The fliR gene contributes to the virulence of S. marcescens in a Drosophila intestinal infection model

Serratia marcescens is an opportunistic bacterium that infects a wide range of hosts including humans. It is a potent pathogen in a septic injury model of Drosophila melanogaster since a few bacteria directly injected in the body cavity kill the insect within a day. In contrast, flies do not succumb to ingested bacteria for days even though some bacteria cross the intestinal barrier into the hemolymph within hours. The mechanisms by which S. marcescens attacks enterocytes and damages the intestinal epithelium remain uncharacterized. To better understand intestinal infections, we performed a genetic screen for loss of virulence of ingested S. marcescens and identified FliR, a structural component of the flagellum, as a virulence factor. Next, we compared the virulence of two flagellum mutants fliR and flhD in two distinct S. marcescens strains. Both genes are required for S. marcescens to escape the gut lumen into the hemocoel, indicating that the flagellum plays an important role for the passage of bacteria through the intestinal barrier. Unexpectedly, fliR but not flhD is involved in S. marcescens-mediated damages of the intestinal epithelium that ultimately contribute to the demise of the host. Our results therefore suggest a flagellum-independent role for fliR in bacterial virulence.


Scientific Reports
| (2022) 12:3068 | https://doi.org/10.1038/s41598-022-06780-w www.nature.com/scientificreports/ in the lumen through the Dual Oxidase (DuOx) enzyme 14,15 . Additionally, at least three resilience mechanisms may contribute to maintain the homeostasis of the intestinal epithelium: (i) the secretion of Immune Response Catalase (IRC) limits the detrimental effect of ROS on intestinal epithelial cells (Enterocytes = ECs) 16 ; (ii) the proliferation of Intestinal Stem Cells (ISCs) compensates EC cell death [17][18][19][20] ; (iii) the extrusion of EC cytoplasm within hours of ingestion eliminates intracellular toxins and damaged organelles 21 . Even though the fly intestine harbors sophisticated defense mechanisms, some microorganisms, such as Serratia marcescens or Pseudomonas aeruginosa, are able to resist, survive, damage and cross the intestinal barrier 10,22,23 . S. marcescens is a Gram-negative entomopathogen and also a human opportunistic bacterium associated with nosocomial infections [24][25][26] . The pathogenicity of S. marcescens relies on multiple virulence factors such as the pore-forming toxin hemolysin [27][28][29] , the serralysin protease [30][31][32] or a phospholipase 33 . S. marcescens is a potent pathogen in the septic injury model of Drosophila. When introduced directly in the hemocoel, a few bacterial cells are sufficient to kill the fly within a day. The bacteria proliferate rapidly in the hemolymph causing bacteremia followed by death. Upon detection of the bacteria, the Immune deficiency (IMD) signaling pathway stimulates the secretion of AMPs by fat body cells. However, this systemic immune response does not affect S. marcescens since IMD-deficient flies are as susceptible as wild-type flies to septic injury 10 . However, in the oral infection model, S. marcescens invades and damages the intestinal epithelium and causes EC cell death; yet, the flies do not succumb to the infection for days. This delay is likely accounted by resilience mechanisms such as ISC compensatory proliferation 20 . In the midgut, the bacteria trigger the local release of AMPs by the IMD signaling pathway 10,12,13 and are thought to induce the local secretion of ROS through the DuOx enzyme 15 . Interestingly, a low but significant number of bacteria can cross the intestinal barrier and manage to reach the hemolymph. In contrast to the septic injury model, S. marcescens is not able to proliferate in the hemocoel as it is controlled by phagocytosis. Indeed, phagocytosis-impaired flies are highly susceptible to the oral infection as ingested bacteria proliferate in the hemolymph 10 . In keeping with this cellular control of bacteria that have escaped in the hemocoel, the bacteria do not trigger the systemic immune response, which monitors short peptidoglycan fragments released by bacteria during their divisions 34 . Thus, under normal conditions, bacteria that have crossed the intestinal barrier do not appear to contribute to the virulence of this pathogen in the oral infection model.
