Drosophila melanogaster mitigates gastro-oral infections by stimulating pathogen expulsion

Drosophila melanogaster mitigates gastro-oral infections by stimulating pathogen expulsion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Drosophila melanogaster mitigates gastro-oral infections by stimulating pathogen expulsion Shreya Verma, Sushovan Bhattacharyya, Meghana Tare, Sandhya Amol Marathe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8473557/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 25 You are reading this latest preprint version Abstract Background Food-borne pathogens, particularly belonging to the Enterobacteriaceae family, are a major cause of gastrointestinal infections in humans. While Drosophila melanogaster has been widely explored to study antimicrobial responses, most studies rely on septic injury or direct injection, routes that bypass the gut. A coordinated set of antimicrobial defenses acts to counteract the invading bacteria, with some variations depending on the entry route into the host. Herein, we tracked the dynamics of Enterobacteriaceae pathogens in Drosophila using a natural infection route. Results Most bacterial species were cleared effectively within 48 hours post-infection (hpi). We did not observe any significant mortality, indicating robust infection control. At 4 hpi, a substantial increase in reactive oxygen species (ROS) production was observed, followed by a decrease at 24 hpi and a resurgence at 48 hpi, suggesting the importance of ROS in bacterial clearance. However, flies lacking the dual oxidase ( Duox ) gene showed unchanged survival rates, suggesting ROS alone is not enough for infection control. We further show that the shedding of bacteria could be attributed to increased TRPA1 expression, a ROS-sensing receptor that triggers intestinal contractions in flies infected with S . Typhimurium and E. cloacae at 4 and 24 hpi. Conclusions Our findings reveal that the host can distinguish and respond to various bacterial species in a well-synchronized, gut-localized, and pathogen-specific manner. This also illustrates the reliability of natural infection route models in unravelling and understanding the complexities of host–pathogen interaction. Enteric pathogens Gut immunity Reactive oxygen species Bacterial shedding TRPA1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background In view of thevast research conducted on host-pathogen interactions, the mechanisms underlying variations in the individual responses to similar Gram-negative bacterial infections remain less understood. To explore this, we utilized Drosophila melanogaster as a hostmodel organism to understand the immune responses that are coordinated and regulated at the whole organism level. The increasing use of Drosophila in the studies of infectious diseases and control is due to its degree of genetic and molecular conservation with vertebrates, particularly in innate immune cascades, transduction pathways, and transcriptional regulators. 1 Moreover, ithas been used to study the pathogenesis of a wide variety of microorganisms, as it also serves as a model of low microbiome complexity. 2 To combat bacterial infections, Drosophila relies solely on its innate immune system, as it is devoid of adaptive immune response for its defense. 3 The innate responses are categorized as cellular and humoral, where cellular responses are phagocytosis and encapsulation, while the humoral responses include the activation of evolutionarily conserved immune response cascades, the immune deficiency (IMD), and Toll pathways. 4 In cellular responses, hemocytes produce reactive oxygen species (ROS) during bacterial infections. The major source of ROS is nicotinamide adenine dinucleotide phosphate (NADPH) oxidases ( Nox ) 5 and dual oxidases ( Duox )derived from hemocytes. 6 In Drosophila , ROS production as early as 30 minutes after pathogen ingestion is a conserved response to infection. However, ROS can have a destructive impact on gut mucosal cells; therefore, its activity must be tightly regulated to minimize host tissue injury. 7 While many previous studies have highlighted the role of ROS in the gut beyond their antibacterial activity, emerging evidence suggests that ROS can modulate gut functions. 8 The ROS produced in response to bacterial infection triggers visceral spasms mediated by TRPA1 channels. These visceral spasms induce intestinal contractions, expediting bacterial elimination from the host. 9 Notably, TRPA1 is also involved in inflammatory bowel diseases, where it affects the gut inflammation and has dual roles: pro- and anti-inflammatory roles, 10 making it an attractive target to treat inflammatory disorders. The interaction between oxidative stress and the mechanosensory pathway seems to be conserved, as Drosophila TRPA1 is a homolog of mammalian TRP receptors responsive to stressful conditions. 11 For infection studies, usually flies are infected with bacteria, either through the septic injury method, where bacteria are directly introduced into the body cavity, bypassing the epithelial barriers, or through the oral infection route, where bacteria are fed to the flies. A few pathogens, such as Serratia marcescens (S. marcescens), Escherichia coli (E. coli) , Salmonella enterica subspecies enterica serovarTyphimurium ( S . Typhimurium), and Enterobacter cloacae (E. cloacae), have been extensively studied in Drosophila using a septic injury model to assess the biological responses encompassing survival rates, change in gut morphology, alterations in triglyceride levels, activation of IMD pathway, and bacterial load. 12-14 However, this does not represent the gut-specific immunocompetence, relevant to the natural route of infection. To address this gap, we developed a Drosophila oral infection model in this study by feeding the flies bacterial suspensions prepared in sucrose for a brief duration. This natural infection model enabled us to track the fate of orally ingested bacteria and characterize the gut immune responses to specific Gram-negative bacteria, including E. coli, S . Typhimurium, Klebsiella pneumoniae (K. pneumoniae), and E. cloacae . Although traditionally, Drosophila culture is also supplemented with preservatives like Methyl Paraben (Nipagin) and Propionic acid to ensure healthy conditions for the cultures, these preservatives may affect the results of oral infection studies. To circumvent this, we investigated the effect of Enterobacteriaceae infections through oral administration to flies cultured without preservatives. Our study aims to investigate how Drosophila mounts distinct immune responses to specific pathogens during natural infection, focusing on bacterial expulsion mediated by ROS and TRPA1. We observe that this interplay between oxidative stress and sensory receptor orchestrates a pathogen-specific gut strategy, minimizing gut damage while clearing the bacteria. Results Infection of Drosophila with different bacteria caused species-specific colonization and clearance To ensure that antimicrobial agents, propionic acid, and nipagin, typically added to fly food, do not impact the bacterial survival and clearance, we tested the antibacterial activity of these agents specifically against the pathogens used in the study. The results indicated that these agents, at a concentration generally added to fly food, exhibited antibacterial activity against E. coli, S . Typhimurium, K. pneumoniae, and E. cloacae (Supplementary file 1). Thus, in all further infection experiments, the flies were fed on the food without adding these agents. The presence of bacteria and the duration of their persistence in Drosophila were monitored using fluorescence microscopy and colony forming unit (CFU) analysis after feeding the flies RFP/m-cherry-tagged bacteria. We observed a strong red fluorescence in the fly's abdomen immediately after bacterial feeding (Figure 1A and 1B), indicating the ingestion of the bacteria. 15 Within 24 hours post-infection (hpi), the fluorescence reduced to a basal level in the flies fed with E. coli, a non-pathogenic species, to Drosophila, 16 with a subsequent reduction in bacterial burden within the fly body (Figure 1C). This indicates efficient elimination of E. coli by the Drosophila . Similar results were not obtained with other bacterial species. Within 24 hpi, though the fluorescence signal reduced to basal levels for K. pneumoniae and E. cloacae ,their numbers remained sufficiently high in the fly body. The K. pneumoniae numbers dropped by a factor of 5, while those of E. cloacae increased 5-fold. At 24 hpi, the S . Typhimurium fluorescence and numbers remained close to those of the ingested. By 48 hpi, the numbers of K. pneumoniae and S . Typhimurium dropped close to those of E. coli . However, for E. cloacae , bacterial numbers decreased gradually and persisted up to 72 hpi Despite the presence of E. cloacae in Drosophila till 72 hpi and other pathogens till 48 hpi, the survivability of infected flies was not compromised up to 30 days post-infection (dpi) (Supplementary file 2). This suggested that oral infection was efficiently controlled by the host's innate immune responses. Bacterial infection triggers host intestinal ROS production in a dynamic pattern It has been previously reported that ROS production is stimulated by natural bacterial infection through the expression of Nox and Duox enzymes, which help clear the pathogen in the Drosophila . 17 Therefore, the ROS production was examined in the fly gut and the carcass, accounting for localized and systemic oxidative stress, respectively. ROS levels in the fly gut were found to be elevated promptly after the infection (after 4 hours of feeding) across all infection groups (Figure 2A), suggesting rapid immune sensing. However, at 24 hpi, the ROS levels in pathogen-fed flies were species-specific, remaining unchanged in some and decreasing in others in the gut (Figure 2B). Nevertheless, by 48 hpi, ROS resurge was observed in the guts for all pathogen-infected flies (Figure 2C). Flies infected with E. coli did not exhibit any surge at either 4 hpi or at 48 hpi, underscoring that ROS activation depends upon bacterial pathogenicity. Notably, no significantly enhanced levels were observed in the carcass, indicating the localized immune response. We next assessed the induction of Nox and Duox transcript levels in the fly guts. At 24 hpi, Nox and Duox levels are unaffected in the guts of bacteria-fed flies, except in K. pneumoniae- infected flies, where Duox was upregulated, but this may not have been sufficient to induce detectable ROS in the gut (Figure 2D). By 48 hpi, Nox and Duox were upregulated (Figure 2D-E), consistent with ROS production in the gut (Figure 2B–2C). Interestingly, Duox levels were found to be induced to a greater extent as compared tothose of Nox , suggesting that Duox is the major contributor to ROS production. Altogether, these results demonstrate that infection by these bacteria triggers ROS production in the fly gut primarily through the activation of Duox , and to a lesser extent via Nox . To further confirm the role of ROS in bacterial clearance from the host, we conducted a survival assay in flies having ubiquitously silenced Duox by RNA interference( Actin Gal-4>DuoxIR ). The Duox knockout flies infected with the pathogens did not exhibit a significant change in their survival (Supplementary file 3)as compared to their respective control flies ( Actin Gal-4>UASGFP). Enteric infection in Drosophila did not compromise gut barrier integrity ROS is known to impair the enterocytes, affecting the intestinal integrity. Thus, the impact of altered ROS on the intestinal integrity of infected flies was monitored using a Smurf assay. The Smurf phenotype is characterized by the leakage of an ingested blue dye from the gut lumen into the body cavity resulting in a whole-body blue coloration and indicating loss of gut barrier integrity. Breaching of gut integrity has been shown to be closely associated with disruption of intestinal homeostasis. 18 None of the infected flies exhibited Smurf phenotype(Figure 3B, C, D, E, F). We next assessed their ability to clear the dye from their gastrointestinal tract. All the flies cleared the dye from their gut, with no residual dye visible in their abdomens (Figure 3B′, C′, D′, E′, F′),illustrating the proper gut functioning and intestinal motility. Species-specific Toll and Imd pathway activation of AMPs after infection Given the intact intestinal barrier, we evaluated the activation of Drosophila AMPs in response to infection to confirm whether local immune responses remained low, consistent with previous findings. 