Ubiquitin C-Terminal Hydrolase L5 Plays an Essential Role in the Fly Innate Immune Defense against Bacterial Infection

Background : Drosophila ubiquitin carboxy-terminal hydrolase L5 (Uch-L5) functions as a critical component of the 26S proteasome to mediate degradation of polyubiquitinated proteins. It was recently shown to modulate tissue/organ development by targeting the Smoothened protein in the hedgehog pathway. However, whether it plays a role in controlling organismal immune response remains largely unknown. Methods : Reverse transcription plus quantitative polymerase chain reaction (RT-qPCR), dual-luciferase, and Western blot assays were used to explore the potential function of Uch-L5 in the innate immune regulation in cultured Drosophila S2 cells. Further genetic manipulations and bacterial infections were conducted to confirm the findings in vivo . Results : Silencing of Uch-L5 antagonizes the immune deficiency (IMD) but not the Toll innate immune signaling both in vitro and in vivo . Moreover, Uch-L5 positively contributes to the Drosophila innate immune response via its N-terminal Uch domain, which is the catalytical triad executing its deubiquitinase activity. Conclusions : Our studies shed light on a novel function of the deubiquitinase Uch-L5 in governing the anti-microbial defense in Drosophila .


Introduction
Innate immunity reacts quickly and efficiently upon recognition of extracellular pathogenic invasion.This constitutes the first line of defense of most organisms [1].Pattern recognition receptors (PRRs) have been suggested to be mainly responsible for recognizing pathogenic stimuli, in order to trigger downstream signaling transductions, thereby inducing expressions of various immune effectors [2,3].Several pioneering studies have demonstrated the pivotal roles of adaptor proteins and correlative modulators in the signal transductions from transmembrane and intracellular receptors to secreted effectors [reviewed in [1][2][3][4][5][6]].Identification and characterization of novel adaptors/modulators has always been one of the hotspots in the basic research of innate immunity.
Drosophila melanogaster (fruit fly) is an excellent animal model for uncovering novel modulators in the innate immune signaling pathways, due to its powerful genetic approaches and worldwide resources of Drosophila mutants and transgenes.The fly innate immune response is mainly governed by two signaling pathways, namely the Toll and the immune deficiency (IMD) pathways [7][8][9], which are similar to the mammalian Myd88 (myeloid differentiation factor 88)-dependent TLR (Toll-like receptor) and TNFR (tumor necrosis factor receptor) signaling pathways, re-spectively.Upon infection by most fungi or Gram-positive bacteria, the Toll receptor-involved signaling is activated in order to trigger the induction of downstream anti-microbial peptides (AMPs), for instance drosomycin (Drs) and metchnikowin (Mtk) [10].On the other hand, the IMD signaling pathway is mostly activated by Gram-negative bacteria and some types of Gram-positive bacteria, resulting in secretion of another set of AMPs like attacins (Att), cecropin (Cec), and diptericin (Dpt) for the host immune defense [11].
In the present study, we focused mainly on exploring the potential role of Uch-L5 in regulating Drosophila innate immunity.We show that silencing of Uch-L5 results in markedly decreased IMD signaling, whereas ectopic expression of Uch-L5 behaves oppositely.The N-terminal Uch domain is both required and sufficient for Uch-L5 positively impacting on IMD signaling, suggesting that Uch-L5 executes its immune function relying on the Dub enzymatical activity.We further provide evidence displaying that Uch-L5 is essential for the immune defense of adult flies upon bacterial infections.Collectively, our results uncover a novel biological role of Uch-L5 in mediating the IMD innate immune reaction in Drosophila.

dsRNA Synthesis and RNAi in S2 Cells
All dsRNAs were synthesized according to methods described previously [33].In brief, DNA templates were amplified using specific primers (Supplementary Table 1).PCR products were then combined with 1/10 volume of 5 M NaCl and 2.5-fold volume of EtOH, followed by centrifugation for 10 min at high speed.The pellet was washed with 75% EtOH and dissolved in RNase/DNase-free water.The T7 in vitro transcription kit (Promega, Cat#P1300, Madison, Wisconson, USA) was used for dsRNA synthesis and purification according to the manufacture's protocol.Purified dsRNAs were diluted in RNase/DNase-free water to a final concentration of 1 µg/µL.The quality of dsRNAs was confirmed by agarose gel electrophoresis (Supplementary Fig. 1A).For gene silencing in S2 cells, dsRNA at the amount of 3 µg was added directly into S2 cells for 48 h.