The difference in the virulence of the bacteria between the septic injury and the oral infection model indicates that the virulence program of S. marcescens is downregulated after its passage from the gut lumen through the midgut epithelium to the hemolymph 10 . How S. marcescens modulates its virulence program according to its infection route remains unknown. Additionally, the virulence factors that the bacteria employ to damage the intestinal epithelium and to cross the fly intestinal barrier are still uncharacterized. To better understand intestinal infection by S. marcescens, we performed a small-scale genetic screen to isolate bacterial mutants displaying an impaired virulence in the Drosophila oral infection model. This screen identified a novel virulence factor, fliR, that is needed for S. marcescens to severely damage the intestinal epithelium and to efficiently kill the flies. Furthermore, this study sheds light on the importance of the flagellum for the dissemination of gut bacteria through the intestinal epithelium into the internal milieu potentially causing systemic infections.

Results
The fliR gene as a novel virulence factor in S. marcescens. To identify new virulence factors for S. marcescens, we partially screened a transposon (mini-Tn5) insertion mutant library generated in the Db10 strain 35 . We examined the survival of eater −/− flies following the oral infection with individual mutant clones. Of note, the eater −/− mutants are phagocytosis-impaired flies proven useful for the screen because of their susceptibility to wild-type Serratia intestinal infection: the bacteria proliferate in the hemolymph and rapidly kill the flies, making easier the selection for less virulent bacterial strains.
We have tested 1348 bacterial mutants and identified a strain (19H12) that exhibited reduced virulence in the intestinal infection ( Supplementary Fig. 1). Sequencing analysis of the 19H12 clone revealed an insertion mutation in the gene fliR, which encodes a structural component of the flagellum and is required for its biosynthesis by participating in the export machinery of its components as well as some virulence factors [36][37][38] . FliR is a protein that forms part of the export gate of the flagellum, a structure embedded within the MS-ring, the basal body that anchors the flagellum to the cytoplasmic membrane and the cell wall. fliR, like flhD, is required for the formation of flagella in S. marcescens, as determined in in vivo studies. The function of fliR in the virulence of Serratia might be dependent on its role in the assembly of the flagellum. The latter is a complex process initiated by the major (class I) regulator FlhDC that controls the expression of several flagellar genes, including fliR 36 .
To validate the implication of fliR in the virulence of the bacteria, and to assess whether it is related to its function in the flagellum apparatus, we designed pKNOCK insertion mutants 39 for the fliR gene as well as for the flhD regulatory gene. These insertion mutants, in addition to a fliR plasmidic rescue (fliR was cloned in the pBB1:lacI:MCS expression plasmid resulting in pBB1:lacI:fliR) 40 , were generated in two different S. marcescens wild-type strains of distinct origins: Db10 (a derivative of a Drosophila isolate from Stockholm, Sweden) 41 and RM66262 (a clinical isolate from Rosario, Argentina) 42 .
After selecting mutants in both the Db10 and the RM66262 backgrounds, we first confirmed that the mutations in fliR or flhD do not alter the growth of the bacteria in the LB medium and in the infection solution (50 mM sucrose + 10% LB) ( Supplementary Fig. 2). We then determined the loss of flagellum-dependent activities in all mutants ( Supplementary Fig. 3): the flagellin expression is lost and the motility is impaired in the flhD and the fliR mutants as shown by western blot, swimming, and swarming assays. Also, the phospholipase of S. marcescens is secreted through the flagellum export system, which is a type 3 secretion system (T3SS) 38 . As expected, we did not detect phospholipase activity for either flhD or fliR mutants as compared to the wild-type  www.nature.com/scientificreports/ wild-type bacteria (Fig. 1C,D). Moreover, we tested a possible role for fliR gene in the virulence of the bacteria in septic injury. We found that fliR mutant bacteria were as virulent as flhD mutant and wild-type S. marcescens when introduced directly in the hemolymph (Fig. 1E). In conclusion, these results reveal a flhD-independent role for fliR in the virulence of S. marcescens in intestinal infection, but not in the septic injury model. Similar results were obtained when monitoring the survival of IMD-deficient (kenny) and DuOx-deficient (silenced in ECs) flies following an oral infection ( Supplementary Fig. 4B,C). These experiments indicate that the contribution of fliR in the virulence of S. marcescens is not related to its interactions with the fly immune system such as eliciting or evading the immune response during intestinal infections. As we had confirmed the role of fliR in the virulence of S. marcescens in two different bacterial strains, we focused only on the RM66262 strain for further investigations.