19 We assessed the expression levels of Cecropin and Defensin in the gut of the infected flies at 24 and 48 hpi (Figure 4A–4B). We found that at 24 hpi, the levels of Cecropin were significantly downregulated among all bacterial infection groups (Figure 4A), except in the E. cloacae infected flies, where they were slightly upregulated by 1.59-fold compared to the control. However, at 48 hpi, Cecropin was mildly upregulated in E. cloacae infections by 1.9-fold , but for other infection groups, the expression levels were either downregulated or similar to that of uninfected control (Figure 4A). Although the IMD pathway primarily defends against Gram-negative bacteria, the Toll pathway also contributes, probably due to the crosstalk between the two pathways, where the effectors of the Toll pathway can modulate the IMD responses and vice versa. 20 Hence, we monitored the expression levels of Defensin (Figure 4B) , a key AMP secreted via the Toll pathwayin the gut of the infected flies. The expression of Defensin remained uninduced or downregulated across all infection groups at both the time points, except for K. pneumoniae -infected guts, where an upregulation was observed at 24 hpi, but not maintained at a high level till 48 hpi. Together, these results suggested that Drosophila AMP levels were selectively unchanged with distinct immune pathways activated against different bacterial pathogens. 21 TRPA1 mediates the bacterial expulsion As ROS showed dynamic changes without Smurf phenotype or AMP induction, we hypothesized that bacterial clearance involves ROS along with other mechanisms and therefore tried to understand how ROS interacts with intestinal contractions involved in bacterial expulsion. Previous studies show that TRPA1 in enteroendocrine cells links ROS to bacterial shedding through ROS-induced defecation. 22 TRPA1, activated by stimuli such as ROS, regulates intestinal muscle activity in Drosophila after bacterial ingestion. 9, 23 To test our hypothesis, we quantified the expression of TRPA1 in the infected fly guts at 4 hpi and 24 hpi (Figure 5A–5C). TRPA1 was mildly upregulated at 4 hpi in the guts of S . Typhimurium and E. cloacae infected flies by 1.9 and 1.4-fold, respectively, while it remained uninduced in the guts of K. pneumoniae -infected flies (Figure 5B). By 24 hpi, the induction became more significant with a 2.5-fold increase in S . Typhimurium infected flies and a 3.3-fold increase in E. cloacae infected flies (Figure 5C), while with K. pneumoniae there was an insignificant induction. The sustained upregulation of TRPA1 likely promoted intestinal contraction, facilitatingbacterial expulsion. Therefore, we assessed the bacteria shed by the infected flies using CFU analysis at 24 and 48 hpi (Figure 5D). We observed that all three pathogens were shed, though the K. pneumoniae shedding does not correlate with TRPA1 expression. E. coli colonies were not recovered in the shedding experiment, indicating the killing of this bacterium by the immune response active in the fly gut. Thus, our data collectively suggest that TRPA1 facilitates intestinal contractions that expel bacteria, demonstrating that a combination of ROS and gut motility is involved in bacterial clearance. Discussion In this study, we employed an ecologically relevant natural feeding route in Drosophila melanogaster infection modelto investigate the gut defense response against a diverse spectrum of Gram-negative bacteria. Here, we highlighted that Drosophila mounts a specific response depending upon the bacterial species it encounters. Unlike previous studies, 24, 3 we excluded antimicrobial additives from the fly food to ensure infection outcomes precisely reflect host–pathogen interactions. We demonstrated that the induction of ROS in response to pathogens possibly leads to intestinal contractions, expelling the ingested bacteria. Our work further emphasises the importance of gut contractions as a non-immune mechanism, beyond their well-established functions like aiding the efficient nutrient absorption and regulating feeding behavior. 25 Leveraging the transparency of the Drosophila abdomen, we monitored the fate of fluorescent bacteria ingested by the flies. Our observation of gradual bacterial clearance within two days of infection highlights the efficiency of the Drosophila innate immune system in resolving infection. This corroborates the findings of an earlier study 26 where it was reported that the number of flies with no CFU increased over time after feeding on Pseudomonas entomophila . Our study shows that clearance and persistent infections are both probable outcomes of the bacterial infection. We observed that E. coli, S. Typhimurium, and K. pneumoniae are effectively cleared from the fly body by 48 hpi. However, E. cloacae persisted until 72 hpi without impairing host homeostasis, with the highest bacterial burden in the infected flies despite no detectable fluorescence signal at this time point. E. cloacae is characterized by lower virulence, which likely contributes to its ability to maintain stable colonization by avoiding significant harm to the host. 27 The discrepancy between the fluorescence and CFU data can be attributed to multiple factors, including bacterial localization from the gut to internal tissues, such as the head, host digestion, and immune response, which can possibly degrade the fluorescent tagged bacteria, thus reducing the fluorescence signals. 28 Notably, we observed that the enteric pathogens used in our study did not compromise the gut barrier integrity and hence, could not establish systemic infection. However, previous studies have reported that bacteria like S. marcescens, when ingested, enter the body cavity but do not kill their host rapidly, unlike in septic injury models, where it can disrupt gut barrier integrity and kill the host within a day after infection. Such findings highlight that different pathogensexhibit different levels of virulence in two infection models, likely influenced by their exposure to midgut defenses. 12 Several infection studies in Drosophila addressing gut immunity help us to understand how the host resists and recovers from microbial infections. 29 Resistance includes the localized production of ROS and AMPs, 30 while resilience depends upon the ability of intestinal epithelium to regenerate and maintain gut homeostasis. In the current work, effective clearance of the blue food from the intestines of infected flies indicated resilience of the intestinal barrier following infection. We demonstrated that a rapid ROS burst is crucial for controlling bacterial growth in the Drosophila gut. We observed dynamic induction of intestinal ROS on infection with S. Typhimurium, K. pneumoniae, and E. cloacae. A ROS surge at 4 hpi is consistent with the pathogen colonization in the flies, except for the non-pathogenic E. coli. This could be due to exposure to bacterial pathogen associated molecular patterns, such as lipopolysaccharide, which are rapidly detected by pathogen recognition receptors. Interestingly, ROS levels declined at 24 hpi, accompanied by the downregulation Nox and Duox, members of the NADPH oxidase family. Previous studies have also discussed the efficacy of the intestinal ROS in blocking the pathogenic bacteria in the anterior section of the intestine, as early as 15 minutes of ingestion. This rapid oxidative burst limits bacterial proliferation, subsequently clearing the bacteria. 23 However, Duox knockout flies did not exhibit increased susceptibility to S. Typhimurium, K. pneumoniae, and E. cloacae, leaving the role of ROS in eliminating these pathogens unclear. Although ROS is important for gut homeostasis, its overproduction can cause damage to intestinal cells. 31 The decline in ROS levels at 24 hpi could be due to the antioxidant activity of the immune system, like the immune-regulated catalase (IRC), 32 which plays a crucial role in mitigating the host damage post-infection. However, this reduction in ROS did not affect bacterial survival. Instead, all the pathogens, except E. cloacae, were cleared within 48 hpi. Later at 48 hpi, a surge in the ROS was again observed throughout the infection groups. This elevated ROS at later stages may have functioned in two ways—by exerting an antimicrobial effect against the persisted bacteria or to induce local AMP response in the gut, thereby activating the IMD pathway. 33 For instance, Erwinia carotovora carotovora 15 ( Ecc15 ) infection activates the Relish (Rel) transcription factor for AMP expression in the fat body. Cecropin,a Rel-activated AMP, is significantly upregulated in Drosophila gut after the oral infection with P. entomophilia. 26 Its losshas a marked impact on infection with E. cloacae and Providencia heimbachii. 13, 34 In our study, we also observed a 2 to 3-fold activation of Rel in the whole body of S . Typhimurium and E. cloacae infected flies at 48 hpi (Supplementary file 4). Contrarily, Cecropin expression either remained uninduced or downregulated across all infection groups, except in the guts of E. cloacae infected flies, where it was mildly upregulated at both 24 and 48 hpi, highlighting its persistence. The reason for the observed lack of robust upregulation of Cecropin in the infected flies in our study remains unclear and needs elucidation. Although our data suggest that ROS contributes to bacterial clearance, it also indicates that ROS alone is insufficient for complete bacterial eradication. 23 An intriguing question is what enables the infected flies to withstand bacterial infection when AMPs are not robustly induced, and gut integrity remains uncompromised? Further, there is a surge in ROS upon infection; still flies lacking the Duox gene exhibit no significant mortality, indicating that ROS does not play a role in clearing the infection. Possibly because ROS alone is insufficient to clear the bacteria. Reportedly, elevated levels of ROS during infection stimulate the TRPA1 receptor in gut enteroendocrine cells, thereby activating the production of neuropeptides, such as Diuretic Hormone 31. 22 TRPA1 stimulation enhances gut motility, increasing defecation rates and promoting the proliferation of epithelial stem cells. These processes work together to facilitate gut clearance and support tissue regeneration following infection. Collectively, we explored the role of the ROS-sensing TRPA1 receptor in bacterial clearance. Our findings highlighted that the activation of TRPA1 correlated with the rapid production of ROS and shedding of S . Typhimurium and E. cloacae . The shedding decreased with time (24 to 48 hpi), consistent with the reduction in internal bacterial load, which was also reduced by 48 hpi, likely suggesting that TRPA1 driven motility remains sustained, leading to bacterial clearance. Notably, K. pneumoniae -infected guts showed no induction of TRPA1, yet the bacteria were shed to significant levels, underscoring the species-specific nature of the host response. It is interesting to note that E. coli was not recovered in the shedding experiment, suggesting that it was effectively eliminated by the host's defenses. We further explored the intestinal muscular contractions of S. Typhimurium and E. cloacae infected fly guts, using light microscopy, and observed the narrowing of the gut lumen at 24 hpi, indicating a responsive contraction mechanism in the Drosophila gut (Supplementary file 5).Hence, our work sheds light on yet to be anatomically characterized intestinal muscular contractions, which appear to be conserved across species, presenting a potential model to study the propulsion of luminal content and maintain intestinal homeostasis. Conclusions Collectively, our findings underscore that the immune system of Drosophila is capable of both discerning between different bacterial species and adapting its response to bacteria in a tissue-specific manner. The differential activation and regulation of pathways in response to pathogens warrant further studies to understand if these responses can be used for repurposing the existing methods of treatment for pathogen clearance. Clinically, gut motility disorders, such as Intestinal Bowel Syndrome and bacterial gastroenteritis, are linked with intestinal muscular contractions. In many instances, drugs are prescribed to control intestinal spasms to relieve the pain, which might delay the clearance of the infection. Our findings suggest that intestinal muscular contractions could help get rid of bacterial infection. Methods Fly stocks Oregon-R flies were used in the study. Flies carrying Duox RNAi (a kind gift from Professor Anurag Sharma, NITTE, India) driven by ubiquitously expressed Actin-Gal4 was used to knock down Duox to investigate the role of ROS in