ABSTRACT
When Drosophila flies feed on Pseudomonas aeruginosa strain PA14, some bacteria cross the intestinal barrier and start proliferating inside the hemocoel. This process is limited by hemocytes through phagocytosis. We have previously shown that the PA14 quorum-sensing regulator RhlR is required for these bacteria to elude the cellular immune response. RhlI synthesizes the auto-inducer signal that activates RhlR. Here, we compare the null mutant phenotypes of rhlR and rhlI in a variety of infection assays in Drosophila and in the nematode Caenorhabditis elegans. Surprisingly, in Drosophila, unlike ΔrhlR mutants, ΔrhlI mutants are only modestly attenuated for virulence and are poorly phagocytosed and opsonized in a Thioester-containing Protein4-dependent manner. Likewise, ΔrhlI but not ΔrhlR mutants colonize the digestive tract of C. elegans and kill it as efficiently as wild-type PA14. Thus, RhlR has an RhlI-independent function in eluding detection or counter-acting the action of the immune system. In contrast to the intestinal infection model, Tep4 mutant flies are more resistant to PA14 in a septic injury model, which also depends on rhlR. Thus, the Tep4 putative opsonin can either be protective or detrimental to host defense depending on the infection route.
INTRODUCTION
Drosophila melanogaster is a powerful genetic model organism for the study of innate immunity that has been intensely investigated during the past 25 years (Buchon, Silverman et al., 2014). It thus represents an informative system in which to study host-pathogen interactions using either systemic infection or so-called “natural” infection paradigms, such as oral infection (Bier & Guichard, 2012, Ferrandon, 2013, Igboin, Griffen et al., 2012, Limmer, Quintin et al., 2011b). Genetic analysis has allowed the detailed dissection of the Drosophila systemic immune response to microbial infections (Lemaitre & Hoffmann, 2007). In addition to melanization which is mediated by the protease-mediated cleavage of prophenol oxidase into active phenol oxidase (PO), two major NF-kappaB pathways, Toll and Immune deficiency (IMD), regulate the induction of the expression of genes that encode potent antimicrobial peptides, which are active against most bacteria and fungi (Binggeli, Neyen et al., 2014, Dudzic, Kondo et al., 2015, Ferrandon, Imler et al., 2007, Ganesan, Aggarwal et al., 2010, Lemaitre & Hoffmann, 2007). The Drosophila systemic immune response is so effective, especially in the case of Gram-negative bacterial infections, that a second arm of host defense, the cellular immune response, has remained comparatively less well studied (El Chamy, Matt et al., 2017, Pean & Dionne, 2014). Indeed, blocking cellular immunity through saturation of the phagocytic apparatus with inert particles does not yield a strong susceptibility phenotype to flies infected by Escherichia coli, unless the systemic immune response is at least also partially impaired (Elrod-Erickson, Mishra et al., 2000). Nevertheless, we have found that when two opportunistic pathogens Serratia marcescens and Pseudomonas aeruginosa are fed to Drosophila, the cellular immune response plays a key role in controlling the bacteria that escape from the digestive tract (Limmer, Haller et al., 2011a, Nehme, Liegeois et al., 2007). In both cases, the putative phagocytic receptor Eater plays a crucial role and prevents the development of a rapid bacteremia (Kocks, Cho et al., 2005, Limmer et al., 2011a).
The thioester-containing protein Tep1 opsonizes bacteria in the mosquito species Anopheles gambiae (Levashina, Moita et al., 2001). It is unknown whether opsonization also plays a role in vivo in Drosophila, even though its genome encodes five functional Tep loci and a pseudogene (Tep5) (Bou Aoun, Hetru et al., 2011). The thioester motif is not present in Tep6 and is therefore thought to be nonfunctional. Indeed, Tep6 is required for the establishment of septate junctions in specific parts of the gut, which explains the lethal phenotype of Tep6 null mutants (Batz, Forster et al., 2014, Hall, Bone et al., 2014). Tep6 has also been shown to induce autophagy in macrophages via a non cell-autonomous process that involves epithelial cells in which it is expressed (Lin, Rodrigues et al., 2017). A previous study failed to find a role for the remaining Tep genes (Tep1 to Tep4) in host defense in several models of bacterial or fungal systemic infections (Bou Aoun et al., 2011), although a study reported that Tep3 mutant flies are highly susceptible to the nematode parasite Heterorhabditis bacteriophora (Arefin, Kucerova et al., 2014). Interestingly, a study led in Drosophila cultured S2 cells showed that Tep2 is required for the phagocytosis of the Gram-negative species E. coli, Tep3 is required for the uptake of the Gram-positive Staphylococcus aureus, and unexpectedly that Tep6 is required to phagocytose the dimorphic yeast Candida albicans (Stroschein-Stevenson, Foley et al., 2006).