The flagellum is essential for S. marcescens to traverse the epithelial barrier. The bacteria in the gut lumen of flies are subjected to various stressors such as immune effectors and digestive enzymes. To monitor the survival of the fliR mutant in the digestive tract, we applied an assay consisting in the ingestion of bacteria that constitutively express GFP from a plasmid together with the propidium iodide stain: the GFP label indicates the presence of live bacteria, whereas the propidium iodide penetrates and stains only dead bacteria. We found that the intestinal lumen of w A5001 flies that ingested RM66262 wild-type, flhD or fliR mutants contained only live bacteria marked with GFP ( Fig. 2A,B) as compared to the lumen of flies that have ingested the E. coli control. The absence of propidium iodide staining for the tested S. marcescens strains suggests that the mutants are not killed in the midgut at least at 4 h post-infection, whereas E. coli was killed in the posterior midgut after having passed through the acidic region ( Fig. 2A,B). In addition, we measured the bacterial titer in the midgut of eater flies at 24 h post-infection. We observed that the CFU count of fliR mutants in the intestine is comparable to the values determined for either the flhD mutant or the wild-type bacteria (Fig. 2C). Taken together, these results indicate that flhD and fliR mutants are able to resist to the stressful environment of the midgut as well as wildtype bacteria. Besides motility, adherence to and invasion of host cells are two other important functions of the flagellum 43,44 . Since flagellum mutants are not motile, we tested the ability of both flhD and fliR mutant bacteria to adhere to and invade CHO cells by forcing the contact between bacteria and host cells by centrifugation. As expected, we observed a decreased adhesion and invasion for both mutants in comparison to wild-type bacteria (Supplementary Fig. 5A,B). We also found that the invasion of Drosophila S2 cells by fliR bacteria is highly diminished when compared to wild-type bacteria (Db10) (Supplementary Fig. 5C). Therefore, these impaired functions of the flagellum may affect the ability of S. marcescens to traverse the intestinal epithelium and to cause septicemia. Therefore, to examine the ability of the bacterial mutants to cross the epithelial barrier, we quantified the amount of bacteria present in the hemolymph of phagocytosis-impaired flies 4 h after the beginning of RM66262 ingestion. We showed that both flhD and fliR bacteria were less abundant in the hemolymph as compared to wild-type bacteria (Fig. 2D). However, the ability of the fliR mutant to cross the intestinal barrier was not rescued by the complementation (Fig. 2D). Of note, the latter is carried out under the control of an inducible promoter that requires IPTG. The IPTG used to activate the expression of the fliR gene may have not been able to pass the intestinal barrier.
These results were confirmed upon dissection of wild-type midguts after gentamicin solution feeding to clear previously ingested bacteria remaining in the lumen. As gentamicin is not able to cross eukaryotic membranes, the microbial titer measured in the treated midguts corresponded to bacteria within ECs or adhering to the basal part of the epithelium, which is in contact with hemolymph 10 . We observed less fliR bacterial loads than wild-type ones ( Supplementary Fig. 5D). In conclusion, both flagellum mutants exhibit difficulties to traverse the intestinal barrier. These findings pinpoint a requirement for the flagellum in the passage of the bacteria from the gut lumen to the body cavity.
The fliR gene is required for S. marcescens to impact the homeostasis of the intestinal epithelium. Following the ingestion of S. marcescens, two distinct resilience mechanisms are activated in the intestinal epithelium: the cytoplasmic purge and the compensatory proliferation of ISCs. In the early phase of infection, pore-forming toxins such as hemolysin elicit the extrusion of EC cytoplasm 21 . This short-term cytoplasmic purge prevents the toxic effect of the hemolysin on the ECs and results in a drastic thinning of the intestinal epithelium 3 h post-infection. We examined the induction of the cytoplasmic purge by measuring the thickness of the intestinal epithelium 3 h post-infection. The cytoplasmic purge was triggered in midguts infected with fliR, flhD mutants or the wild-type control, as the thinning of the epithelium (~ 10 µm) occurred in midguts infected with either mutant or control strains ( Fig. 3A and Supplementary Fig. 6A). Thus, both mutants are toxic enough to trigger the cytoplasmic purge in ECs possibly because they secrete equivalent levels of hemolysin.