S2 Cell Transfection and Luciferase Reporter Assay
S2 cells were cultured in a 27 °C incubator using insect medium (Gibco) with 10% FBS (Hyclone).The MycoAlert TM kit (Lonza, Cat#LT07-318, Basel, Halbkanton, Switzerland) was used for mycoplasma detection and validation of the authenticity of S2 cells.Lipofectamine TM 2000 (Thermo Fisher, Cat#11668019, Waltham, Massachusetts, USA) was used for plasmid transfection into S2 cells according to previous protocols [34].The Att-Luc [35] and Drs-Luc [33] reporter systems were utilized to detect the relative activities of the IMD and Toll pathways, respectively.For the IMD pathway, we transfected S2 cells with the plasmid that express Imd, in order to induce the Att-Luc activity.As a positive control, we used the dsRNA targeting kenny, which is one of the pivotal genes of the IMD pathway.For the Toll pathway, the Myd88 expressing plasmid and the pelle dsRNA (positive control) were used.The methods for detecting the Firefly and Renilla luciferase activities were as follows: S2 cells were adequately lysed with 100 µL of passive lysis buffer (Promega, Cat#PR-E1941, Madison, Wisconson, USA).After centrifugation, 30 µL of the supernatant from each sample was added into a 96-well plate containing detection reagents.Firefly and Renilla luciferase activities were detected and finally the ratio of Firefly to Renilla was calculated.Results were analyzed based on data from 3 independent biological replications.

Reverse transcription plus quantitative polymerase chain reaction (RT-qPCR) Assay
S2 cells or fly samples were homogenized in Trizol Reagent (Invitrogen, Cat#15596026, Waltham, Massachusetts, USA).Chloroform solution (1/5 volume) was added into the sample, followed by intense votexing for 15 sec, and centrifugation at high speed for 15 min.The upper layer of the supernatant was transferred into a fresh tube and an equal volume of isopropanol was added.After centrifugation again at high speed for 10 min, the pellet was washed with 75% EtOH and diluted in RNase/DNase-free water.Samples were further incubated with DNase for 30 min, and the quality of RNA was assessed by examining the 260:280 ratio, and the pattern in the agarose gel electrophoresis assay.The first-strand cDNA synthesis kit (Transgen, Cat#AT341-01, Beijing, China) was used to reversetranscribe RNA (1 µg) into cDNA.Quantitative PCR assays (in 3 technical repetitions) were performed using a SYBR Green Master Mix (Thermo Fisher, Cat#A46012, Waltham, Massachusetts, USA) in the Lightcycler 480 PCR platform (Roche, Cat#05015278001, Basel, Halbkanton, Switzerland).Rp49 was used as the internal control.Results were analyzed based on data from 7 independent biological replications.The primers used in RT-qPCR experiments are shown in Supplementary Table 1.

Infection, Fly Survival, and Bacterial Burden Assay
Infection experiments were performed as previously described [36].In brief, overnight bacterial cultures were harvested and diluted in sterile PBS at a concentration of OD 600 = 1.The Serratia marcescens strain (#1.1215) was obtained from China General Microbiological Culture Collection Center (CGMCC) and the Ecc15 was a kind gift from Dr. Dominique Ferrandon's laboratory (Institut de Biologie Moleculaire et Cellulaire, France).For injection, male flies were collected and anesthetizsed on the fly pad with CO 2 .Diluted bacteria (4.6 nL), or the same volume of PBS, was injected into each fly with a nanoliter injector (Nanoject III, Drummond, Cat#3-000-207, Broomall, Pennsylvania, USA).Flies that died within 2 h after bacterial infection were not included in further studies.For survival analysis, flies were transferred to fresh vials and counted for death every day.For bacterial burden experiments, 10 flies were collected, dipped in 75% EtOH, and volatilized with EtOH on the fly pad for several minutes.The flies were then homogenized in 200 µL sterile PBS, followed by serial dilutions.Finally, 100 µL of each diluent was inoculated on an LB agar plate at 30 °C for 12 h before we counted the numbers of the colonies.