In contrast to S. marcescens, P. aeruginosa strain PA14 ultimately manages to establish an exponential infection in the hemocoel four to five days after its ingestion. In a previous study, we showed that a member of the LuxR family of signal receptor-transcriptional regulators in PA14, RhlR, is required to circumvent the cellular immune response (Limmer et al., 2011a). Indeed, rhlR mutants are almost avirulent in an intestinal infection model since they remain at very low levels in the hemolymph and kill the infected flies at a much reduced rate. Interestingly, the cellular immune response remains functional until late stages of a PA14 infection, suggesting that hemocytes are not directly targeted by PA14, unlike what happens with P. aeruginosa strain CHA, which neutralizes Drosophila phagocytosis through the secretion of its ExoS toxin into hemocytes (Avet-Rochex, Bergeret et al., 2005).
RhlR is the major regulator of one of the three known quorum-sensing systems in P. aeruginosa. Quorum-sensing systems play a major role in coordinating the expression of virulence genes in several infection models (Coggan & Wolfgang, 2012, Jimenez, Koch et al., 2012, Schuster, Sexton et al., 2013, Williams & Camara, 2009). However, we have failed to uncover a strong role for the other two P. aeruginosa quorum sensing systems regulated by LasR and MvfR in the Drosophila intestinal infection model (Limmer et al., 2011a). This observation was somewhat unexpected since the LasR-mediated quorum-sensing system appears to function upstream of the RhlR-mediated system. RhlR is activated by binding to an auto-inducer molecule, butanoyl-homoserine lactone (C4-HSL), which is synthesized by the RhlI enzyme. The transcription of the rhlI and rhlR genes is in turn activated by the Las transcriptional regulator LasR (Coggan & Wolfgang, 2012, Jimenez et al., 2012, Schuster et al., 2013, Williams & Camara, 2009). Activation of RhlR takes place when a threshold concentration of C4-HSL is reached, which correlates with a threshold bacterial concentrations reached during exponential growth in in vivo studies.
Here, we report that the virulence phenotype exhibited by ingested PA14 rhlI mutants is strikingly distinct from that exhibited by rhlR mutants. rhlRmutants exhibit severely impaired virulence, whereas rhlI mutants at most modestly impaired virulence, suggesting that RhlR can function independently of activation by C4-HSL. We further establish that RhlR, but not RhlI, is required to elude opsonization by a Tep4-dependent process. Finally, we establish that in contrast to its protective role during PA14 intestinal infections, Tep4 plays the opposite role in a systemic infection model, possibly by preventing the activation of phenol oxidase.
RESULTS
ΔrhlI is more virulent than ΔrhlR in the Drosophila intestinal infection model
RhlR is activated by C4-HSL synthesized by the RhlI enzyme, as shown in numerous in vitro studies (Gambello, Kaye et al., 1993, Latifi, Winson et al., 1995, Pesci, Pearson et al., 1997, Seed, Passador et al., 1995). Thus, we expected that rhlI and rhlR mutants would display the same phenotype. Unexpectedly, however, although a ΔrhlI deletion mutant strain was indeed less virulent than wild-type PA14 when ingested by flies, it was significantly more virulent than ΔrhlR (Fig. 1A, B). Moreover, whereas the ΔrhlR mutant was cleared from the hemolymph, the ΔrhlI mutant proliferated in this compartment, although it appeared to grow less rapidly than wild-type PA14 (Fig. 1C, D). Consistent with this latter result, ΔrhlI but not ΔrhlR triggered a systemic immune response, as monitored by measuring the expression of the Diptericin gene (Fig. S1E-F). Similar results were obtained with independent ΔrhlR and ΔrhlI in frame deletion mutants constructed by another laboratory (Fig. S1) (Hoyland-Kroghsbo, Paczkowski et al., 2017), thereby confirming the correlation between the ΔrhlR and ΔrhlI null genotypes and the differing ΔrhlR and ΔrhlI phenotypes.
We next tested the survival rates of flies in which the cellular response had been ablated by injecting latex beads (LXB) after feeding on wild-type or ΔrhlR or ΔrhlI mutant bacteria. Both ΔrhlR and ΔrhlI killed latex bead-injected flies much faster than PBS-injected control flies, at approximately the same rate, but more slowly than wild-type PA14, with the difference between ΔrhlI and wild-type PA14 at the borderline of statistical significance (p=0.07) (Fig. 1E, F).
It is important to determine whether the apparently enhanced virulence of ΔrhllR and ΔrhlI mutants observed in phagocytosis-impaired flies (compared to flies not injected with latex beads) is simply a reflection of the increased virulence of wild-type PA14 in these immuno deficient flies, or whether the enhanced virulence of the ΔrhlR and ΔrhlI mutants is indicative of the fact that the RhlR-mediated regulatory systems plays an important role in counteracting the cellular immune response. To this end, it is useful to measure the difference in LT50 values of control vs. latex bead-injected flies (LT50[wt-wtLXB]) for each mutant and to compare it to that measured for wild-type PA14. ΔrhlR did recover virulence with a LT50[wt-wtLXB] of 4.7 days, as compared to 2.4 days for wild-type PA14, which corresponds to the level of recovered virulence reported earlier (Fig. 1G) (Limmer et al., 2011a). With a value of 3.5 days, ΔrhlI displayed an intermediate LT50[wt-wtLXB], although the significance of the difference with wild-type PA14 or ΔrhlR could not be assessed as the ΔrhlI values were too spread out (Fig. 1G). Indeed, ΔrhlI mutants consistently tended to display a more variable survival phenotype than the ΔrhlR mutant (Fig. 1B).