Despite several midgut defense mechanisms, the bacteria manage to inflict damages to the epithelium, to stress and to kill ECs via unknown virulence factors 10,20 . Subsequently, ISCs proliferate at 24 h in response to EC stress or death. A phosphohistone H3 (PH3) staining, which marks dividing ISCs in the gut, allows to indirectly monitor the extent of gut damages: an increase in the PH3 level results from an enhanced proliferation of ISC, which may reflect the extent of epithelial damage. Of note, ISC compensatory proliferation in response to EC cell death was previously detected throughout the midgut epithelium of flies that have ingested S. marcescens 20 .
To examine the ability of both mutants to damage the intestinal epithelium, we performed a PH3 staining on eater −/− infected midguts. We detected a significant decrease in the PH3-positive cell count in the midguts infected with fliR mutants as compared to the ones infected with either flhD mutants or wild-type RM66262 bacteria (Fig. 3B). Similar results were obtained following the infection of w A5001 flies with flagellar mutants in    Fig. 6B,C). This finding suggests a diminished efficiency for the fliR bacteria to attack the intestinal cells, a process which is at least partially independent from flhD.

Discussion
Intestinal infection with S. marcescens shares similar features with P. aeruginosa oral infection including the passage through the epithelial barrier and the damages to ECs 10,22,23 . However, the mechanisms used by these two bacterial species to exert these two features has not yet been characterized. Here we have presented evidence that the flagellum of S. marcescens is required for its passage from the gut to the body cavity of the flies. Importantly, we have identified FliR as a novel virulence factor that is needed for the bacteria to severely damage the intestinal epithelium, apparently independently from its major function in building up flagella. Bacteria can cross the intestinal barrier via two distinct strategies: paracellular/extracellular passage by swimming in between the closely apposed enterocytes through the septate junctions or intracellular passage through the intestinal cells. In this study, we showed that the flagellum of S. marcescens plays a crucial role in the passage of bacteria from the gut lumen to the hemolymph as both flagellar mutants flhD and fliR displayed decreased bacterial loads in the body cavity of the fly (Fig. 2D, Supplementary Fig. 5D). Most S. marcescens bacteria remain confined to the gut endoperitrophic compartment as the peritrophic matrix forms an efficient barrier 10 . It remains to be determined whether the flagellum is required for the passage through the peritrophic matrix of the few bacteria that manage to cross it. It has been previously shown that some bacteria were attempting to traverse the epithelium in between ECs at late stages of infection 10 . An open possibility is that for the early passage that occurs within 2 h of feeding, bacteria may cross at the proximal part of the midgut, in the proventriculus region where the peritrophic matrix is synthesized before being reinforced by ECs along the midgut 9,10 . In both cases, the role of the flagellum may be restricted to its motility function. S. marcescens appears to traverse the intestinal barrier more efficiently when they do not express hemolysin and therefore do not trigger the cytoplasmic purge enterocyte defense 21 . This observation suggests the possibility that S. marcescens crosses the epithelial barrier by invading intestinal cells, in keeping with a study that also showed that S. marcescens requires the flagellum to adhere to and invade CHO cells 44 (Supplementary Fig. 5A,B). The lack of adherence and invasion observed for flagellar mutants can be related to the motility function of the flagellum or to the secretion, through the T3SS, of several virulence factors such as the phospholipase or the S. marcescens nuclease 38,45 . We note that in Caulobacter crescentus, the synthesis of the type IV pilus, which plays a primordial role in adherence, depends on flagellar genes for the production of pilin 46 .
Altogether, our results suggest that the escape of a few bacteria into the hemocoel does not contribute to the fatal outcome of the infection as the flhD mutant is as lethal as wild-type bacteria. This result is in keeping with the low bacterial burden detected in the hemolymph throughout the infection, which is limited by hemocytes that phagocytose S. marcescens 10 .