bacterial clearance, and UAS-GFP reporter flies (a kind gift from Professor Subhash C Lakhotia, BHU, India). Drosophila stocks were maintained at 25°C on a 12:12 hour light:dark cycle. Bacterial strains Bacterial strains used in this study were E. coli, S . Typhimurium, S. marcescens, K. pneumoniae, and E. cloacae. The RFP expressing strains of E. coli and S . Typhimuriumwere obtained through their electro-transformation with plasmid pFPV-mcherry (Addgene plasmid #20956) , while those of K. pneumoniae and E. cloacae were obtained by transformation with plasmidpEB2-2ndVal mRFP1(Addgene plasmid #104025) . RFP expressing strains were cultured in Luria Broth medium supplemented with antibiotics for their selection: Ampicillin 100 µg/mL (Himedia, India) was used for E. coli and S. Typhimurium, while Kanamycin 50 µg/mL (Himedia, India) was used for K. pneumoniae and E. cloacae. Fly infection model and survival assays Overnight grown bacteria were subcultured in 10 mL of fresh media containing suitable antibiotics and allowed to grow until the culture OD 600 was between 0.3 and 0.5. These bacterial cultures were centrifuged, and the pellet was resuspended in 500 µL of 5% sucrose solution prepared in sterile phosphate buffered saline (PBS). Whatman filter papers soaked with 200 µL of these bacterial suspensions were placed inside the empty culture vials. Age-matched adult flies (3–5 days old) subjected to a 3-hour starvation were allowed to feed on these bacterial cultures for 4 hours. Flies fed only a 5% sucrose solution prepared in sterile PBS were used as a negative control. Following oral infection, the infected flies were transferred to the standard cornmeal agar devoid of antimicrobials, such as propionic acid and nipagin. The number of dead flies was recorded daily up to 30 days post-infection. Each day, surviving flies were transferred to the new vial containing antimicrobial-free standard cornmeal agar. These data was presented as Kaplan–Meier curves, plotting the percent surviving flies over time. Measurement of fly bacterial load by CFU assay Bacterial colonization in the fly body was determined as the number of CFU at 0-, 24-, 48-, and 72-hpi using the standard plate count method. Infected flies were gently washed thrice with 1 mL of sterile PBS. The washed flies were then homogenized in 500 µL of sterile PBS, using a bead beater at 20 Hz frequency for a 2-minute cycle (20 seconds homogenization, followed by 10 seconds on ice). The homogenate was diluted serially 1:10 and plated onto selective media to estimate bacterial load, as described previously. 35 Evaluation of gut permeability and dye clearance in flies After infecting the flies using the method described above, they were transferred to food containing 2.5% (wt./vol.) bromophenol blue for 12 hours. A fly was categorized as Smurf (observed using a microscope) if the dye could be observed outside the intestine 36 and the blue color was visible throughout its body. For the dye clearance assay, separate sets of flies were used. These flies were fed with the dye containing food for 12 hours as described above. After the examination for Smurf phenotype (if any), the flies were transferred to standard cornmeal agar food to assess their ability to clear the dye from the gut. 37 Photographs of the representative flies were taken using the Optika microscope camera (Optika, Italy). Estimation of ROS Infected fly guts were isolated, and both the gut and the carcass were added to 1.5 mL Eppendorf tubes containing 20 mM Tris Buffer. Tissue samples were ground using a pestle and then centrifuged. The supernatants were transferred to a fresh Eppendorf tube, and protein concentrations of the lysate were quantified using Bradford assay and the lysates amounting to 50 µg/µL protein were incubated with 5 µM DCF-DA for 60 minutes. Following this, fluorescence was measured with excitation at 490 nm and emission at 530 nm. Measurement of bacterial shedding from infected flies As described by Siva-Jothy, 24 the infected flies were individually transferred to an Eppendorf tube containing 50 µL of standard cornmeal agar and subsequently transferred to a fresh tube every 24 hours. The bacterial shedding was quantified at 24 and 48 hours using a serial dilution method by plating the tube content onto appropriate media. Analysis of immune gene expression Total RNA was isolated from 40–50 fly guts and/or 10–15 whole adult flies using the TRIzol reagent (Himedia, India) as specified in an earlier study. 38 Subsequently, 800 ng of total RNA was reverse transcribed to cDNA using Verso cDNA synthesis kit (AB1453A; Thermo Scientific, MA, USA) followed by qPCR reactions using gene-specific primers (Supplementary file 6) Statistical analysis All statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software, MA, USA). For intergroup comparison, an unpaired two-tailed Student’s t -test was used. For experiments involving multiple groups, two-way ANOVA, followed by Dunnett’s Multiple comparison test, was used. All data are presented as mean ± standard deviation. p-value less than 0.05 were considered statistically significant. All experiments were performed with at least three independent biological replicates. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding This research did not receive funding from any public, commercial, or nonprofit agency. Authors' contributions Conception and design of the work: SV, SM, and MT. Methodology + Analysis + Investigation: SV with the help of SM and MT. CFU Analysis: SB. SV has drafted the manuscript, and all authors have provided substantive revisions. All authors read and approved the final manuscript. Acknowledgements We would like to express our sincere gratitude to Birla Institute of Technology and Science, Pilani, for the invaluable infrastructural support. References Younes S, Al-Sulaiti A, Nasser EAA, Najjar H, Kamareddine L. Drosophila as a Model Organism in Host–Pathogen Interaction Studies. Front Cell Infect Microbiol. 2020;10:214. https://doi.org/10.3389/fcimb.2020.00214. Priyadarsini S, Sahoo M, Sahu S, Jayabalan R, Mishra M. An infection of Enterobacter ludwigii affects development and causes age-dependent neurodegeneration in Drosophila melanogaster. Invert Neurosci. 2019;19:13. https://doi.org/10.1007/s10158-019-0233-y. Vodovar N, Vinals M, Liehl P, Basset A, Degrouard J, Spellman P, et al. Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species. Proc Natl Acad Sci USA. 2005;102:11414–9. https://doi.org/10.1073/pnas.0502240102. Zhai Z, Huang X, Yin Y. Beyond immunity: The Imd pathway as a coordinator of host defense, organismal physiology and behavior. Developmental & Comparative Immunology. 2018;83:51–9. https://doi.org/10.1016/j.dci.2017.11.008. Moghadam ZM, Henneke P, Kolter J. From Flies to Men: ROS and the NADPH Oxidase in Phagocytes. Front Cell Dev Biol. 2021;9:628991. https://doi.org/10.3389/fcell.2021.628991. Shaka M, Arias-Rojas A, Hrdina A, Frahm D, Iatsenko I. Lipopolysaccharide -mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster. PLoS Pathog. 2022;18:e1010825. https://doi.org/10.1371/journal.ppat.1010825. Buchon N, Broderick NA, Poidevin M, Pradervand S, Lemaitre B. Drosophila Intestinal Response to Bacterial Infection: Activation of Host Defense and Stem Cell Proliferation. Cell Host & Microbe. 2009;5:200–11. https://doi.org/10.1016/j.chom.2009.01.003. Sun Y, Wang X, Li L, Zhong C, Zhang Y, Yang X, et al. The role of gut microbiota in intestinal disease: from an oxidative stress perspective. Front Microbiol. 2024;15:1328324. https://doi.org/10.3389/fmicb.2024.1328324. Benguettat O, Jneid R, Soltys J, Loudhaief R, Brun-Barale A, Osman D, et al. The DH31/CGRP enteroendocrine peptide triggers intestinal contractions favoring the elimination of opportunistic bacteria. PLoS Pathog. 2018;14:e1007279. https://doi.org/10.1371/journal.ppat.1007279. Landini L, Souza Monteiro De Araujo D, Titiz M, Geppetti P, Nassini R, De Logu F. TRPA1 Role in Inflammatory Disorders: What Is Known So Far? IJMS. 2022;23:4529. https://doi.org/10.3390/ijms23094529. Ogawa N, Kurokawa T, Fujiwara K, Polat OK, Badr H, Takahashi N, et al. Functional and Structural Divergence in Human TRPV1 Channel Subunits by Oxidative Cysteine Modification. Journal of Biological Chemistry. 2016;291:4197–210. https://doi.org/10.1074/jbc.M115.700278. Nehme NT, Liégeois S, Kele B, Giammarinaro P, Pradel E, Hoffmann JA, et al. A Model of Bacterial Intestinal Infections in Drosophila melanogaster. PLoS Pathog. 2007;3:e173. https://doi.org/10.1371/journal.ppat.0030173. Carboni AL, Hanson MA, Lindsay SA, Wasserman SA, Lemaitre B. Cecropins contribute to Drosophila host defense against a subset of fungal and Gram-negative bacterial infection. Genetics. 2022;220:iyab188. https://doi.org/10.1093/genetics/iyab188. Wang L, Lin J, Yu J, Yang K, Sun L, Tang H, et al. Downregulation of Perilipin1 by the Immune Deficiency Pathway Leads to Lipid Droplet Reconfiguration and Adaptation to Bacterial Infection in Drosophila . The Journal of Immunology. 2021;207:2347–58. https://doi.org/10.4049/jimmunol.2100343. Tracy C, Krämer H. Escherichia coli Infection of Drosophila. BIO-PROTOCOL. 2017;7. https://doi.org/10.21769/BioProtoc.2256. Keller R, Pedroso MZ, Ritchmann R, Silva RM. Occurrence of Virulence-Associated Properties in Enterobacter cloacae . Infect Immun. 1998;66:645–9. https://doi.org/10.1128/IAI.66.2.645-649.1998. Ramond E, Jamet A, Ding X, Euphrasie D, Bouvier C, Lallemant L, et al. Reactive Oxygen Species-Dependent Innate Immune Mechanisms Control Methicillin-Resistant Staphylococcus aureus Virulence in the Drosophila Larval Model. mBio. 2021;12:e00276-21. https://doi.org/10.1128/mBio.00276-21. Galenza A, Foley E. A glucose-supplemented diet enhances gut barrier integrity in Drosophila . Biology Open. 2021;10:bio056515. https://doi.org/10.1242/bio.056515. Zane F, Bouzid H, Sosa Marmol S, Brazane M, Besse S, Molina JL, et al. Smurfness‐based two‐phase model of ageing helps deconvolve the ageing transcriptional signature. Aging Cell. 2023;22:e13946. https://doi.org/10.1111/acel.13946. De Gregorio E. The Toll and Imd pathways are the major regulators of the immune response in Drosophila. The EMBO Journal. 2002;21:2568–79. https://doi.org/10.1093/emboj/21.11.2568. Hanson MA, Dostálová A, Ceroni C, Poidevin M, Kondo S, Lemaitre B. Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach. eLife. 2019;8:e44341. https://doi.org/10.7554/eLife.44341. Du EJ, Ahn TJ, Kwon I, Lee JH, Park J-H, Park SH, et al. TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway. PLoS Genet. 2016;12:e1005773. https://doi.org/10.1371/journal.pgen.1005773. Tleiss F, Montanari M, Milleville R, Pierre O, Royet J, Osman D, et al. Spatial and temporal coordination of Duox/TrpA1/Dh31 and IMD pathways is required for the efficient elimination of pathogenic bacteria in the intestine of Drosophila larvae. eLife. 2024;13:RP98716. https://doi.org/10.7554/eLife.98716.3. Siva-Jothy JA, Prakash A, Vasanthakrishnan RB, Monteith KM, Vale PF. Oral Bacterial Infection and Shedding in Drosophila melanogaster. JoVE. 2018;:57676. https://doi.org/10.3791/57676. Mace OJ, Tehan B, Marshall F. Pharmacology and physiology of gastrointestinal enteroendocrine cells. Pharmacology Res & Perspec. 2015;3:e00155. https://doi.org/10.1002/prp2.155. Paulo TF, Akyaw PA, Paixão T, Sucena É. Evolution of resistance and disease tolerance mechanisms to oral bacterial infection in Drosophila melanogaster . Open Biol. 2025;15:240265. https://doi.org/10.1098/rsob.240265. Hidalgo BA, Silva LM, Franz M, Regoes RR, Armitage SAO. Decomposing virulence to understand bacterial clearance in persistent infections. Nat Commun. 2022;13:5023. https://doi.org/10.1038/s41467-022-32118-1. Bell KS, Ko S, Ali S, Bognar B, Khmelkov M, Rau N, et al. Tissue-Specific Fluorescent Protein Turnover in Free-Moving Flies. Insects. 2025;16:583. https://doi.org/10.3390/insects16060583. Erkosar B, Leulier F. Transient adult microbiota, gut homeostasis and longevity: Novel insights from the Drosophila model. FEBS Letters. 2014;588:4250–7. https://doi.org/10.1016/j.febslet.2014.06.041. Tafesh-Edwards G, Eleftherianos I. The role of Drosophila microbiota in gut homeostasis and immunity. Gut Microbes. 2023;15:2208503. https://doi.org/10.1080/19490976.2023.2208503. Kuraishi T, Hori A, Kurata S. Host-microbe interactions in the gut of Drosophila melanogaster. Front Physiol. 2013;4. https://doi.org/10.3389/fphys.2013.00375. Prakash A, Monteith KM, Vale PF. Mechanisms of damage prevention, signalling, and repair impact disease tolerance. 2021. https://doi.org/10.1101/2021.10.03.462916. Wu S-C, Liao C-W, Pan R-L, Juang J-L. Infection-Induced Intestinal Oxidative Stress Triggers Organ-to-Organ Immunological Communication in Drosophila. Cell Host & Microbe. 