Finally, both the ΔrhlR and ΔrhlI mutant strains yielded similarly shaped survival curves when used to infect flies with an impaired cellular defense. These survival curves were less steep than those obtained with wild-type PA14, as measured by their Hill coefficients (Fig. 1H), suggesting that quorum-sensing is involved in determining the shape of the survival curve. This reflects a collective property of flies placed in the same vial, which succumb less synchronously during infections when the rhl quorum sensing system is missing.
rhlI and wild-type PA14, but not rhlR, strains colonize the C. elegans digestive tract
The nematode C. elegans is a well-established model host to study P. aeruginosa pathogenesis (Irazoqui, Urbach et al., 2010, Pukkila-Worley & Ausubel, 2012, Tan, Mahajan-Miklos et al., 1999a). The C. elegans intestinal infection model shares some key features with the Drosophila model, at least during the initial stages of the infection. For instance, in both models, ingested bacteria are exposed in the gut lumen to antimicrobial peptides and to reactive oxygen species generated by the Dual oxidase enzyme {Ha, 2005 #1857}{Chavez, 2009 #3616}. However, in contrast to ingested P. aeruginosa in Drosophila, PA14 are not known to escape from the gut compartment during the nematode infection. We therefore tested whether the differences that we observed in the virulence of ΔrhlR and ΔrhlI mutants in the Drosophila intestinal infection assay were reflected in a well-established C. elegans – P. aeruginosa nematode “slow killing” survival assay (McEwan, Feinbaum et al., 2016, Tan et al., 1999a). Indeed, two independently constructed in frame rhlR deletion mutants in the PA14 background were dramatically less virulent than two independently constructed rhlI deletion mutants in their ability to kill C. elegans (Fig. 2A)
The primary C. elegans immune response occurs in intestinal epithelial cells and because the worms are transparent, host-pathogen interactions can be easily visualized in the intestinal lumen. Thus, in addition to the C. elegans survival assay (Fig. 2A), we also used a quantitative assay (Figs. 2B-E; see Materials and Methods) that monitors the accumulation of live bacterial cells in the intestine of the nematodes using PA14, ΔrhlR, and ΔrhlI expressing GFP to monitor the level of intestinal colonization. Live wild-type P. aeruginosa PA14 cells start accumulating in the intestine 24-48 hours post infection. Consistent with the Drosophila infection assays described in Fig. 1, two independent ΔrhlR mutants in the PA14 background colonized the C. elegans intestine at significantly lower levels than two independent ΔrhlI mutants. Indeed, in this colonization assay, the ΔrhlI mutants were indistinguishable from wild-type PA14, similar to the results in the nematode killing assay. An alternative explanation for these results is that C. elegans preferentially feeds on the ΔrhlI mutant compared to the ΔrhlR mutant and simply overwhelms the immune response with a larger number of ingested cells. However, this possibility was ruled out by monitoring the pumping (feeding) rate of C. elegans feeding on wild-type, ΔrhlR and ΔrhlI mutants. C. elegans pumped at the same rate on all three strains (Fig. S2).
Phagocytosis protects Drosophila against invasion of its hemocoel by wild-type PA14 during the early phase of the infection
In the Drosophila intestinal infection model, flies constantly feed on PA14 present on a filter. A defining feature of this model is that even though bacteria are able to rapidly cross the intestinal barrier to penetrate the hemocoel, as had been previously described for Serratia marcescens (Nehme et al., 2007), the PA14 titer in the hemolymph remains low for the first few days of the infection. After this initial incubation period, there is an exponential proliferation of PA14 in the hemocoel, which coincides with the activation of the systemic immune response. Using bacteria expressing different colored fluorescent proteins, we previously showed that S. marcescens continuously crosses the intestinal barrier during the infection (Nehme et al., 2007). Fig. 3A-B shows that when flies that have been feeding on dsRed-labeled PA14 bacteria for four days were switched to a filter laced with GFP-labeled PA14, the green bacteria progressively replaced the red bacteria both in the gut and hemocoel compartments. We conclude that P. aeruginosa, like S. marcescens, continuously crosses the intestinl barrier during the infection.
Next, we asked at what time periods during an infection is phagocytosis important in preventing PA14 growth in the hemolymph. To this end, we saturated the phagocytic apparatus of hemocytes by injecting latex beads into flies at different time points during infection. As expected, blocking phagocytosis one day prior to the infection led to an earlier demise of the PA14-infected flies compared to PBS-injected control flies. Similar results were found when latex beads were injected four hours or one day after infection, although in the latter case the difference was not significant (its value was nevertheless similar to that obtained by injection one day prior to infection at -1 day; Fig. 3C). In contrast to injecting the latex beads one day after infection, the injection of latex beads four or six days after the beginning of the ingestion of wild-type PA14 did not modify the survival rate of flies. That is, the LT50 values were similar to those of control (PBS-injected) flies, consistent with the conclusion that starting about four days after infection the cellular immune response no longer plays a major role in limiting a wild-type PA14 infection.