Statistical Analysis
All statistical analyses were performed by using GraphPad Prism 9 (v.9.4.0,Dotmatics, Boston, MA, USA).Data were shown as means plus standard errors.Statistical significance was determined by using the ANOVA or Mann-Whitney tests except for survival assays, in which the Log-Rank test (Kaplan-Meier method) was used.The p value of less than 0.05 was considered statistically significant.* p < 0.05, ** p < 0.01, *** p < 0.001, ns (not significant) p > 0.05.

Uch-L5 Positively Modulates IMD Signaling in Drosophila S2 Cells
We examined the potential role of Uch-L5, a previously described Dub modulating Hh signaling [31], in affecting the innate immune reaction in Drosophila.To do this, we first designed 3 types of dsRNAs that targeted different regions of the coding sequence of Uch-L5 (referred to as Uch-L5 dsRNAs #1, #2, and #3, respectively, Supplementary Fig. 1A).Cultured Drosophila S2 cells were treated with these Uch-L5 dsRNAs (dsRNA that targeted GFP was used as the control), and the knockdown efficiency was monitored by RT-qPCR assays (Supplementary Fig. 1B).As illustrated in Fig. 1A, silencing of Uch-L5 resulted in drastic decreases (by ~75% to ~80%) of the Att-Luc activities upon Imd over-expression, indicating that Uch-L5 is a potentially positive regulator in the IMD signaling pathway in cultured S2 cells.Further, we performed RT-qPCR assays to detect the endogenous inductions of the AMPs that are downstream of the IMD pathway, including AttA, Dpt, and CecA1.We obtained consistent results: down-regulation of Uch-L5 significantly prevented the Imd-driven transcription of several AMP genes (Fig. 1B-D).

Uch-L5 is Dispensable for Affecting Toll Signaling
As mentioned in the Introduction, the Drosophila innate immune response is mostly governed by two signaling cascades, the Toll and the IMD pathways.We then conducted another luciferase reporter assay (Drs-Luc) in S2 cells, as previously reported, aiming to test whether Uch-L5 is also involved in affecting Toll signaling.However, we did not observe apparent alterations in the Myd88-driven Drs-Luc activities when Uch-L5 was knocked down, compared to that in the control (Supplementary Fig. 2A).Similar results were obtained when we looked at the transcript levels of Drs and Mtk, two well-known downstream AMP genes in the Toll pathway (Supplementary Fig. 2B,C).These data indicated that Drosophila Dub Uch-L5 is specifically involved in controlling the IMD other than the Toll innate immune signaling pathway in S2 cell cultures.

Uch-L5 Relies on the N-Terminal Uch Domain to Regulate IMD Signaling
We collected more evidence on the functional role of Uch-L5 in controlling IMD innate immunity by constructing a plasmid expressing Myc-tagged Uch-L5 in S2 cells.By again using the Att-Luc reporter system, we observed that ectopic expression of Myc-Uch-L5 enhanced IMD sig-naling in a dose-dependent manner (Fig. 2A).Accordingly, co-transfection of Uch-L5 expressing plasmid resulted in increased inductions (by ~32% to ~136%) of the IMDdownstream AMPs upon Imd over-expression (Fig. 2B-D), which indicated a positive role of Uch-L5 in modulating IMD signaling.
A recent study that explored the biochemical characteristic of Uch-L5 demonstrated that Uch-L5 harbors a typical Uch domain at its N-terminus, which is the catalytical region for its Dub enzymatical activity [31].We therefore constructed two kinds of plasmids that expressed truncated forms of Uch-L5, including Uch-L5 UD (Uch domain) and Uch-L5 CTD (C-terminal domain) (Fig. 2E), and transfected them into S2 cells for Att-Luc reporter assay.As illustrated in Fig. 2F, over-expression of Uch-L5 UD , but not Uch-L5 CTD , mimicked the positive contribution of Uch-L5 FL (full-length Uch-L5) to IMD signaling.Taken together, these data suggested that the N-terminal Uch domain is both required and sufficient for Uch-L5 to benifit Drosophila IMD signaling.