2012;11:410–7. https://doi.org/10.1016/j.chom.2012.03.004. Kylsten P, Samakovlis C, Hultmark D. The cecropin locus in Drosophila; a compact gene cluster involved in the response to infection. The EMBO Journal. 1990;9:217–24. https://doi.org/10.1002/j.1460-2075.1990.tb08098.x. Chambers MC, Jacobson E, Khalil S, Lazzaro BP. Consequences of chronic bacterial infection in Drosophila melanogaster. PLoS ONE. 2019;14:e0224440. https://doi.org/10.1371/journal.pone.0224440. Akhmetova K, Balasov M, Chesnokov I. Drosophila STING protein has a role in lipid metabolism. eLife. 2021;10:e67358. https://doi.org/10.7554/eLife.67358. Livingston DBH, Patel H, Donini A, MacMillan HA. Active transport of brilliant blue FCF across the Drosophila midgut and Malpighian tubule epithelia. 2019. https://doi.org/10.1101/771675. Narwal S, Singh A, Tare M. Analysis of α-syn and parkin interaction in mediating neuronal death in Drosophila model of Parkinson’s disease. Front Cell Neurosci. 2024;17:1295805. https://doi.org/10.3389/fncel.2023.1295805. Additional Declarations No competing interests reported. Supplementary Files Vermaetal.Supplementaryfile.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Jan, 2026 Reviews received at journal 19 Jan, 2026 Reviews received at journal 19 Jan, 2026 Reviewers agreed at journal 19 Jan, 2026 Reviews received at journal 18 Jan, 2026 Reviews received at journal 18 Jan, 2026 Reviews received at journal 18 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviews received at journal 14 Jan, 2026 Reviews received at journal 13 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 11 Jan, 2026 Reviewers agreed at journal 11 Jan, 2026 Reviewers agreed at journal 11 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers invited by journal 09 Jan, 2026 Editor invited by journal 31 Dec, 2025 Editor assigned by journal 30 Dec, 2025 Submission checks completed at journal 30 Dec, 2025 First submitted to journal 29 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8473557","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":573299789,"identity":"6611eeb1-d881-4204-b83f-c164c022b549","order_by":0,"name":"Shreya Verma","email":"","orcid":"","institution":"Birla Institute of Technology and Science, Pilani","correspondingAuthor":false,"prefix":"","firstName":"Shreya","middleName":"","lastName":"Verma","suffix":""},{"id":573299790,"identity":"09117254-f969-43cd-9083-cc8c121ffb4e","order_by":1,"name":"Sushovan Bhattacharyya","email":"","orcid":"","institution":"Birla Institute of Technology and Science, Pilani","correspondingAuthor":false,"prefix":"","firstName":"Sushovan","middleName":"","lastName":"Bhattacharyya","suffix":""},{"id":573299791,"identity":"0a2e01d4-78e3-465b-9f61-44e4a941c2bb","order_by":2,"name":"Meghana Tare","email":"","orcid":"","institution":"Birla Institute of Technology and Science, Pilani","correspondingAuthor":false,"prefix":"","firstName":"Meghana","middleName":"","lastName":"Tare","suffix":""},{"id":573299797,"identity":"558dea00-d5e3-43ef-9949-f825e3a09cc3","order_by":3,"name":"Sandhya Amol Marathe","email":"data:image/png;base64,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","orcid":"","institution":"Birla Institute of Technology and Science, Pilani","correspondingAuthor":true,"prefix":"","firstName":"Sandhya","middleName":"Amol","lastName":"Marathe","suffix":""}],"badges":[],"createdAt":"2025-12-29 13:38:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8473557/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8473557/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100368648,"identity":"ed1b9db9-c49d-4975-9ecd-42097da913d6","added_by":"auto","created_at":"2026-01-16 07:58:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13841832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduction in fluorescence intensity and internal bacterial load after natural infection with RFP-tagged bacteria.\u003c/strong\u003eFlies fed 5% sucrose solution served as the control.\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003e RFP expressing bacteria were observed in the fly abdomens immediately after oral infection. \u003cstrong\u003e(A′) \u003c/strong\u003eThe fluorescent signal gradually diminished between 24hpi and \u003cstrong\u003e(Aʺ)\u003c/strong\u003e 48 hpi. \u003cstrong\u003e(B)\u003c/strong\u003e Representative graph depicting a decline in the fluorescence intensity over the time (in Arbitrary units [AU]). \u003cstrong\u003e(C)\u003c/strong\u003eInternal bacterial counts were obtained by plating the homogenates of two flies. \u003cem\u003eE. coli\u003c/em\u003e, a commensal bacterium in \u003cem\u003eDrosophila\u003c/em\u003e, was used as the reference control for statistical analyses of bacterial clearance. The number of CFU per fly obtained at each time point after oral infection represents the mean of three independent measurements. Error bars indicate Standard Deviation (SD). Statistical significance is denoted as follows: ****, p \u0026lt; 0.0001; and ns, not significant.\u003c/p\u003e","description":"","filename":"Vermaetal.Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8473557/v1/213ea88786810cbb1827d0cb.png"},{"id":100171094,"identity":"d19f005c-4eb3-491e-8b3e-2f9dbfad7a02","added_by":"auto","created_at":"2026-01-13 16:39:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1648785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic ROS production and regulation by ROS generating enzymes in infected flies at mRNA level.\u003c/strong\u003e \u003cstrong\u003e(A and C)\u003c/strong\u003e Surges in ROS production were observed in the gut of the infected flies at 4 and 48 hpi, respectively, \u003cstrong\u003e(B)\u003c/strong\u003e with a transient decrease at 24 hpi. No ROS elevation was observed in the carcass of the infected flies. \u003cstrong\u003e(D-E)\u003c/strong\u003e A quantitative real time PCR (qRT-PCR) analysis of \u003cem\u003eNox\u003c/em\u003e and \u003cem\u003eDuox \u003c/em\u003etranscripts was done using total RNA extracted from the gut lysate recovered at \u003cstrong\u003e(D)\u003c/strong\u003e 24 and \u003cstrong\u003e(E)\u003c/strong\u003e 48 hpi. \u003cem\u003eNox\u003c/em\u003eand, primarily, \u003cem\u003eDuox\u003c/em\u003e at 48 hpi are responsible for the elevated ROS levels in the gut lysate. Data is represented as the mean ± SD from three independent biological replicates. Statistical significance is denoted as follows: *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001 and ns, not significant.\u003c/p\u003e","description":"","filename":"Vermaetal.Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8473557/v1/22343c18ef28c63b1917f7f1.png"},{"id":100171093,"identity":"464d3220-3a11-4b60-8c60-2ba8f3f8deed","added_by":"auto","created_at":"2026-01-13 16:39:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11561109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaintenance of gut integrity and effective intestinal dye clearance in the infected flies.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eFlies fed with 0.6% SDS exhibited a Smurf phenotype, acting as a positive control. Representatives of intact intestinal barrier integrity and clearance of the dye of control and infected flies. \u003cstrong\u003e(B, C, D, E, F)\u003c/strong\u003e No spreading of blue dye in the abdomen was observed in the infected flies within 12 hpi. \u003cstrong\u003e(B′, C′, D′, E′, F′)\u003c/strong\u003e Both control and infected flies cleared the dye from their gut after an additional 12 hours of feeding on standard cornmeal agar.\u003c/p\u003e","description":"","filename":"Vermaetal.Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8473557/v1/ab39b172b6a0af49163925b3.png"},{"id":100171092,"identity":"3b2e64ec-23fc-4e3f-9745-8a0ed07c322f","added_by":"auto","created_at":"2026-01-13 16:39:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":500013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathogen-specific induction of AMPs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003efollowing infection\u003c/strong\u003e. \u003cstrong\u003e(A-B)\u003c/strong\u003e mRNA transcript levels of \u003cem\u003eCecropin\u003c/em\u003e and \u003cem\u003eDefensin\u003c/em\u003e in the guts of control and infected flies were determined by qRT-PCR using \u003cem\u003erp49\u003c/em\u003e gene as an internal control. Relative expression was calculated against control gut samples. Each biological replicate consisted of 45 guts. Data is represented as the mean ± SD from three independent biological replicates. Significance levels are denoted as follows: *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001 and ns, not significant.\u003c/p\u003e","description":"","filename":"Vermaetal.Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8473557/v1/f0a569a83a7ea4d4064d160d.png"},{"id":100171097,"identity":"c075c889-d831-4844-a24f-fe374d053afa","added_by":"auto","created_at":"2026-01-13 16:39:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7062742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathogen-specific regulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTRPA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transcript levels and its impact on bacterial clearance. (A)\u003c/strong\u003eIndividual flies were incubated in tubes with cornmeal agar for 24 hours and were transferred to a different tube for an additional 24 hours. Bacteria shed onto the agar were calculated by CFU assay. \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eTRPA1\u003c/em\u003e transcript levels in the guts following bacterial infection at 4 and \u003cstrong\u003e(C)\u003c/strong\u003e 24 hpi were quantified using qRT-PCR relative to uninfected control, using \u003cem\u003erp49 \u003c/em\u003egene as an internal control. \u003cstrong\u003e(D)\u003c/strong\u003e Bacteria shed by infected flies at 24 and 48 hpi. \u003cem\u003eE. coli \u003c/em\u003ewas used as a reference control and did not yield colonies, so no statistical analysis was applied for \u003cem\u003eE. coli \u003c/em\u003eshedding data. Data is represented as the mean ± SD from three independent biological replicates. Statistical significance for \u003cem\u003eTRPA1\u003c/em\u003e expression was assessed by comparing to uninfected controls. Significance levels are denoted as follows: *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; and ns, not significant.\u003c/p\u003e","description":"","filename":"Vermaetal.Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8473557/v1/4319ed3b9f229d9c6647a5c9.png"},{"id":100382466,"identity":"154b821b-8d34-46d4-b0e4-8e8af3c8ae5b","added_by":"auto","created_at":"2026-01-16 10:42:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29563142,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8473557/v1/1a28b776-d4e4-4e66-a2a0-648476725f37.pdf"},{"id":100171095,"identity":"9dcdd53c-88d3-40df-aabc-96d4af78d6f0","added_by":"auto","created_at":"2026-01-13 16:39:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2354760,"visible":true,"origin":"","legend":"","description":"","filename":"Vermaetal.Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8473557/v1/b4442a25fcb83bb61acda75b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Drosophila melanogaster mitigates gastro-oral infections by stimulating pathogen expulsion","fulltext":[{"header":"Background","content":"\u003cp\u003eIn view of thevast research conducted on host-pathogen interactions, the mechanisms underlying variations in the individual responses to similar Gram-negative bacterial infections remain less understood. To explore this, we utilized\u003cem\u003e\u0026nbsp;Drosophila melanogaster\u0026nbsp;\u003c/em\u003eas a hostmodel organism to understand the immune responses that are coordinated and regulated at the whole organism level. The increasing use of \u003cem\u003eDrosophila\u003c/em\u003e in the studies of infectious diseases and control is due to its degree of genetic and molecular conservation with vertebrates, particularly in innate immune cascades, transduction pathways, and transcriptional regulators.\u003csup\u003e1\u003c/sup\u003eMoreover, ithas been used to study the pathogenesis of a wide variety of microorganisms, as it also serves as a model of low microbiome complexity.\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo combat bacterial infections, \u003cem\u003eDrosophila\u003c/em\u003e relies solely on its innate immune system, as it is devoid of adaptive immune response for its defense.\u003csup\u003e3\u003c/sup\u003e The innate responses are categorized as cellular and humoral, where cellular responses are phagocytosis and encapsulation, while the humoral responses include the activation of evolutionarily conserved immune response cascades, the immune deficiency (IMD), and Toll pathways.