In contrast to wild-type PA14, ΔrhlR bacteria were kept in check by phagocytosis at least up to day four and to some extent up to six days after infection (Fig. 3D, F). Phagocytosis was efficient against ΔrhlI bacteria for approximately four days (Fig. 3E, F). These data suggest that ΔrhlR bacteria are constantly kept in check by the cellular immune response when penetrating the hemocoel, whereas wild-type PA14 ultimately escape this immune surveillance. ΔrhlI bacteria display an intermediate phenotype, with a somewhat decreased virulence in flies in which phagocytosis was blocked at day 4 (Fig. 3F).
A recent study has reported that hemocytes are recruited to the gut after the ingestion of bacteria (Ayyaz, Li et al., 2015). We confirmed this finding in the case of P. aeruginosa oral infection, with a significant recruitment observed at four hours after the beginning of the infection with either wild-type PA14 or Δrhl mutants (Fig. 3G-H, Fig. S3). While hemocytes remained associated with the midgut for at least three days after the beginning of the ingestion of wild-type PA14 or ΔrhlR bacteria, they were not at three days in the case of ΔrhlI bacteria (Fig. 3H). While some ingested bacteria could be detected in the hemocytes recruited to the gut, this phenomenon was not reproducible enough to allow reliable quantification.
Drosophila Tep4 is required for host defense against ingested PA14
Our previous work had shown that flies mutant for the putative phagocytic receptor gene Eater are more susceptible to PA14 ingestion and display a phenotype similar to that obtained by latex bead-mediated ablation of the phagocytic capacity of hemocytes (Limmer et al., 2011a). Thioester-containing proteins have been reported to be required for phagocytosis in mosquitoes and also in cultured Drosophila S2 cells (Levashina et al., 2001, Stroschein-Stevenson et al., 2006). We therefore tested mutations affecting the Tep2, Tep3, and/or Tep4 genes (Bou Aoun et al., 2011). In the case of Tep1, since no mutants were available, we tested an RNAi transgene expressed either in hemocytes or in the fat body. However, we did not observe any change in the virulence of ingested PA14 (Fig. S4A, B). Tep4 and triple Tep2-Tep3-Tep4 mutants displayed increased susceptibility to PA14 ingestion, in contrast to Tep3 and double Tep2-Tep3 mutants that displayed respectively a somewhat decreased or wild-type susceptibility (Fig. 4A, D-E). Of note, uninfected Tep3 mutants fed on a sucrose solution displayed an enhanced fitness when compared to wild-type or other Tep mutant lines (Fig. S4C). We conclude from these data that Tep4, but not other thioester-containing proteins, is required for host defense against ingested PA14.
Next, we found that ΔrhlR became as virulent as wild-type PA14 when ingested by Tep4 or Tep2-Tep3-Tep4 mutants, which was not observed with the Tep2 and Tep2-Tep3 mutant strains (Fig. 4B, D-E). Interestingly, the injection of latex beads in Tep4 flies only modestly increased the virulence of rhlR bacteria when compared to PBS-injected Tep4 flies (Fig. S4 D, E), suggesting that phagocytosis of PA14 is severely affected in the Tep4 mutant. ΔrhlI bacteria behaved like ΔrhlR bacteria when ingested by Tep4 (Fig. 4C, E), similarly to flies injected with latex beads (Fig. 1E), although ΔrhlR recovered virulence to a much larger extent (3.1 days) than ΔrhlI (0.9 days) when ingested by Tep4 flies. Hence, the behavior of ΔrhlR mutant PA14 is similar in eater and Tep4 mutant flies, thereby raising the possibility that both fly genes are involved in the same process, in keeping with an unchanged phenotype of Tep4 when phagocytosis was blocked by the injection of latex beads (Fig. S4DC).
Phagocytosis of PA14 bacteria is impaired in Tep4 mutant hemocytes
To quantitatively monitor the uptake of PA14, we used assays that relied on larval hemocytes. First, we injected heat-killed, pHrodo®-labeled bacteria in wild-type or Tep4 third-instar larvae. The dye becomes fluorescent when placed in an acidic environment such as that encountered in phagosomes. After 45 minutes, the larvae were bled and a phagocytic index was established. Wild-type hemocytes ingested significantly more PA14 or ΔrhlR bacteria than Tep4 hemocytes (Fig. 5A). There were however no significant differences between heat-killed wild-type PA14 and rhlR bacteria uptake by wild-type hemocytes on the one hand, or Tep4 hemocytes on the other (Fig. 5A). This was not necessarily unexpected as heat-killing likely inactivates rhlR-dependent virulence factors and might also alter the surface of bacteria. We therefore modified the assay with live bacteria and used an antibody we had raised against PA14 to differentially immuno-stain the bacteria, prior to and after permeabilization of the fixed cells. As before, Tep4 hemocytes exhibited a decreased uptake of bacteria compared to wild-type hemocytes. However, in the case of live bacteria, ΔrhlR bacteria were significantly better phagocytosed than wild-type PA14 bacteria when injected into wild-type or Tep4 larvae. ΔrhlI exhibited an intermediate phenotype in this assay and was not significantly different from either wild-type or ΔrhlR bacteria (Fig. 5B). We conclude that this assay is not sensitive enough to discriminate between these two bacterial mutant strains. We obtained similar results using the ΔrhlR and ΔrhlI strains generated by another laboratory (Fig. S1G).