Uch-L5 Mediates the IMD Innate Immune Defense in Adult Flies
We next sought to decipher the immune function of Uch-L5 in vivo.To this end, we collected w 1118 flies (referred to as the wild-type control), key c02831 flies (key loss-of-function mutant as the positive control), and Uch-L5 j2B8 flies (Uch-L5 loss-of-function mutant, isogenized with w 1118 ) for infection experiments using the Gramnegative bacteria S. marcescens (Serratia marcescens), to activate the host IMD innate immune defense.Six hours after infection, we detected decreases (by ~34% to ~56%) of AttA, Dpt, and CecA1 in the Uch-L5-defective flies relative to those in the wild-type control (Fig. 3A-C).To confirm our results, we subjected these flies to infection with another type of Gram-negative bacteria, Ecc15 (Erwinia carotovora carotovora 15).Similar reduction trends were observed when we compared the expression levels of AMPs in the Uch-L5 and key loss-of-function mutants to those in the wild-type flies (Fig. 3A-C).In addition, we noted compara-ble survival rates of these flies (w 1118 , key c02831 , and Uch-L5 j2B8 ) after injection of sterile PBS buffer (Fig. 4A).However, the Uch-L5 loss-of-function mutant flies succumbed much faster than did the wild-type controls upon injection of either S. marcescens (Fig. 4B) or Ecc15 (Fig. 4C), indicating that Uch-L5 plays an essential role in the host defense against bacterial infections.
To test the involvement of Uch-L5 in affecting the proliferation of injected pathogens (S. marcescens or Ecc15), we performed CFU (colony-forming-units) assays at different time points (0, 6, and 12 h) after injection.As shown in Fig. 4D,E, the burdens of S. marcescens and Ecc15 in Uch-L5 j2B8 adults were significantly higher than those in the wild-type flies.In summary, our in vivo data strongly support the notion that Uch-L5 plays a critical role in the host defense against bacterial pathogens in Drosophila.

Silencing of Uch-L5 in Fat Body Leads to Immune Defects
Fat body is one of the main responsible immune tissues/organs during systemic infection in Drosophila.As the Uch-L5 transcript is relatively abundant in the fat body cells, according to the high-throughput sequencing or array data in the FlyBase website (https://flybase.org/reports/FBgn0011327), we sought to investigate the functional role of Uch-L5 in Drosophila fat body.Two different Uch-L5 RNAi (RNA interference) flies (Uch-L5 RNAi #1 and #2, for detailed information, see Materials and Methods) were crossed with the fat body-specific driver c564-gal4.The Tub-gal80 ts strain was used in order to allow gene RNAi at adult stage (Supplementary Fig. 3A).The progenies, including c564 ts >Uch-L5 RNAi #1 and c564 ts >Uch-L5 RNAi #2, were collected and subjected to S. marcescens or Ecc15 infection (c564 ts >+ was used as the wild-type control).Six hours after S. marcescens or Ecc15 infection, the inductions of AttA, Dpt, and CecA1 in Uch-L5 RNAi flies were prominently decreased compared to those in the control group (Fig. 5A-C).These results suggested that Uch-L5 functions in fat body to modulate IMD signaling in response to bacterial challenge.Consistently, we observed that the Uch-L5 RNAi flies were less resistant to S. marcescens or Ecc15 infection: they displayed higher mortality than did the wildtype control (Fig. 5D-F).Moreover, the bacterial burdens at various time points after infection were remarkably elevated by the loss of Uch-L5 in fat body (Fig. 5G,H).