\u003csup\u003e4\u003c/sup\u003e In cellular responses, hemocytes produce reactive oxygen species (ROS) during bacterial infections. The major source of ROS is nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (\u003cem\u003eNox\u003c/em\u003e)\u003csup\u003e5\u0026nbsp;\u003c/sup\u003eand dual oxidases (\u003cem\u003eDuox\u003c/em\u003e)derived from hemocytes.\u003csup\u003e6\u003c/sup\u003e In \u003cem\u003eDrosophila\u003c/em\u003e, ROS production as early as 30 minutes after pathogen ingestion is a conserved response to infection. However, ROS can have a destructive impact on gut mucosal cells; therefore, its activity must be tightly regulated to minimize\u0026nbsp;host tissue injury.\u003csup\u003e7\u003c/sup\u003e While many previous studies have highlighted the role of ROS in the gut beyond their antibacterial activity, emerging evidence suggests that ROS can modulate gut functions.\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe ROS produced in response to bacterial infection triggers visceral spasms mediated by TRPA1 channels. These visceral spasms induce intestinal contractions, expediting bacterial elimination from the host.\u003csup\u003e9\u003c/sup\u003e Notably, \u003cem\u003eTRPA1\u003c/em\u003e is also involved in inflammatory bowel diseases, where it affects the gut inflammation and has dual roles: pro- and anti-inflammatory roles,\u003csup\u003e10\u003c/sup\u003e making it an attractive target to treat inflammatory disorders. The interaction between oxidative stress and the mechanosensory pathway seems to be conserved, as \u003cem\u003eDrosophila\u0026nbsp;\u003c/em\u003eTRPA1 is a homolog of mammalian TRP receptors responsive to stressful conditions.\u003csup\u003e11\u003c/sup\u003e For infection studies, usually flies\u0026nbsp;are infected with bacteria, either through the septic injury method, where bacteria are directly introduced into the body cavity, bypassing the epithelial barriers, or through the oral infection route, where bacteria are fed to the flies. A few pathogens, such as \u003cem\u003eSerratia marcescens (S. marcescens), Escherichia coli (E. coli)\u003c/em\u003e, \u003cem\u003eSalmonella enterica\u0026nbsp;\u003c/em\u003esubspecies enterica serovarTyphimurium (\u003cem\u003eS\u003c/em\u003e. Typhimurium), and \u003cem\u003eEnterobacter cloacae (E. cloacae),\u0026nbsp;\u003c/em\u003ehave been extensively studied in \u003cem\u003eDrosophila\u003c/em\u003e using a septic injury model to assess the biological responses encompassing survival rates, change in gut morphology, alterations in triglyceride levels, activation of IMD pathway, and bacterial load.\u003csup\u003e12-14\u003c/sup\u003e However, this does not represent the gut-specific immunocompetence, relevant to the natural route of\u0026nbsp;infection. To address this gap, we developed a \u003cem\u003eDrosophila\u003c/em\u003e oral infection model in this study\u0026nbsp;by feeding the flies bacterial suspensions prepared in sucrose for a brief duration. This natural infection model enabled us to track the fate of orally ingested bacteria and characterize the gut immune responses to specific Gram-negative bacteria, including \u003cem\u003eE. coli, S\u003c/em\u003e. Typhimurium,\u003cem\u003e\u0026nbsp;Klebsiella pneumoniae (K. pneumoniae),\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE. cloacae\u003c/em\u003e. Although traditionally, \u003cem\u003eDrosophila\u003c/em\u003e culture is also supplemented with preservatives like Methyl Paraben (Nipagin) and Propionic acid to ensure healthy conditions for the cultures, these preservatives may affect the results of oral infection studies. To circumvent this, we investigated the effect of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e infections through oral administration to flies cultured without preservatives.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur study aims to investigate how \u003cem\u003eDrosophila\u003c/em\u003e mounts distinct immune responses to specific pathogens during natural infection, focusing on bacterial expulsion mediated by ROS and TRPA1. We observe that this interplay between oxidative stress and sensory receptor orchestrates a pathogen-specific gut strategy, minimizing gut damage while clearing the bacteria.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eInfection of \u003cem\u003eDrosophila\u003c/em\u003e with different bacteria caused species-specific colonization and clearance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ensure that antimicrobial agents, propionic acid, and nipagin, typically added to fly food, do not impact the bacterial survival and clearance, we tested the antibacterial activity of these agents specifically against the pathogens used in the study. The results indicated that these agents, at a concentration generally added to fly food, exhibited antibacterial activity against \u003cem\u003eE. coli, S\u003c/em\u003e. Typhimurium,\u003cem\u003e\u0026nbsp;K. pneumoniae,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE. cloacae\u0026nbsp;\u003c/em\u003e(Supplementary file 1). Thus, in all further infection experiments, the flies were fed on the food without adding these agents.\u003c/p\u003e\n\u003cp\u003eThe presence of bacteria and the duration of their persistence in \u003cem\u003eDrosophila\u003c/em\u003e were monitored using fluorescence microscopy and\u0026nbsp;colony forming unit (CFU) analysis after feeding the flies RFP/m-cherry-tagged bacteria. We observed a strong red fluorescence in the fly's abdomen immediately after bacterial feeding (Figure 1A and 1B), indicating the ingestion of the bacteria.\u003csup\u003e15\u003c/sup\u003e Within 24 hours post-infection (hpi), the fluorescence reduced to a basal level in the flies fed with \u003cem\u003eE. coli,\u003c/em\u003e a non-pathogenic species, to \u003cem\u003eDrosophila,\u003c/em\u003e\u003csup\u003e16\u003c/sup\u003e with a subsequent reduction in bacterial burden within the fly body (Figure 1C). This indicates efficient elimination of \u003cem\u003eE. coli\u003c/em\u003e by the \u003cem\u003eDrosophila\u003c/em\u003e. Similar results were not obtained with other bacterial species. Within 24 hpi, though the fluorescence signal reduced to basal levels for \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eE. cloacae\u003c/em\u003e,their numbers remained sufficiently high in the fly body. The \u003cem\u003eK. pneumoniae\u003c/em\u003e numbers dropped by a factor of 5, while those of \u003cem\u003eE. cloacae\u003c/em\u003e increased 5-fold. At 24 hpi, the \u003cem\u003eS\u003c/em\u003e. Typhimurium fluorescence and numbers remained close to those of the ingested. By 48 hpi, the numbers of \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e. Typhimurium dropped close to those of \u003cem\u003eE. coli\u003c/em\u003e. However, for \u003cem\u003eE. cloacae\u003c/em\u003e, bacterial numbers decreased gradually and persisted up to 72 hpi\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite the presence of \u003cem\u003eE. cloacae\u0026nbsp;\u003c/em\u003ein \u003cem\u003eDrosophila\u003c/em\u003e till 72 hpi and other pathogens till 48 hpi, the survivability of infected flies was not compromised up to 30 days post-infection (dpi) (Supplementary file 2). This suggested that oral infection was efficiently controlled by the host's innate immune responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial infection triggers host intestinal ROS production in a dynamic pattern\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt has been previously reported that\u0026nbsp;ROS production is stimulated by natural bacterial infection through the expression of Nox and Duox enzymes, which help clear the pathogen in the \u003cem\u003eDrosophila\u003c/em\u003e.\u003csup\u003e17\u003c/sup\u003e Therefore, the ROS production was examined in the fly gut and the carcass, accounting for localized and systemic oxidative stress, respectively. ROS levels in the fly gut were found to be elevated promptly after the infection (after 4 hours of feeding) across all infection groups (Figure 2A), suggesting rapid immune sensing. However, at 24 hpi, the ROS levels in pathogen-fed flies were species-specific, remaining unchanged in some and decreasing in others in the gut (Figure 2B). Nevertheless, by 48 hpi, ROS resurge was observed in the guts for all pathogen-infected flies (Figure 2C). Flies infected with \u003cem\u003eE. coli\u003c/em\u003e did not exhibit any surge at either 4 hpi or at 48 hpi, underscoring that ROS activation depends upon bacterial pathogenicity. Notably, no significantly enhanced levels were observed in the carcass, indicating the localized immune response.\u003c/p\u003e\n\u003cp\u003eWe next assessed the induction of \u003cem\u003eNox\u0026nbsp;\u003c/em\u003eand \u003cem\u003eDuox\u0026nbsp;\u003c/em\u003etranscript levels in the fly guts. At 24 hpi, \u003cem\u003eNox\u003c/em\u003e and \u003cem\u003eDuox\u003c/em\u003e levels are unaffected in the guts of bacteria-fed flies,\u0026nbsp;except in \u003cem\u003eK. pneumoniae-\u003c/em\u003einfected flies, where \u003cem\u003eDuox\u003c/em\u003e was upregulated,\u0026nbsp;but this\u0026nbsp;may not have been sufficient to induce detectable ROS in the gut\u0026nbsp;(Figure 2D). By 48 hpi, \u003cem\u003eNox\u003c/em\u003e and \u003cem\u003eDuox\u003c/em\u003e were upregulated (Figure 2D-E),\u0026nbsp;consistent with ROS production in the gut (Figure 2B–2C). Interestingly, \u003cem\u003eDuox\u003c/em\u003e levels were found to be induced to a greater extent as compared tothose of \u003cem\u003eNox\u003c/em\u003e, suggesting that \u003cem\u003eDuox\u003c/em\u003e is the major contributor to ROS production. Altogether, these results demonstrate that infection by these bacteria triggers ROS production in the fly gut primarily through the activation of \u003cem\u003eDuox\u003c/em\u003e, and to a lesser extent \u003cem\u003evia Nox\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo further confirm the role of ROS in bacterial clearance from the host, we\u0026nbsp;conducted a survival assay in flies having ubiquitously silenced \u003cem\u003eDuox\u0026nbsp;\u003c/em\u003eby RNA interference(\u003cem\u003eActin Gal-4\u0026gt;DuoxIR\u003c/em\u003e). The \u003cem\u003eDuox\u0026nbsp;\u003c/em\u003eknockout flies infected with the pathogens did not exhibit a significant change in their survival (Supplementary file 3)as compared to their respective control flies (\u003cem\u003eActin Gal-4\u0026gt;UASGFP).\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnteric infection in \u003cem\u003eDrosophila\u003c/em\u003e did not compromise gut barrier integrity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eROS is known to impair the enterocytes, affecting the intestinal integrity. Thus, the impact of altered ROS on the intestinal integrity of infected flies was monitored using a Smurf assay. The Smurf phenotype is characterized by the leakage of an ingested blue dye from the gut lumen into the body cavity resulting in a whole-body blue coloration and indicating loss of gut barrier integrity. Breaching of gut integrity has been shown to be closely associated with disruption of intestinal homeostasis.\u003csup\u003e18\u003c/sup\u003eNone of the infected flies exhibited Smurf phenotype(Figure 3B, C, D, E, F). We next assessed their ability to clear the dye from their gastrointestinal tract. All the flies cleared the dye from their gut, with no residual dye visible in their abdomens (Figure 3B′, C′, D′, E′, F′),illustrating the proper gut functioning and intestinal motility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecies-specific Toll and Imd pathway activation of AMPs after infection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the intact intestinal barrier, we evaluated the activation of \u003cem\u003eDrosophila\u003c/em\u003e AMPs in response to infection to confirm whether local immune responses remained low, consistent with previous findings.\u003csup\u003e19\u003c/sup\u003e We assessed the expression levels of \u003cem\u003eCecropin\u003c/em\u003e and \u003cem\u003eDefensin\u0026nbsp;\u003c/em\u003ein the gut of the infected flies at\u0026nbsp;24 and 48 hpi (Figure 4A–4B). We found that at 24 hpi, the levels of \u003cem\u003eCecropin\u0026nbsp;\u003c/em\u003ewere significantly downregulated among all bacterial infection groups (Figure 4A), except in the \u003cem\u003eE. cloacae\u003c/em\u003e infected flies, where they were slightly upregulated by 1.59-fold compared to the control. However, at 48 hpi, \u003cem\u003eCecropin\u003c/em\u003e was mildly upregulated in \u003cem\u003eE. cloacae\u003c/em\u003e infections by 1.9-fold\u003cem\u003e,\u003c/em\u003e but for other infection groups, the expression levels were either downregulated or similar to that of uninfected control (Figure 4A).