensitive to reactive oxygen species (ROS) produced by the immune cells. Notably, AMPs-sensitive mutants remained virulent in a Drosophila mutant that lacks AMPs, while pathogens sensitive to oxidative stress retained their virulence in a fruit fly mutant devoid of oxidative species. These results suggest that the ability of P. alcalifaciens to resist two major host immune molecules, namely AMPs and ROS, is the major virulence mechanism. Overall, our systematic analysis of P. alcalifaciens virulence factors has identified the major defense mechanisms of the fruit fly against this pathogen and the bacterial mechanisms to combat these immune responses.

microbiology↗