Tep4 opsonizes rhlR better than rhlI or wild-type bacteria
We next designed an experiment to assess whether Tep4 functions as an opsonin, i.e., that it is deposited on the surface of bacteria to facilitate its detection and subsequent ingestion by hemocytes. To this end, we collected the hemolymph from either wild-type or Tep4 larvae and incubated it with live bacteria. These bacteria were then retrieved and injected into either naive wild-type or Tep4 mutant larvae prior to bleeding these injected larvae to be able to count the ingested bacteria as described above (Fig. 6A). Wild-type PA14 were poorly phagocytosed in this assay (medians of phagocytic index lower than 10, Fig. 6C), when the opsonized bacteria were first incubated in the hemolymph of Tep4-containing wild-type larvae and then secondarily injected into wild-type or Tep4 larvae (although the former yielded a significantly increased phagocytic index, as compared to bacteria incubated first in Tep4 hemolymph; Fig. 6C). In contrast, bacteria that had been first incubated in Tep4 mutant hemolymph were hardly ingested when re-injected into Tep4 larvae (Fig. 6E). Re-injection of these bacteria into wild-type larvae recipients modestly increased the phagocytic index (Fig. 6E), which was nevertheless lower than that of bacteria that had been pre-incubated with wild-type hemolymph (Fig. 6C). When these experiments were performed using ΔrhlR bacteria that were injected in Tep4 larvae, it made a major difference whether these mutant bacteria had first been pre-exposed to wild-type or Tep4 hemolymph. Tep4-dependent opsonization led to a massive uptake of bacteria (median phagocytic value of 162), whereas nonopsonized bacteria (pre-incubated with Tep4 mutant hemolymph) were hardly ingested (median phagocytic value of 12; Fig. 6D-E). As expected, nonopsonized bacteria that were then injected in wild-type recipients were much better phagocytosed (median phagocytic value of 214), presumably because Tep4 was circulating in the wild-type hemolymph (Fig. 6B). They were nevertheless ingested less efficiently than opsonized bacteria (median phagocytic value of 376; Fig. 6B). Finally, ΔrhlI bacteria were also opsonized by Tep4, but significantly less than ΔrhlR bacteria (Fig. 6B, D). Again, they displayed a phenotype that was intermediate to that of wild-type PA14 on the one hand, and ΔrhlR on the other. We conclude that PA14 and, to a lesser extent, ΔrhlI bacteria are less efficiently opsonized and subsequently phagocytosed than ΔrhlR bacteria, which are therefore unable to elude the cellular immune response.
Tep4 plays an adverse role in a PA14 systemic infection model in Drosophila
A recent study has reported that Tep4 mutants are more resistant to a systemic infection with the entomopathogenic bacterium Photorhabdus luminescens in a septic injury model (Shokal & Eleftherianos, 2017). By injecting several doses of PA14, from 10 to 1000 CFUs, directly into the thorax of flies, we also consistently found that Tep4 mutants survived better than wild-type flies in this systemic infection model (Fig. 7A). This difference in survival between Tep4 and wild-type flies was largely attenuated when ΔrhlR bacteria were injected, thereby establishing again a relationship of altered virulence of these bacteria in a Drosophila Tep4 mutant background (Fig. 7B). Using the steady-state expression of the antibacterial peptide gene Diptericin as a read-out of the activation of the IMD pathway that regulates the systemic immune response against Gram-negative bacteria, we found no significant difference of expression between wild-type and Tep4 (Fig. 7C). As a higher level of induction of the IMD pathway is unlikely to account for the increased resistance of Tep4 mutants against PA14 infection, we tested whether the phenol oxidase cascade was more efficiently activated in this mutant background, as previously reported (Shokal & Eleftherianos, 2017). Indeed, we found that pro-phenol oxidase was cleaved to some extent in Tep4 but not in wild-type flies. These data further suggest that wild-type PA14 bacteria elude detection by the factors that trigger the prophenol oxidase cascade, and that Tep4 plays a role in this process of evasion from the melanization response.
DISCUSSION
In this study, we analyzed the interactions of P. aeruginosa with Drosophila from the dual perspective of both pathogen and host. Our data lead us to propose a model in which RhlR plays a pivotal role in virulence by diminishing the ability of the cellular immune arm of the host defense response to detect P. aeruginosa once the bacteria have reached the internal body cavity of the insect after crossing the intestinal barrier. Surprisingly, RhlR function in eluding opsonization by Tep4 is at least partially independent of the C4-HSL producing enzyme RhlI. These results as well as those of another study (Mukherjee, Moustafa et al., 2017) show that RhlR can function independently of C4-HSL, but they do not formally establish that RhlR is functioning independently from a quorum-sensing system in this function. Furthermore, we establish a dual function for the putative opsonin Tep4, which plays opposite roles in host defense depending on the infection route.