\u003c/p\u003e\n\u003cp\u003eAlthough the IMD pathway primarily defends\u0026nbsp;against Gram-negative bacteria, the Toll pathway also contributes, probably due to the crosstalk between the two pathways, where the effectors of the Toll pathway can modulate the IMD responses and vice versa.\u003csup\u003e20\u003c/sup\u003e Hence, we monitored the expression levels of \u003cem\u003eDefensin\u0026nbsp;\u003c/em\u003e(Figure 4B)\u003cem\u003e,\u0026nbsp;\u003c/em\u003ea key AMP secreted via the Toll pathwayin the gut of the infected flies. The expression of \u003cem\u003eDefensin\u003c/em\u003e remained uninduced or downregulated across all infection groups at both the time points, except for \u003cem\u003eK. pneumoniae\u003c/em\u003e-infected guts, where an upregulation was observed at 24 hpi, but not maintained at a high level till 48 hpi.\u003c/p\u003e\n\u003cp\u003eTogether, these results suggested that \u003cem\u003eDrosophila\u003c/em\u003e AMP levels were selectively unchanged\u0026nbsp;with distinct immune pathways activated against different bacterial pathogens.\u003csup\u003e21\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTRPA1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mediates the bacterial expulsion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs ROS showed dynamic changes without Smurf phenotype or AMP induction, we hypothesized that bacterial clearance involves ROS along with other mechanisms and therefore tried to understand how ROS interacts with intestinal contractions involved in bacterial expulsion.\u0026nbsp;Previous studies show that TRPA1 in enteroendocrine cells links ROS to bacterial shedding through ROS-induced defecation.\u003csup\u003e22\u003c/sup\u003e TRPA1, activated by stimuli such as ROS, regulates intestinal muscle activity in \u003cem\u003eDrosophila\u003c/em\u003e after bacterial ingestion.\u003csup\u003e9, 23\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo\u0026nbsp;test our hypothesis, we quantified the expression of \u003cem\u003eTRPA1\u0026nbsp;\u003c/em\u003ein the infected fly guts at 4 hpi and 24 hpi (Figure 5A–5C). \u003cem\u003eTRPA1\u0026nbsp;\u003c/em\u003ewas mildly upregulated at 4 hpi in the guts of \u003cem\u003eS\u003c/em\u003e. Typhimurium and \u003cem\u003eE. cloacae\u003c/em\u003e infected flies by 1.9 and 1.4-fold, respectively, while it remained uninduced in the guts of \u003cem\u003eK. pneumoniae\u003c/em\u003e-infected flies (Figure 5B). By 24 hpi, the induction became more significant with a 2.5-fold increase in \u003cem\u003eS\u003c/em\u003e. Typhimurium infected flies and a 3.3-fold increase in \u003cem\u003eE. cloacae\u003c/em\u003e infected flies (Figure 5C), while with \u003cem\u003eK.\u0026nbsp;\u003c/em\u003e\u003cem\u003epneumoniae\u0026nbsp;\u003c/em\u003ethere was an insignificant induction.\u003c/p\u003e\n\u003cp\u003eThe sustained\u0026nbsp;upregulation of \u003cem\u003eTRPA1\u0026nbsp;\u003c/em\u003elikely promoted intestinal contraction, facilitatingbacterial expulsion. Therefore, we assessed the bacteria shed by the infected flies using CFU analysis at 24 and 48 hpi (Figure 5D). We observed that all three pathogens were shed, though the \u003cem\u003eK. pneumoniae\u003c/em\u003e shedding does not correlate with \u003cem\u003eTRPA1\u0026nbsp;\u003c/em\u003eexpression. \u003cem\u003eE. coli\u003c/em\u003e colonies were not recovered in the shedding experiment, indicating the killing of this bacterium by the immune response active in the fly gut.\u003c/p\u003e\n\u003cp\u003eThus,\u0026nbsp;our data collectively suggest that \u003cem\u003eTRPA1\u003c/em\u003e facilitates intestinal contractions that expel bacteria, demonstrating that a combination of ROS and gut motility is involved in bacterial clearance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we employed an ecologically relevant natural feeding route in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e infection modelto investigate the gut defense response against a diverse spectrum of Gram-negative bacteria. Here, we highlighted that \u003cem\u003eDrosophila\u0026nbsp;\u003c/em\u003emounts a specific response\u0026nbsp;depending upon the bacterial species it encounters. Unlike previous studies,\u003csup\u003e24, 3\u003c/sup\u003e we excluded antimicrobial additives from the fly food to ensure infection outcomes precisely reflect host–pathogen interactions. We demonstrated that the induction of ROS in response to pathogens possibly leads to intestinal contractions, expelling the ingested bacteria. Our work further emphasises the importance of gut contractions as a non-immune mechanism, beyond their well-established functions like aiding the efficient nutrient absorption and regulating feeding behavior.\u003csup\u003e25\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eLeveraging the transparency of the \u003cem\u003eDrosophila\u0026nbsp;\u003c/em\u003eabdomen, we monitored the fate of fluorescent bacteria ingested\u0026nbsp;by the flies. Our observation of gradual bacterial clearance within two days of infection highlights the efficiency of the \u003cem\u003eDrosophila\u003c/em\u003e innate immune system in resolving infection. This corroborates the findings of an earlier study\u003csup\u003e26\u003c/sup\u003e where it was reported that the number of flies with no CFU increased over time after feeding on \u003cem\u003ePseudomonas entomophila\u003c/em\u003e. Our study shows that clearance and persistent infections are both probable outcomes of the bacterial infection. We observed that \u003cem\u003eE. coli, S.\u003c/em\u003e Typhimurium, and \u003cem\u003eK. pneumoniae\u003c/em\u003e are effectively cleared from the fly body by 48 hpi.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, \u003cem\u003eE. cloacae\u003c/em\u003e persisted until 72 hpi without impairing host homeostasis, with the highest bacterial burden in the infected flies despite no detectable fluorescence signal at this time point. \u003cem\u003eE. cloacae\u003c/em\u003e is characterized by lower virulence, which likely contributes to its ability to maintain stable colonization by avoiding significant harm to the host.\u003csup\u003e27\u003c/sup\u003e The discrepancy between the fluorescence and CFU data can be attributed to multiple factors, including bacterial localization from the gut to internal tissues, such as the head, host digestion, and immune response, which can possibly degrade the fluorescent tagged bacteria, thus reducing the fluorescence signals.\u003csup\u003e28\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eNotably, we observed that the\u0026nbsp;enteric pathogens used in our study did not compromise the gut barrier integrity and hence, could not establish systemic infection. However, previous studies have reported that bacteria like \u003cem\u003eS. marcescens,\u0026nbsp;\u003c/em\u003ewhen ingested, enter the body cavity but do not kill their host rapidly, unlike in septic injury models, where it can disrupt gut barrier integrity and kill the host within a day after infection. Such findings highlight that different pathogensexhibit different levels of virulence in two infection models, likely influenced by their exposure to midgut defenses.\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSeveral infection studies in \u003cem\u003eDrosophila\u0026nbsp;\u003c/em\u003eaddressing gut immunity help us to understand how the host resists and recovers from microbial infections.\u003csup\u003e29\u003c/sup\u003e Resistance includes the\u0026nbsp;localized production of ROS and AMPs,\u003csup\u003e30\u003c/sup\u003e while resilience depends upon the ability of intestinal epithelium to regenerate and maintain gut homeostasis.\u0026nbsp;In the current work, effective clearance of the blue food from the intestines of infected flies indicated resilience of the intestinal barrier following infection.\u003c/p\u003e\n\u003cp\u003eWe demonstrated that a rapid ROS burst is crucial for controlling bacterial growth in the \u003cem\u003eDrosophila\u003c/em\u003e gut. We observed dynamic induction of intestinal ROS on infection with\u0026nbsp;\u003cem\u003eS.\u003c/em\u003e Typhimurium, \u003cem\u003eK. pneumoniae,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE. cloacae.\u003c/em\u003e A ROS\u0026nbsp;surge at 4 hpi is consistent with the pathogen colonization in the flies, except for the non-pathogenic \u003cem\u003eE. coli.\u0026nbsp;\u003c/em\u003eThis could be due to exposure to bacterial pathogen associated molecular patterns, such as lipopolysaccharide, which are rapidly detected by pathogen recognition receptors. Interestingly, ROS levels declined at 24 hpi, accompanied by the downregulation \u003cem\u003eNox\u0026nbsp;\u003c/em\u003eand \u003cem\u003eDuox,\u003c/em\u003e members of the NADPH oxidase family. Previous studies have also discussed the efficacy of the intestinal ROS in blocking the pathogenic bacteria in the anterior section of the intestine, as early as 15 minutes of ingestion. This rapid oxidative burst limits bacterial proliferation, subsequently clearing the bacteria.\u003csup\u003e23\u003c/sup\u003e However, \u003cem\u003eDuox\u0026nbsp;\u003c/em\u003eknockout flies did not exhibit increased susceptibility to \u003cem\u003eS.\u003c/em\u003e Typhimurium, \u003cem\u003eK. pneumoniae,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE. cloacae,\u0026nbsp;\u003c/em\u003eleaving the role of ROS in eliminating these pathogens unclear. Although ROS is important for gut homeostasis, its overproduction can cause damage to intestinal cells.\u003csup\u003e31\u003c/sup\u003e The decline in ROS levels at 24 hpi could be due to the antioxidant activity of the immune system, like the immune-regulated catalase (IRC),\u003csup\u003e32\u003c/sup\u003e which plays a crucial role in mitigating the host damage post-infection. However, this reduction in ROS did not affect bacterial survival. Instead, all the pathogens, except \u003cem\u003eE. cloacae,\u0026nbsp;\u003c/em\u003ewere cleared within 48 hpi.\u003c/p\u003e\n\u003cp\u003eLater at 48 hpi, a surge in the ROS was\u0026nbsp;again observed throughout the infection groups. This elevated ROS at later stages may have functioned in two ways—by exerting an antimicrobial effect against the persisted bacteria or to induce local AMP response in the gut, thereby activating the IMD pathway.\u003csup\u003e33\u003c/sup\u003e For instance, \u003cem\u003eErwinia carotovora carotovora 15\u003c/em\u003e (\u003cem\u003eEcc15\u003c/em\u003e) infection activates the Relish (Rel) transcription factor for AMP expression in the fat body. Cecropin,a Rel-activated AMP, is significantly upregulated in \u003cem\u003eDrosophila\u003c/em\u003e gut after the oral infection with \u003cem\u003eP. entomophilia.\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e Its losshas a marked impact on infection with \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eProvidencia heimbachii.\u003c/em\u003e\u003csup\u003e13, 34\u003c/sup\u003e In our study, we also observed a 2 to 3-fold activation of Rel in the whole body of \u003cem\u003eS\u003c/em\u003e. Typhimurium and \u003cem\u003eE. cloacae\u003c/em\u003e infected flies at 48 hpi (Supplementary file 4). Contrarily, \u003cem\u003eCecropin\u0026nbsp;\u003c/em\u003eexpression either remained uninduced or downregulated across all infection groups, except in the guts of \u003cem\u003eE. cloacae\u0026nbsp;\u003c/em\u003einfected flies, where it was mildly upregulated at both 24 and 48 hpi, highlighting its persistence. The reason for the observed lack of robust upregulation of \u003cem\u003eCecropin\u0026nbsp;\u003c/em\u003ein the infected flies in our study remains unclear and needs elucidation.\u0026nbsp;Although our data suggest that ROS contributes to bacterial clearance, it also indicates that ROS alone is insufficient for complete bacterial eradication.\u003csup\u003e23\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAn intriguing question\u0026nbsp;is what enables the infected flies to withstand bacterial infection when AMPs are not robustly induced, and gut integrity remains uncompromised? Further, there is a surge in ROS upon infection; still flies lacking the \u003cem\u003eDuox\u003c/em\u003e gene exhibit no significant mortality, indicating that ROS does not play a role in clearing the infection. Possibly because ROS alone is insufficient to clear the bacteria. Reportedly, elevated levels of ROS during infection stimulate the TRPA1 receptor in gut enteroendocrine cells, thereby activating the production of neuropeptides, such as Diuretic Hormone 31.