In this article, we have generated a set of single and compound immune module-deficient fly lines in a controlled genetic background. Using various assays, we confirmed the validity of our lines, revealing that each module can be activated independently. We did, however, observe a higher Toll activation in ∆Mel flies upon systemic infection with dead bacteria. Future studies may reveal if this higher Toll activity in ∆Mel flies involves a regulatory pathway, or perhaps reflects higher persistence of microbial elicitors (e.g. peptidoglycan) that activate the Toll pathway. We also observed a lesser Imd activation in ∆Phag flies at 6 hr post-infection, which could reflect a role for hemocytes to stimulate the Imd systemic response. In this study, we used NimC11; eater1 double mutants to assess the cellular response. These flies have defects in phagocytosis, but also adhesion and sessility (Melcarne et al., 2019b). NimC11; eater1 larvae also have increased hemocyte number at the larval stage, but adults rapidly lose Hml-positive cells (Melcarne, 2020). Despite these limitations, we believe that the mutations we have used here are among the best available to assess the role of these four modules to host defense. Surprisingly, we could produce a fly line lacking the four main defense mechanisms of the systemic immune response, indicating that none of these modules is essential for survival. Theoretically, ∆ITPM flies are almost completely immune deficient, but they can still clot wounds, activate the JNK and JAK–STAT pathways that mediate the wound healing response, and retain constitutive immune defense molecules that could also provide a certain degree of protection. Consistent with several studies (Capilla et al., 2017; Carvalho et al., 2014; Rämet et al., 2002), we show that Toll and melanization synergistically contribute to wound healing in adults. However, the observation that ∆Phag, ∆Mel or ∆IMD, ∆Mel double mutant flies are also more susceptible to clean injury than ∆Mel flies indicates that the Imd pathway and phagocytosis also contribute to wound healing. Thus, although immune-deficient fly lines for the four modules are viable, the use of compound mutation and ∆ITPM flies reveals a clear role of the immune system in response to wounding and lifespan maintenance.
Use of single- and double-module-deficient flies provides key insights on the mechanisms used by Drosophila to combat infection. We could confirm previous studies revealing the role of Imd against Gram-negative bacteria, Toll against Gram-positive bacteria and virulent fungi, and an importance of melanization and phagocytosis against specific pathogens (Charroux and Royet, 2009; Defaye et al., 2009; Garg and Wu, 2014; Nehme et al., 2011). Surprisingly, melanization was a consistently important module in survival to virus infection. It seems unlikely that PPO activity taking place in the hemolymph could combat viral agents that are intracellular. We instead speculate this contribution of melanization to surviving virus infection could be due to its role in wound healing, a role in infected cell clearance, or perhaps autotoxic contributions of melanization reaction intermediates that fail to convert in phenoloxidase-deficient flies. We additionally recovered a role of Imd signaling (i.e. Relish) in antiviral defense, which was expected given recent characterizations of cGLR–Sting–Relish antiviral immunity (Ai et al., 2024; Cai et al., 2022; Goto et al., 2018). However, susceptibilities of RelE20 flies were paralleled by ∆AMP14 in all cases, suggesting AMPs contribute to this susceptibility. While Sting regulates a number of genes likely important for antiviral defense (Goto et al., 2018), the susceptibility we observe here could be a direct action of AMPs on enveloped viruses as described in some studies (Feng et al., 2020; Huang et al., 2013; Yasin et al., 2004), or an indirect effect, such as a failure to regulate gut microbes after the damage induced by viral replication (Marra et al., 2021). We additionally used dual modes of infection for the fungus B. bassiana, finding an importance of melanization and Toll signaling in both infection modes. However, our study reveals that Bomanin effectors explain most of the Toll contribution upon septic injury but not natural infection, for B. bassiana. This observation is in line with different importance of effectors or modules according to the route of infection (Martins et al., 2013).
Our double-module mutant analysis reveals that most modules contribute additively to host defense. This is consistent with these modules functioning independently. However, we noticed instances of synergy between two pathways, notably including Toll and Imd. Synergy between Toll and Imd can be explained by the fact that many immune-inducible regulated genes receive input from both the Imd and Toll pathways, including genes like Metchnikowin, Drosomycin, and Transferrin1 (De Gregorio et al., 2002). Finally, we also observed rarer cases of synergistic susceptibility in flies deficient for Toll and Melanization or Imd and Melanization.
Our study confirms that the Toll and Imd humoral modules provide a broad role against certain classes of pathogens, Imd for Gram-negative bacteria and DAP-type containing Gram-positive bacteria, and Toll for Fungi and Gram-positive bacteria. Use of AMP and Bomanin mutants revealed that this can be largely explained by the effectors they control. In contrast, the contribution of phagocytosis and melanization appears to be critical to a more specific and diverse set of pathogens. We speculate that phagocytes or melanization are critical to handle bacteria that resist HDPs (Hanson et al., 2019), or can hide from them (Touré et al., 2023). The melanization reaction is a source of ROS that is potent against pathogens such as S. aureus that have been shown to be sensitive to ROS and resistant to Toll and Imd defenses (Dudzic et al., 2019; Ford and King, 2021; Needham et al., 2004; Ramond et al., 2021). These two modules play important roles in immune-related processes such as encapsulation (melanization), the uptake of bacteria escaping from the gut, or tissue homeostasis (phagocytosis) (Braun et al., 1998; Melcarne et al., 2019b; Nehme et al., 2007), which were not assessed here. Collectively, our study validates, with minor discrepancies, many studies that have assessed the contribution of these modules individually (e.g. Apidianakis et al., 2005; Binggeli et al., 2014; Lamiable et al., 2016; Lemaitre et al., 1996; Lemaitre et al., 1995; Nehme et al., 2011). Our mutant lines can now be used to analyze the contribution of these immune modules in resistance to other pathogens, notably wasps, nematodes, microsporidia, and protozoans, or in other contexts such as mating and local infection.