Discussion
Drosophila Uch-L5 belongs to the Uch sub-family of Dubs [31,37].Dub-mediated cleavage of ubiquitin/polyubiquitin from ubiquitinated substrates has been widely studied and demonstrated to be involved in a broad range of cellular processes [reviewed in [38][39][40]].However, our knowledge regarding the biological function of the fly Uch-L5 is incomplete.In the present study, we carried out a series of investigations using both in vitro and in vivo models.We showed that Uch-L5 behaves as a positive modulator in the fly IMD innate immune defense against bacterial stimuli.Our data shed light on a previously undescribed role of Uch-L5 in controlling organismal innate immunity.
How does Uch-L5 execute its essential role in regulating the IMD signaling pathway?A recent study by Zhou and colleagues illustrated that Uch-L5 depends on its Nterminal Uch domain to positively contribute to Hh signaling [31], highlighting the critical requirement of the Dub catalytical triad for Uch-L5 functioning.Indeed, when we utilized several truncated forms of Uch-L5 expressing plasmids and conducted Att-Luc reporter assays in cultured S2 cells, we observed that Uch-L5 without the Uch domain no longer promoted IMD signaling.It is of interest to note that the Dub enzymatical activity of Drosophila Uch-L5 is somehow autoinhibited by the CTD via oligomerization, which can be alleviated by the association of a co-factor such as Rpn13 [31,37].However, we would like to con-clude that this is not the case for the regulatory role of Uch-L5 in innate immune regulation, based on the following reasons: (1) ectopic expression of Uch-L5 without CTD showed a similar contribution to the IMD signaling as that of the Uch-L5 FL in S2 cells; and (2) co-expression of Rpn13 hardly affected the beneficial role of Uch-L5 in IMD signaling (Supplementary Fig. 4A).It seems that Uch-L5 relies on the same Uch domain to participate in different signaling cascades, but its CTD largely functions in distinct manners.Nevertheless, it would be worthwhile to generate corresponding Uch-L5 transgenic flies to confirm these hypotheses in vivo.
Recently, numerous efforts have been made to decipher the critical role of Dubs in regulating IMD signaling.To date, only Imd, Tak1, and Dredd in the canonical IMD pathway, have been clearly demonstrated to be modified by ubiquitination/deubiquitination.Whether the other key factors also involve a ubiquitin-mediated modulation remains a mystery.Uch-L5 might therefore target a factor, or some of these factors, to enhance IMD signaling.The 48th lysine (K48)-linked ubiquitination has been suggested to primarily mediate protein recognition and degradation by the 26S proteasome, whereas the 63rd lysine (K63)-linked ubiquitination commonly governs signal transduction [41].According to our current knowledge and the results of the present study, we speculate that the Dub Uch-L5 may inhibit the K48-linked ubiquitination of its substrate(s), thus improving its (their) stability.Future biochemical approaches, for instance co-immunoprecipitation and ubiquitination assays, will greatly help us further understand the molecular mechanism by which Uch-L5 benefits the IMD innate immune defense in Drosophila.
On the other hand, it is somehow lagged off that no Dubs have been shown to play a role in the Toll innate immune defense of Drosophila.This may be due to the fact that ubiquitination modification in Toll signaling is not as important as that in IMD signaling.Even though we failed to observe in the present study any involvement of Uch-L5 in controlling the Toll pathway, we cannot exclude the possibility that the Toll pathway is regulated by other Dubs.One strategy would be to explore Dubs that can physically associate with MyD88, a pivotal Toll downstream adaptor protein that undergoes ubiquitination [33].

Conclusions
We have demonstrated a critical regulatory role of the Dub Uch-L5 in Drosophila IMD innate immunity.The present study has provided insight into the understanding of the precise dynamic modulation of IMD signaling in response to bacterial infection.

Fig. 1 .
Fig. 1.Silencing of ubiquitin carboxy-terminal hydrolase L5 (Uch-L5) reduces immune deficiency (IMD) signaling in Drosophila S2 cells.(A) S2 cells were pretreated with indicated dsRNAs.48 h later, cells were transfected with various combinations of expressing plasmids, followed by dual-luciferase assays.(B-D) S2 cells were pretreated with dsRNAs as in (A) for 48 h.Cells were then transfected with indicated expressing plasmids for 36 h and harvested for RT-qPCR assays to detect the mRNA levels of AttA (B), Dpt (C), and CecA1 (D).In (A-D), each dot represents one independent replicate and data are shown as means plus standard errors.* p < 0.05, ** p < 0.01, *** p < 0.001.