\u003csup\u003e22\u003c/sup\u003e TRPA1 stimulation enhances gut motility, increasing defecation rates and promoting the proliferation of epithelial stem cells. These processes work together to facilitate gut clearance and support tissue regeneration following infection.\u0026nbsp;Collectively, we explored the role of the ROS-sensing TRPA1 receptor in bacterial clearance. Our findings highlighted that the activation of \u003cem\u003eTRPA1\u003c/em\u003e correlated with the rapid production of ROS and shedding of \u003cem\u003eS\u003c/em\u003e. Typhimurium and \u003cem\u003eE. cloacae\u003c/em\u003e. The shedding decreased with time (24 to 48 hpi), consistent with the reduction in internal bacterial load, which was also reduced by 48 hpi, likely suggesting that \u003cem\u003eTRPA1\u003c/em\u003e driven motility remains sustained, leading to bacterial clearance.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Notably, \u003cem\u003eK. pneumoniae\u003c/em\u003e-infected guts showed no induction of \u003cem\u003eTRPA1,\u0026nbsp;\u003c/em\u003eyet the bacteria were shed to significant levels, underscoring the species-specific nature of the host response. It is interesting to note that \u003cem\u003eE. coli\u003c/em\u003e was not recovered in the shedding experiment, suggesting that it was effectively eliminated by the host's defenses.\u003c/p\u003e\n\u003cp\u003eWe further explored the intestinal muscular contractions of \u003cem\u003eS.\u003c/em\u003e Typhimurium and \u003cem\u003eE. cloacae\u0026nbsp;\u003c/em\u003einfected\u0026nbsp;fly guts, using light microscopy, and observed the narrowing of the gut lumen at 24 hpi, indicating a responsive contraction mechanism in the \u003cem\u003eDrosophila\u003c/em\u003e gut (Supplementary file 5).Hence, our work sheds light on yet to be anatomically characterized intestinal muscular contractions, which appear to be conserved across species, presenting a potential model to study the propulsion of luminal content and maintain intestinal homeostasis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCollectively, our findings underscore that the immune system of \u003cem\u003eDrosophila\u003c/em\u003e is capable of both discerning between different bacterial species and adapting its response to bacteria in a tissue-specific manner. The differential activation and regulation of pathways in response to pathogens warrant further studies to understand if these responses can be used for repurposing the existing methods of treatment for pathogen clearance. Clinically, gut motility disorders, such as Intestinal Bowel Syndrome and bacterial gastroenteritis, are linked with intestinal muscular contractions. In many instances, drugs are prescribed to control intestinal spasms to relieve the pain, which might delay the clearance of the infection. Our findings suggest that intestinal muscular contractions could help get rid of bacterial infection.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eFly stocks\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOregon-R\u003c/em\u003e flies were used in the study. Flies carrying \u003cem\u003eDuox\u0026nbsp;\u003c/em\u003eRNAi\u0026nbsp;(a kind gift from Professor Anurag Sharma, NITTE, India) driven by ubiquitously expressed \u003cem\u003eActin-Gal4\u003c/em\u003e was used to knock down \u003cem\u003eDuox\u003c/em\u003e to investigate the role of ROS in bacterial clearance, and UAS-GFP reporter flies (a kind gift from Professor Subhash C Lakhotia, BHU, India). \u003cem\u003eDrosophila\u0026nbsp;\u003c/em\u003estocks were maintained at 25°C on a 12:12 hour light:dark cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial strains used in this study were\u003cem\u003eE. coli, S\u003c/em\u003e. Typhimurium, \u003cem\u003eS. marcescens, K. pneumoniae,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE. cloacae.\u0026nbsp;\u003c/em\u003eThe RFP expressing strains of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eand \u003cem\u003eS\u003c/em\u003e. Typhimuriumwere obtained through their electro-transformation with plasmid pFPV-mcherry (Addgene plasmid #20956)\u003cem\u003e,\u003c/em\u003e while those of \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eE. cloacae\u003c/em\u003e were obtained by transformation with plasmidpEB2-2ndVal mRFP1(Addgene plasmid #104025)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eRFP expressing strains were cultured in Luria Broth medium supplemented with antibiotics for their selection: Ampicillin 100 µg/mL (Himedia, India) was used for \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;S.\u0026nbsp;\u003c/em\u003eTyphimurium, while Kanamycin 50 µg/mL (Himedia, India) was used for \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eE. cloacae.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFly infection model and survival assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight grown bacteria were subcultured in 10 mL of fresh media containing suitable antibiotics\u0026nbsp;and allowed to grow until the culture OD\u003csub\u003e600\u003c/sub\u003e was between 0.3 and 0.5. These bacterial cultures were centrifuged, and the pellet was resuspended in 500 µL of 5% sucrose solution prepared in sterile phosphate buffered saline (PBS). Whatman filter papers soaked with 200 µL of these bacterial suspensions were placed inside the empty culture vials. Age-matched adult flies (3–5 days old) subjected to a 3-hour starvation were allowed to feed on these bacterial cultures for 4 hours. Flies fed only a 5% sucrose solution prepared in sterile PBS were used as a negative control. Following oral infection, the infected flies were transferred to the standard cornmeal agar devoid of antimicrobials, such as\u0026nbsp;propionic acid and nipagin.\u003c/p\u003e\n\u003cp\u003eThe number of dead flies was recorded daily up to 30 days post-infection. Each day, surviving flies were transferred to the new vial containing antimicrobial-free standard cornmeal agar. These data was presented as Kaplan–Meier curves, plotting the percent surviving flies over time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of fly bacterial load by CFU assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial colonization in the fly body was determined as the number of CFU at 0-, 24-, 48-, and 72-hpi using the standard plate count method. Infected flies were gently washed thrice with 1 mL of sterile PBS. The washed flies were then homogenized in 500 µL of sterile PBS, using a bead beater at 20 Hz frequency for a 2-minute cycle (20 seconds homogenization, followed by 10 seconds on ice). The homogenate was diluted serially 1:10 and plated onto selective media to estimate\u0026nbsp;bacterial load, as described previously.\u003csup\u003e35\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of gut permeability and dye clearance in flies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter infecting the flies using the method described above, they were transferred to food containing 2.5% (wt./vol.) bromophenol blue for 12 hours. A fly was categorized as Smurf (observed using a microscope) if the dye could be observed outside the intestine\u003csup\u003e36\u003c/sup\u003e and the blue color was visible throughout its body.\u003c/p\u003e\n\u003cp\u003eFor the dye clearance assay, separate sets of flies were used. These flies were fed with the dye containing food for 12 hours as described above. After the examination for Smurf phenotype (if any), the flies were transferred to standard cornmeal agar food to assess their ability to clear the dye from the gut.\u003csup\u003e37\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ePhotographs of the representative flies were taken using the Optika microscope camera (Optika, Italy).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstimation of ROS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInfected fly guts were isolated, and both the gut and the carcass were added to 1.5 mL Eppendorf tubes containing 20 mM Tris Buffer. Tissue samples were ground using a pestle and then centrifuged. The supernatants were transferred to a fresh Eppendorf tube, and protein concentrations of the lysate were quantified using Bradford assay and the lysates amounting to 50 µg/µL protein were incubated with 5 µM DCF-DA for 60 minutes. Following this, fluorescence was measured with excitation at 490 nm and emission at 530 nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of bacterial shedding from infected flies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs described by Siva-Jothy,\u003csup\u003e24\u003c/sup\u003e the infected flies were individually transferred to an Eppendorf tube containing 50 µL of standard cornmeal agar and subsequently transferred to a fresh tube every 24 hours. The bacterial shedding was quantified at 24 and 48 hours using a serial dilution method by plating the tube content onto appropriate media.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of immune gene expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from 40–50 fly guts and/or 10–15 whole adult flies using the TRIzol reagent (Himedia, India) as specified in an earlier study.\u003csup\u003e38\u003c/sup\u003e Subsequently, 800 ng of total RNA was reverse transcribed to cDNA using Verso cDNA synthesis kit (AB1453A; Thermo Scientific, MA, USA) followed by qPCR reactions using gene-specific primers (Supplementary file 6)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software, MA, USA). For intergroup comparison, an unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test was used. For experiments involving multiple groups, two-way ANOVA, followed by Dunnett’s Multiple comparison test, was used. All data are presented as mean ± standard deviation. p-value less than 0.05 were considered statistically significant. All experiments were performed with at least three independent biological replicates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive funding from any public, commercial, or nonprofit agency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design of the work: SV, SM, and MT. Methodology + Analysis + Investigation: SV with the help of SM and MT. CFU Analysis: SB. SV has drafted the manuscript, and all authors have provided substantive revisions. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to Birla Institute of Technology and Science, Pilani, for the invaluable infrastructural support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYounes S, Al-Sulaiti A, Nasser EAA, Najjar H, Kamareddine L. Drosophila as a Model Organism in Host\u0026ndash;Pathogen Interaction Studies. Front Cell Infect Microbiol. 2020;10:214. https://doi.org/10.3389/fcimb.2020.00214.\u003c/li\u003e\n\u003cli\u003ePriyadarsini S, Sahoo M, Sahu S, Jayabalan R, Mishra M. An infection of Enterobacter ludwigii affects development and causes age-dependent neurodegeneration in Drosophila melanogaster. Invert Neurosci. 2019;19:13. https://doi.org/10.1007/s10158-019-0233-y.\u003c/li\u003e\n\u003cli\u003eVodovar N, Vinals M, Liehl P, Basset A, Degrouard J, Spellman P, et al. \u003cem\u003eDrosophila\u003c/em\u003e host defense after oral infection by an entomopathogenic \u003cem\u003ePseudomonas\u003c/em\u003e species. Proc Natl Acad Sci USA. 2005;102:11414\u0026ndash;9. https://doi.org/10.1073/pnas.0502240102.\u003c/li\u003e\n\u003cli\u003eZhai Z, Huang X, Yin Y. Beyond immunity: The Imd pathway as a coordinator of host defense, organismal physiology and behavior. Developmental \u0026amp; Comparative Immunology. 2018;83:51\u0026ndash;9. https://doi.org/10.1016/j.dci.2017.11.008.\u003c/li\u003e\n\u003cli\u003eMoghadam ZM, Henneke P, Kolter J. From Flies to Men: ROS and the NADPH Oxidase in Phagocytes. Front Cell Dev Biol. 2021;9:628991. https://doi.org/10.3389/fcell.2021.628991.\u003c/li\u003e\n\u003cli\u003eShaka M, Arias-Rojas A, Hrdina A, Frahm D, Iatsenko I. Lipopolysaccharide -mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster. PLoS Pathog. 2022;18:e1010825. https://doi.org/10.1371/journal.ppat.1010825.\u003c/li\u003e\n\u003cli\u003eBuchon N, Broderick NA, Poidevin M, Pradervand S, Lemaitre B. Drosophila Intestinal Response to Bacterial Infection: Activation of Host Defense and Stem Cell Proliferation. Cell Host \u0026amp; Microbe. 2009;5:200\u0026ndash;11. https://doi.org/10.1016/j.chom.2009.01.003.\u003c/li\u003e\n\u003cli\u003eSun Y, Wang X, Li L, Zhong C, Zhang Y, Yang X, et al. The role of gut microbiota in intestinal disease: from an oxidative stress perspective. Front Microbiol. 2024;15:1328324. https://doi.org/10.3389/fmicb.2024.1328324.