Here we showed that fliR mutants are less virulent in the intestinal infection model when compared to flhD mutant bacteria in two distinct S. marcescens strains (Fig. 1A-D). This difference in the virulence observed On the one hand, the analysis of the epithelial thickness revealed a normal induction of the cytoplasmic purge following the infection with the fliR mutant bacteria ( Fig. 3A and Supplementary Fig. 6A). This purge is triggered in response to the release of the hemolysin pore-forming toxin by S. marcescens. Therefore, the role of fliR in the virulence of the bacteria is not related to the secretion of hemolysin. Indeed, hemolysin is secreted by a T5SS 27,49,50 . On the other hand, we showed that the fliR strain is likely to induce less damage to the intestinal epithelium as the proliferation rate of ISCs is diminished in the midguts infected with fliR as compared to the ones infected with flhD bacteria (Fig. 3B and Supplementary Fig. 6B,C). This finding strongly suggests that FliR is implicated in the attack and the death of the ECs independently from its function in the flagellum. An attractive hypothesis is that FliR may be needed for the formation of a distinct secretory apparatus required for the secretion of unknown virulence factors that may directly attack and kill the fly intestinal cells (Fig. 4).

Methods
Fly strains. Flies were raised at 25 °C with 60% humidity on a semi-solid standard medium composed of 50 L of sterile water containing 3.2 kg of cornmeal, 2.4 kg of sugar, 580 g of yeast brewer's dry powder, 240 g of agar and 260 g of 4-hydroxybenzoate sodium salt (Merck). The different fly strains used in the experiments were: w A5001 and eater −/−51 .
Bacterial strains and culture. Two strains of S. marcescens were used: Db10 41 and RM66262 42 . The different mutants were generated by the pKNOCK plasmid insertion technique 39 . This plasmid carries antibiotic resistance to chloramphenicol (20 µg/mL) or to gentamicin (15 µg/mL). The fliR; pBB1:lacI:fliR (fliR; pBB1::fliR) strain expresses a wild-type copy of fliR under the control of an Isopropyl ß-D-1-thiogalactopyranoside (IPTG)inducible promoter. The bacteria were cultured overnight on LB agar plates or in liquid medium at 37 °C with the corresponding antibiotics.
Infection experiments and survival. The oral infection and the septic injury were performed at 25 °C essentially as described in Nehme et al. 10 . Bacterial pellet was diluted in 50 mM sucrose solution and 10% LB for Bacterial loads. The bacterial titer of the intestine was measured 24 h post-infection. A single midgut was dissected and homogenized in 100 µL of PBS. The bacterial titer in the hemolymph was determined 4 h postinfection. The hemolymph was retrieved from five flies using a Nanoject II microinjector (Drummond) and collected in 10 µL of PBS. A serial dilution was applied on the samples, then each dilution was plated on LB-agar plates with ampicillin for the RM66262 wild-type strain and mutants thereof.
Staining and imaging. To perform a propidium iodide staining, the flies were fed for 4 h with a solution containing 50 mM sucrose, 10% LB, bacteria that constitutively express GFP from a plasmid (OD 600 of 10) and 50 µg/mL of propidium iodide. The midguts were dissected in PBS, fixed with 8% PFA then washed three times with PBS.
To measure epithelial thinning, midguts were dissected and fixed as described above. Actin staining was performed by incubating the samples for 1h30 in 10 µM of FITC-labeled phalloidin (Sigma-Aldrich #P5282). The epithelium thickness was measured using the FIJI software. The PH3 staining was performed at 24 h postinfection (OD 600 of 10). The midguts were dissected in PBS, fixed with 8% PFA, incubated with the PH3 antibody (Millipore, ref 09-797) overnight at 4 °C, then stained with an anti-rabbit FITC-labeled antibody (Abcam #6717) overnight at 4 °C or 2 h at room temperature. All stained midguts were mounted in the Vectashield mounting medium (Vector Laboratories). The samples were observed and imaged using a LSM780 confocal microscope (Zeiss).

Statistical analysis.
All graphs and statistical tests were performed using GraphPad Prism. The statistical test used for the survival curves was Log-rank. Mann-Whitney, one-way ANOVA or Kruskal Wallis tests were performed for all other experiments (as specified in figure legends). The number of stars (*) represents the P values P ≥ 0.05 (ns), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) and P < 0.0001 (****).

Data availability
All data and materials are available upon request.