A rhlI-independent function of rhlR
P. aeruginosa is a pathogen that uses complex signaling mechanisms to adapt to its environment, and its three quorum-sensing regulators (LasR, RhlR and MvfR) appear to be involved in the regulation of a variety of virulence-related functions (Coggan & Wolfgang, 2012, Jimenez et al., 2012, Schuster et al., 2013). In vitro studies, sometimes complemented by in vivo experiments, have revealed that these quorum-sensing systems are intricately intertwined. It was thus unexpected that only the P. aeruginosa RhlR regulator appears to play a critical role for virulence in a Drosophila intestinal infection model and not the two other quorum sensor regulators (Limmer et al., 2011a). Here, we report that ΔrhlR null mutants consistently display virulence levels that are much weaker than those observed with ΔrhlI mutants (Fig. 1B), do not proliferate in the hemolymph in contrast to ΔrhlI mutants, and are phagocytosed and opsonized in a Tep4-dependent process more efficiently than ΔrhlI and wild-type bacteria. These observations suggest that RhlR functions at least partially independently from RhlI and presumably independently from C4-HSL activation. In contrast, ΔrhlI mutants exhibit an impaired virulence in both wild-type and Tep4 immuno-deficient flies, similarly to ΔrhlR mutants. Both ΔrhlI and ΔrhlR mutants display survival curves with shallow slopes. Thus, the partially overlapping phenotypes of ΔrhlI and ΔrhlR in flies with impaired cellular immunity suggests that RhlR partially functions together with RhlI as a classical quorum-sensing regulator in a process that remains to be delineated. Interestingly, it appears that RhlR controls gene expression for biofilm formation both in a C4-HSLdependent and C4-HSL independent manner (Mukherjee et al., 2017). However, this putative conventional function of RhlR plays a minor role in virulence of PA14 in Drosophila, as shown by the weak virulence-related phenotypes of ΔrhlI mutants documented in our study.
One explanation for the RhlI-independent activity of RhlR is that it gets activated in a C4-HSL-independent manner by an as yet unidentified quorum-sensing compound. Of note, RhlR does not appear to be activated by 3-oxo-C12-HSL (Mukherjee et al., 2017), the LasR ligand. In any case, lasR and lasI mutant bacteria display only a modestly decreased virulence phenotype in the Drosophila infection model (Fig. S5). The diketopiperazines (DKPs) represent a candidate family of RhlR-activating compounds (Holden, Ram Chhabra et al., 1999); however, at least one study failed to detect any interaction of these compounds with LuxR proteins (Campbell, Lin et al., 2009). The resolution of this issue will require testing mutants that affect the synthesis of DKPs.
Another hypothesis to consider is that RhlR may function independently of auto-inducer molecules. RhlR forms dimers in the presence or absence of C4-HSL (Ledgham, Ventre et al., 2003). Further studies reported that RhlR functions as a repressor when unbound to C4-HSL (Anderson, Zimprich et al., 1999, Medina, Juarez et al., 2003). Interestingly, RhlR dimers seem to bind its target DNA sequence with an altered conformation (Medina et al., 2003). Finally, transcriptomics studies on lasR-rhlR double mutants also revealed several target genes that appear to be repressed by either LasR or RhlR (Schuster, Lostroh et al., 2003, Wagner, Bushnell et al., 2003). Thus, a repressor function for RhlR unbound to C4-HSL cannot be excluded at this stage. A limitation of all of the studies related to the C4-HSLrelated activity of RhlR is that they were performed in vitro and not in vivo.
Finally, our studies on the inactivation of the cellular immune response at different time points of the infection further support a quorum sensing-independent role of RhlR. Our study revealed that phagocytosis is required only when very few bacteria are present in the hemolymph, that is, during the first days of the infection. A caveat here is that we cannot exclude the possibility that C4-HSL or other cryptic autoinducers might be produced by the bacteria present in large numbers in the gut compartment. However, if autoinducers, including C4-HSL, were produced in the intestinal lumen and were able to cross the digestive barrier, it is difficult to understand why they would not immediately activate RhlR resulting in fullblown bacteremia without the observed lag before the exponential proliferation phase in the hemocoel. This hypothesis also does not account for why the rhlI reduced virulence phenotype is much weaker than that of rhlR, unless this reflects the differing opsonization properties of these mutants.
The function of RhlR in bypassing host defenses in C. elegans?
Except for fungal invasion of the epidermis by nematophagous fungi, how C. elegans senses infections remains poorly understood (Zugasti, Bose et al., 2014). The finding that ΔrhlR mutants are much less virulent than ΔrhlI mutants in a C. elegans infection model, and that ΔrhlR mutants fail to colonize the intestinal tract of worms might be due either to an impaired escape from detection by the immune system or to a defective resistance to its action. It is not clear, however, that RhlR-mediated regulation of the production of toxic phenazines, as shown in the case of PA14-mediated “fast killing” of C. elegans (Mukherjee et al., 2017), is the reason the ΔrhlR mutant is so dramatically impaired in the C. elegans “slow killing” assay used in our study in Fig. 2A. The major difference in the fast and slow C. elegans killing assay is the composition of the agar medium in which the P. aeruginosa is grown and on which the killing assays are performed (Mahajan-Miklos, Tan et al., 1999, Tan et al., 1999a, Tan, Rahme et al., 1999b). Fast killing is mediated by phenazines (Cezairliyan, Vinayavekhin et al., 2013, Mahajan-Miklos et al., 1999) whereas slow killing is multi-factorial (Feinbaum, Urbach et al., 2012), and PA14 mutants deficient in the production of phenazines do not exhibit a significant killing defect in the slow killing assay (Tan et al., 1999b). In contrast to Drosophila, no cellular host defense has been detected in C. elegans and is unlikely to be involved in the immune response to intestinal infection. The use of ΔrhlR mutants will open the way to the identification of the relevant host defense systems that are circumvented by wild-type PA14 bacteria. In any case, the lack of a significant phenotype of the ΔrhlI mutants in the C. elegans killing and intestinal proliferation assays is striking. This can be exploited in future studies to help elucidate the underlying rhlI-independent mechanisms involved in RhlR-mediated regulation of virulence.