\u003c/li\u003e\n\u003cli\u003eBenguettat O, Jneid R, Soltys J, Loudhaief R, Brun-Barale A, Osman D, et al. The DH31/CGRP enteroendocrine peptide triggers intestinal contractions favoring the elimination of opportunistic bacteria. PLoS Pathog. 2018;14:e1007279. https://doi.org/10.1371/journal.ppat.1007279.\u003c/li\u003e\n\u003cli\u003eLandini L, Souza Monteiro De Araujo D, Titiz M, Geppetti P, Nassini R, De Logu F. TRPA1 Role in Inflammatory Disorders: What Is Known So Far? IJMS. 2022;23:4529. https://doi.org/10.3390/ijms23094529.\u003c/li\u003e\n\u003cli\u003eOgawa N, Kurokawa T, Fujiwara K, Polat OK, Badr H, Takahashi N, et al. Functional and Structural Divergence in Human TRPV1 Channel Subunits by Oxidative Cysteine Modification. Journal of Biological Chemistry. 2016;291:4197\u0026ndash;210. https://doi.org/10.1074/jbc.M115.700278.\u003c/li\u003e\n\u003cli\u003eNehme NT, Li\u0026eacute;geois S, Kele B, Giammarinaro P, Pradel E, Hoffmann JA, et al. A Model of Bacterial Intestinal Infections in Drosophila melanogaster. PLoS Pathog. 2007;3:e173. https://doi.org/10.1371/journal.ppat.0030173.\u003c/li\u003e\n\u003cli\u003eCarboni AL, Hanson MA, Lindsay SA, Wasserman SA, Lemaitre B. Cecropins contribute to \u003cem\u003eDrosophila\u003c/em\u003e host defense against a subset of fungal and Gram-negative bacterial infection. Genetics. 2022;220:iyab188. https://doi.org/10.1093/genetics/iyab188.\u003c/li\u003e\n\u003cli\u003eWang L, Lin J, Yu J, Yang K, Sun L, Tang H, et al. Downregulation of Perilipin1 by the Immune Deficiency Pathway Leads to Lipid Droplet Reconfiguration and Adaptation to Bacterial Infection in \u003cem\u003eDrosophila\u003c/em\u003e. The Journal of Immunology. 2021;207:2347\u0026ndash;58. https://doi.org/10.4049/jimmunol.2100343.\u003c/li\u003e\n\u003cli\u003eTracy C, Kr\u0026auml;mer H. Escherichia coli Infection of Drosophila. BIO-PROTOCOL. 2017;7. https://doi.org/10.21769/BioProtoc.2256.\u003c/li\u003e\n\u003cli\u003eKeller R, Pedroso MZ, Ritchmann R, Silva RM. Occurrence of Virulence-Associated Properties in \u003cem\u003eEnterobacter cloacae\u003c/em\u003e. Infect Immun. 1998;66:645\u0026ndash;9. https://doi.org/10.1128/IAI.66.2.645-649.1998.\u003c/li\u003e\n\u003cli\u003eRamond E, Jamet A, Ding X, Euphrasie D, Bouvier C, Lallemant L, et al. Reactive Oxygen Species-Dependent Innate Immune Mechanisms Control Methicillin-Resistant Staphylococcus aureus Virulence in the \u003cem\u003eDrosophila\u003c/em\u003e Larval Model. mBio. 2021;12:e00276-21. https://doi.org/10.1128/mBio.00276-21.\u003c/li\u003e\n\u003cli\u003eGalenza A, Foley E. A glucose-supplemented diet enhances gut barrier integrity in \u003cem\u003eDrosophila\u003c/em\u003e. Biology Open. 2021;10:bio056515. https://doi.org/10.1242/bio.056515.\u003c/li\u003e\n\u003cli\u003eZane F, Bouzid H, Sosa Marmol S, Brazane M, Besse S, Molina JL, et al. Smurfness‐based two‐phase model of ageing helps deconvolve the ageing transcriptional signature. Aging Cell. 2023;22:e13946. https://doi.org/10.1111/acel.13946.\u003c/li\u003e\n\u003cli\u003eDe Gregorio E. The Toll and Imd pathways are the major regulators of the immune response in Drosophila. The EMBO Journal. 2002;21:2568\u0026ndash;79. https://doi.org/10.1093/emboj/21.11.2568.\u003c/li\u003e\n\u003cli\u003eHanson MA, Dost\u0026aacute;lov\u0026aacute; A, Ceroni C, Poidevin M, Kondo S, Lemaitre B. Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach. eLife. 2019;8:e44341. https://doi.org/10.7554/eLife.44341.\u003c/li\u003e\n\u003cli\u003eDu EJ, Ahn TJ, Kwon I, Lee JH, Park J-H, Park SH, et al. TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway. PLoS Genet. 2016;12:e1005773. https://doi.org/10.1371/journal.pgen.1005773.\u003c/li\u003e\n\u003cli\u003eTleiss F, Montanari M, Milleville R, Pierre O, Royet J, Osman D, et al. Spatial and temporal coordination of Duox/TrpA1/Dh31 and IMD pathways is required for the efficient elimination of pathogenic bacteria in the intestine of Drosophila larvae. eLife. 2024;13:RP98716. https://doi.org/10.7554/eLife.98716.3.\u003c/li\u003e\n\u003cli\u003eSiva-Jothy JA, Prakash A, Vasanthakrishnan RB, Monteith KM, Vale PF. Oral Bacterial Infection and Shedding in Drosophila melanogaster. JoVE. 2018;:57676. https://doi.org/10.3791/57676.\u003c/li\u003e\n\u003cli\u003eMace OJ, Tehan B, Marshall F. Pharmacology and physiology of gastrointestinal enteroendocrine cells. Pharmacology Res \u0026amp; Perspec. 2015;3:e00155. https://doi.org/10.1002/prp2.155.\u003c/li\u003e\n\u003cli\u003ePaulo TF, Akyaw PA, Paix\u0026atilde;o T, Sucena \u0026Eacute;. Evolution of resistance and disease tolerance mechanisms to oral bacterial infection in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. Open Biol. 2025;15:240265. https://doi.org/10.1098/rsob.240265.\u003c/li\u003e\n\u003cli\u003eHidalgo BA, Silva LM, Franz M, Regoes RR, Armitage SAO. Decomposing virulence to understand bacterial clearance in persistent infections. Nat Commun. 2022;13:5023. https://doi.org/10.1038/s41467-022-32118-1.\u003c/li\u003e\n\u003cli\u003eBell KS, Ko S, Ali S, Bognar B, Khmelkov M, Rau N, et al. Tissue-Specific Fluorescent Protein Turnover in Free-Moving Flies. Insects. 2025;16:583. https://doi.org/10.3390/insects16060583.\u003c/li\u003e\n\u003cli\u003eErkosar B, Leulier F. Transient adult microbiota, gut homeostasis and longevity: Novel insights from the \u003cem\u003eDrosophila\u003c/em\u003e model. FEBS Letters. 2014;588:4250\u0026ndash;7. https://doi.org/10.1016/j.febslet.2014.06.041.\u003c/li\u003e\n\u003cli\u003eTafesh-Edwards G, Eleftherianos I. The role of \u003cem\u003eDrosophila\u003c/em\u003e microbiota in gut homeostasis and immunity. Gut Microbes. 2023;15:2208503. https://doi.org/10.1080/19490976.2023.2208503.\u003c/li\u003e\n\u003cli\u003eKuraishi T, Hori A, Kurata S. Host-microbe interactions in the gut of Drosophila melanogaster. Front Physiol. 2013;4. https://doi.org/10.3389/fphys.2013.00375.\u003c/li\u003e\n\u003cli\u003ePrakash A, Monteith KM, Vale PF. Mechanisms of damage prevention, signalling, and repair impact disease tolerance. 2021. https://doi.org/10.1101/2021.10.03.462916.\u003c/li\u003e\n\u003cli\u003eWu S-C, Liao C-W, Pan R-L, Juang J-L. Infection-Induced Intestinal Oxidative Stress Triggers Organ-to-Organ Immunological Communication in Drosophila. Cell Host \u0026amp; Microbe. 2012;11:410\u0026ndash;7. https://doi.org/10.1016/j.chom.2012.03.004.\u003c/li\u003e\n\u003cli\u003eKylsten P, Samakovlis C, Hultmark D. The cecropin locus in Drosophila; a compact gene cluster involved in the response to infection. The EMBO Journal. 1990;9:217\u0026ndash;24. https://doi.org/10.1002/j.1460-2075.1990.tb08098.x.\u003c/li\u003e\n\u003cli\u003eChambers MC, Jacobson E, Khalil S, Lazzaro BP. Consequences of chronic bacterial infection in Drosophila melanogaster. PLoS ONE. 2019;14:e0224440. https://doi.org/10.1371/journal.pone.0224440.\u003c/li\u003e\n\u003cli\u003eAkhmetova K, Balasov M, Chesnokov I. Drosophila STING protein has a role in lipid metabolism. eLife. 2021;10:e67358. https://doi.org/10.7554/eLife.67358.\u003c/li\u003e\n\u003cli\u003eLivingston DBH, Patel H, Donini A, MacMillan HA. Active transport of brilliant blue FCF across the \u003cem\u003eDrosophila\u003c/em\u003e midgut and Malpighian tubule epithelia. 2019. https://doi.org/10.1101/771675.\u003c/li\u003e\n\u003cli\u003eNarwal S, Singh A, Tare M. Analysis of \u0026alpha;-syn and parkin interaction in mediating neuronal death in Drosophila model of Parkinson\u0026rsquo;s disease. Front Cell Neurosci. 2024;17:1295805. https://doi.org/10.3389/fncel.2023.1295805.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Enteric pathogens, Gut immunity, Reactive oxygen species, Bacterial shedding, TRPA1","lastPublishedDoi":"10.21203/rs.3.rs-8473557/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8473557/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFood-borne pathogens, particularly belonging to the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family, are a major cause of gastrointestinal infections in humans. While\u003cem\u003e Drosophila melanogaster \u003c/em\u003ehas been widely explored to study antimicrobial responses, most studies rely on septic injury or direct injection, routes that bypass the gut. A coordinated set of antimicrobial defenses acts to counteract the invading bacteria, with some variations depending on the entry route into the host. Herein, we tracked the dynamics of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e pathogens in \u003cem\u003eDrosophila \u003c/em\u003eusing a natural infection route.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMost bacterial species were cleared effectively within 48 hours post-infection (hpi). We did not observe any significant mortality, indicating robust infection control. At 4 hpi, a substantial increase in reactive oxygen species (ROS) production was observed, followed by a decrease at 24 hpi and a resurgence at 48 hpi, suggesting the importance of ROS in bacterial clearance. However, flies lacking the \u003cem\u003edual oxidase\u003c/em\u003e (\u003cem\u003eDuox\u003c/em\u003e) gene showed unchanged survival rates, suggesting ROS alone is not enough for infection control. We further show that the shedding of bacteria could be attributed to increased \u003cem\u003eTRPA1\u003c/em\u003eexpression, a ROS-sensing receptor that triggers intestinal contractions in flies infected with \u003cem\u003eS\u003c/em\u003e. Typhimurium and \u003cem\u003eE. cloacae \u003c/em\u003eat 4 and 24 hpi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings reveal that the host can distinguish and respond to various bacterial species in a well-synchronized, gut-localized, and pathogen-specific manner. This also illustrates the reliability of natural infection route models in unravelling and understanding the complexities of host–pathogen interaction.\u003c/p\u003e","manuscriptTitle":"Drosophila melanogaster mitigates gastro-oral infections by stimulating pathogen expulsion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 16:39:05","doi":"10.21203/rs.3.rs-8473557/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-20T06:04:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-20T01:37:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T19:42:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106331423075322225622242474935328050562","date":"2026-01-19T14:09:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T01:34:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-18T15:33:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-18T10:48:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83042463428964203763919626771529146228","date":"2026-01-15T03:10:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-15T02:22:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T15:31:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240657824835506282451059201085630304492","date":"2026-01-12T13:29:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305513441620392121062011043161989554008","date":"2026-01-12T07:47:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77423256261624185864361343069562373281","date":"2026-01-12T00:41:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42382272216603427841038214397101666846","date":"2026-01-11T18:00:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286645686833335626299043699937576902024","date":"2026-01-11T09:32:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5951891774623980624360590094639176487","date":"2026-01-09T22:13:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40233114191753215453369908727811466676","date":"2026-01-09T16:44:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76276905567363499960505038015392516973","date":"2026-01-09T15:08:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110014791384672399361045835838921983630","date":"2026-01-09T14:41:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324045915945410234114723417113233956010","date":"2026-01-09T14:35:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-09T14:19:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-31T13:41:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-31T02:56:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-31T02:56:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-12-29T13:15:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f8c9a7e-df76-4096-9697-969718afd179","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T05:55:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 16:39:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8473557","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8473557","identity":"rs-8473557","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}