While some immune modules play a predominant role against some pathogens, other pathogens are handled by multiple modules. We hypothesize that pathogens that have intermediate levels of virulence, killing only a fraction of wild-type flies, may better reveal the role of multiple modules. Indeed, the stochasticity in survival analysis partly stems from the arms race occurring between the pathogen and host immunity, as shown for Pr. rettgeri (Duneau et al., 2017a). In this condition, any small change in the immune system may tip the outcome of the arm race toward lethality or survival. In this line of thinking, it is notable that multiple modules were important to survive Pr. rettgeri infection. Previous studies have revealed a major role of the Imd pathway-regulated AMP Diptericin A against this bacterium (Hanson et al., 2023; Hanson et al., 2019; Unckless et al., 2016). However, Duneau et al. showed that survival patterns to Pr. rettgeri bifurcate into two outcomes based on time taken to fully activate systemic defenses (Duneau et al., 2017a), and showed a role for a Toll-PO SP cascade regulating serine protease in defense against this microbe (Duneau et al., 2017b). We may speculate that melanization, although not as uniquely critical as Diptericin, might tip the balance of this arms race toward host lethality, resulting in comparable survival phenotypes. The observation that DPhag flies expressed less DptA (Figure 1) can also explain their susceptibility. Future studies will clarify how these multiple modules can contribute to host survival according to additive or Achilles dynamics – the concept that microbes have generic or specific weaknesses that host effectors can target (Hanson, 2024). It will also be important to consider the distinct roles of resistance, tolerance, and resilience in host defense (Howick and Lazzaro, 2017; Wukitch et al., 2023; Duneau et al., 2025).
We observed a good correlation between survival analysis and pathogen growth in single-module flies for Pr. rettgeri, S. aureus, and C. albicans. This indicates that these immune modules mostly contribute to resistance mechanisms that target pathogen growth. Our study did not reveal key early contributions of phagocytosis or melanization to control pathogen growth, despite their quasi-immediate activation; higher S. aureus growth at 2 hr in DPhag flies was paralleled by DImd flies, and we observed lesser Imd activation in DPhag flies. Melanization, while being critical to survive S. aureus infection, impacts bacterial growth beginning only after the 6-hr time point. This indicates that the melanization microbicidal activity in vivo takes place slower than the blackening reaction seen from bled hemolymph. Interestingly, ∆Phag and also ∆Mel flies could suppress C. albicans yeast growth, but ultimately some individuals succumb to infection with lower PLUD levels. We confirmed ∆Phag PLUD results using both an isogenic and a second wild-type genetic background, suggesting this lower PLUD is genuine. Susceptibility to fungal infection independent of fungal proliferation has also been reported using an A. fumigatus septic infection model and relies on the protection offered by Bomanins from pathogen-secreted toxins (Xu et al., 2023). It is tempting to speculate, based on the modules involved, that the loss of tolerance to C. albicans we observe has to do with wound repair or accumulating damage, such as what has been reported in beetles that suffer renal failure (Khan et al., 2017; Li et al., 2020), or flies with autotoxic trachea degradation upon stress pathway disruption (Rommelaere et al., 2024). Thus, this tolerance effect could rely on pathogen-mediated or autotoxic damage, which may be elucidated in a future study.
Here, we have provided a single and double mutant analysis of Drosophila immune module functions. Our study provides several insights on what modules are most important to survive infection by defined pathogens. However, we also highlight the collective contribution of modules to defense even when one module is of an outsized importance. We extend our comprehension of innate immune responses by revealing higher complexity, implicating multiple host defense modules in survival to various germs, including some with more cryptic contributions. As illustrated by our previous characterizations of AMP function (Carboni et al., 2022; Hanson et al., 2019), the melanization response (Dudzic et al., 2015), and stress-induced Turandot proteins (Rommelaere et al., 2024), a combinatorial mutation approach to deciphering immune functions can be extended even to the broad level of immune modules. Of note, we were unable to systematically sample all genotype-by-pathogen interactions equally. We have therefore been highly conservative in our reporting of major effects. There are likely many important interactions not discussed in our study. Future investigations may highlight important biology that is apparent in our data, but which we may not have mentioned here. To this end, we have deposited our isogenic immunity fly stocks in the Vienna Drosophila Resource Centre to facilitate their use. Beyond immunity, our tools can also be of use to study various questions at the cutting edge of aging, memory, neurodegeneration, cancer, and more, where immune genes have been implicated repeatedly. We hope that this set of lines will be useful to the community to better characterize the Drosophila host defense.