RhlR counteracts the cellular host defense by eluding detection by Tep4
Our phagocytosis and opsonization data are consistent with a model in which RhlR controls the expression of gene products that mask the site being recognized by Tep4 or a Tep4-associated protein, presumably on the cell wall. Alternatively, RhlR may actively inhibit the uptake of opsonized bacteria. The masking or inhibition of ingestion processes may be sensitive to heat, as wild-type heat-killed bacteria appeared to be more efficiently taken up by hemocytes than live ones (compare median values for PA14WT in Fig. 5A to those in 5B). Alternatively, the processes may be unstable and require permanent maintenance that can no longer be achieved when the bacterial cells are killed.
Insect thioester-containing proteins belong to the complement family, and have been shown to be involved in the opsonization of bacteria in mosquitoes. In Drosophila, Tep2 has been reported to be required for the uptake of Escherichia coli, a Gram-negative bacterium, by cultured S2 cells (Stroschein-Stevenson et al., 2006), a finding confirmed in vivo (Shokal, Kopydlowski et al., 2017). In contrast, we find no involvement of Tep2 in our in vivo intestinal infection model with P. aeruginosa but do detect a requirement for Tep4 in phagocytosis and opsonization assays. Given that the structure of mosquito thioester containing protein 1 is similar to that of complement family C3, a well-described opsonin, our data are compatible with a model of direct opsonization of bacteria by Tep4.
A host factor plays opposite roles in host defense against the same pathogen according to the infection route
The finding that Tep4 plays a protective function in the intestinal infection model whereas it is detrimental in the case of a direct systemic infection is paradoxical. This may actually represent two faces of the same phenomenon. PA14 may have developed a stealth strategy to avoid detection by the immune system of living organisms and thus may actively hide any features that might reveal its presence. We propose here that RhlR plays a critical role in a program that renders PA14 furtive, in keeping with our finding that bacteria likely need to be alive to escape phagocytosis efficiently (Fig. 5). As a result of RhlR action, only a few sites would be available on the surface of the wild-type bacteria for Tep4 direct or indirect binding. There, Tep4 would mediate opsonization and then subsequent phagocytosis of the bacteria. Our data in the systemic infection model are compatible with the possibility that Tep4 competes for these sites with Pattern Recognition Receptors (PRRs) that activate the prophenol oxidase cascade since PO activation occurs only in the Tep4 mutant background. It is likely that small peptidoglycan (PGN) fragments released by proliferating bacteria represent a major trigger of the IMD pathway in addition to large PGN fragments directly sensed by PGRP-LC, thereby accounting for the apparent normal expression of Diptericin when flies are challenged with injected PA14. In contrast, we have previously established that some fungi and Gram-positive bacteria trigger the phenol oxidase cascade through defined PRRs (Matskevich, Quintin et al., 2010). The situation is less clear as regards Gram-negative bacteria. While the original characterization of PGRP-LE suggested that it triggers the phenol oxidase activation cascade (Takehana, Katsuyama et al., 2002), and acts non cellautonomously (Takehana, Yano et al., 2004), subsequent studies have documented a role for PGRP-LE as an intracellular sensor for PGN fragments (Bosco-Drayon, Poidevin et al., 2012, Ferrandon et al., 2007, Yano, Mita et al., 2008). Thus, further work will be required to identify whether Tep4 actually competes with PRRs in the detection of pathogens.
Our results further suggest that the cellular immune response is a relevant defense when a few bacteria enter the hemocoel after escaping from the digestive tract in the intestinal infection model, but not in the septic injury model. Conversely, melanization mediated by activated phenol oxidase appears to be somewhat effective after injection but not ingestion of PA14.
A major challenge will be to establish how RhlR influences the surface properties of PA14 or alternatively inhibits the uptake of opsonized bacteria.
Finally, ΔrhlR mutants exhibit reduced dissemination capacities in a rodent lung infection model when compared to ΔrhlI or wild-type PA14(Mukherjee et al., 2017). By analogy to our findings in the Drosophila intestinal infection model, it will be interesting to determine whether the complement system restricts the systemic escape of ΔrhlR mutants from the mouse lung into the periphery.
MATERIALS AND METHODS
Many methods employed in this study have been described in detail in Haller et al. (2014).
C. elegans killing and intestinal accumulation assays
The “slow-killing” of C. elegans by P. aeruginosa was monitored using automated image analysis as previously described (McEwan et al., 2016, Stroustrup, Ulmschneider et al., 2013). To monitor the accumulation of P. aeruginosa in the C. elegans intestine, wild-type (N2) animals were used for all experiments. Worms were reared on non-pathogenic E. coli OP50 on nematode growth media at 25°C. Synchronized L4 worms (fourth larval stage) were transferred to slow kill (SK) nematode growth media agar plates containing a lawn of P. aeruginosa PA14::GFP strains. Post infection at 24 and 48 hours approximately 20 worms were picked onto a 2% agar pad that contain the paralyzing agent levamisole (1mM). The worms were imaged in the GFP channel using a Zeiss Apotome microscope with the same exposure time for all the worms on wild type PA14 and the ΔrhlR and ΔrhlI mutants. Post acquisition the images were processed using ImageJ software and the area and fluorescence intensity was measured. The relative fluorescence intensity is plotted and a non-parametric Mann-Whitney test was used to determine statistical significance.
Opsonization assay of live bacteria
Overnight cultures of PA14, ΔrhlR, and ΔRhlI mutants were concentrated to OD10 in PBS. Twenty third instar larvae were bled in 150 µL of bacterial resuspended in PBS at aOD of 10 and incubated at room temperature for 30 to 45 min. Samples were centrifuged at 500 rcf for 15 min and the pellet (containing larval debris) was removed. A second centrifugation was performed at 3500 rcf for 15 min to retrieve bacteria in the pellet, that was re-suspended in 10 µL PBS. A5001 or tep4 third instar larvae were injected with 32.2 nL of the live bacteria solutions, using a Nanoject apparatus (Drummont). After 60-90 min of incubation, one larva was bled in each well of an 8-well pattern microscopy slide that contained PBS. The cells were left to settle to the bottom for 30 min and were then fixed in 4 % paraformaldehyde for 15 min, in a humid chamber. The samples were washed twice in PBS and they were stained with a 1/500 diluted rabbit antiserum against PA14 in a PBS solution with 2 % BSA for 2 hours at room temperature. The cells were incubated with a FITC-labeled goat anti-rabbit secondary antibody (Invitrogen) in a PBS solution with 2 % BSA for 2 hours at room temperature. After a 20 min permeabilization step in a PBS solution with 0.1 % Triton X-100 and 2 % BSA, a second round of staining with a 1/500 diluted rabbit antiserum against PA14 in a PBS solution with 0.1 % Triton X-100 and 2 % BSA was performed for 2 hours at room temperature. The samples were then incubated with a Cy3-labeled goat anti-rabbit secondary antibody (Invitrogen) in a PBS solution with 0.1 % Triton X-100 and 2 % BSA for 2 hours at room temperature. The slides were mounted in Vectashield with DAPI (Vector Laboratories) and analyzed using a Zeiss Axioskope 2 fluorescent microscope. 40 to 50 cells per larva were analyzed: the number of red fluorescent bacteria that were not green fluorescent was counted for each DAPI-positive hemocyte, and the phagocytosis index was calculated (% of phagocytes containing at least 1 only-green bacterium)×(mean number of only-green bacteria per positive cell). We used the nonparametric Mann-Whitney test for statistical analysis.
Phenol oxidase cleavage assay
The procedure was performed as described (Leclerc, Pelte et al., 2006), except that hemolymph loads were not adjusted by measuring the protein content of the extracted hemolymph. The antibody used has been generated by Dr. H. M. Müller against Anopheles phenol oxidases (Muller, Dimopoulos et al., 1999). The ratio of cleaved to noncleaved form was performed by densitometry scanning.
Statistical analysis
All statistical analysis were performed on Graphpad Prism version 5 (Graphpad software Inc., San Diego, CA). Details are indicated in the legend of each figure.
AUTHOR CONTRIBUTIONS
SH, AF, SL, and DF conceived the Drosophila experiments and analyzed the data. SH performed the majority of these experiments, with later work performed by AF with an input from SL; SS performed the precursor experiments that led to this work. Work on the septic injury model was performed by JC, with some help from ZL. ΔlasR, ΔlasI, ΔrhlR and ΔrhlI mutants were constructed by ED and SY, except when indicated otherwise. AH and FMA conceived the C. elegans experiments and analyzed the data, which were obtained by AH. SH, DF, and FMA wrote the manuscript, with inputs from other co-authors.
CONFLICT OF INTEREST
The authors report no conflict of interests.
ACKNOWLEDGEMENTS
We are grateful to J. Nguyen, to Gaétan Caravello, and D. McEwan for help with some experiments, and to W. M. Yamba and J. Bourdeau for expert technical help. We thank Dr. A. Filloux and Dr. B. Bassler for the gift of PA14 mutant strains, the Bloomington Stock Center for Drosophila stocks. Dr. S. Niehus provided valuable advice. We thank Dr. B. Bassler for sharing her work with us prior to publication. This work has been funded by CNRS, University of Strasbourg, Fondation pour la Recherche Médicale (Equipe FRM to D.F.), Agence Nationale de la Recherche (DROSOGUT, ANR-11-EQPX-0022) and US NIH grant R01 AI085581 awarded to F.M.A.