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An infection-induced RhoB-Beclin 1-Hsp90 complex enhances clearance of uropathogenic Escherichia col

Issuing time:2021-05-11 16:24

Abstract

Host cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/− and RhoB−/− mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.

Introduction

Many anti-bacterial pathways are involved in host defenses against pathogens. Autophagy serves as a process induced by invading pathogens1, and selectively targets invasive bacteria2, such as Mycobacterium tuberculosis, Shigella flexneri, Group A streptococcus, and Salmonella3,4,5,6,7, for autolysosome-dependent degradation. However, the detailed mechanisms involved in autophagy-mediated bacterial clearance are diverse.

Uropathogenic Escherichia coli (UPEC), a Gram-negative intracellular pathogenic bacterium, is the leading cause of urinary tract infections (UTIs), which have a high incidence and frequent recurrence rate in women8. UPEC is reported to reside in autophagosomes during infection9. However, ATG16L1 (an autophagy protein) deficient mice lead to rapid clearance of UPEC, through inducing architectural alterations in superficial urothelial cells and enhancing IL-1β-mediated hyperinflammatory response in macrophages10,11,12,13. On the other hand, significantly increased UPEC bacterial loads are observed in mice with ATG3 deficiency in mouse bladder superficial epithelium14. The role of autophagy in UPEC clearance remains unclear.

Lots of host factors responsible for sensing invasive pathogens and initiating anti-bacterial effects are induced during bacterial infections. It can be triggered through recognition of pathogen-associated molecular patterns (PAMPs) by a variety of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs)15,16,17. RhoB, a member of the small Rho GTPases family, is rapidly and transiently upregulated by cell exposure to LPS or inflammatory cytokines, which may be dependent on the NF-κB pathway18,19,20. RhoB contributes to multiple cellular functions, such as protein trafficking, maintaining endothelial barrier integrity, modulating cell migration and adhesion18, and induction of cytokines that regulate inflammatory events21,22. In addition, post-translational modification of RhoB promotes lysosomal translocation and degradation of mTORC1, resulting in an increase of DNA damage-induced autophagy23. Beclin 1 is the central component of PI3K complex and mainly responsible for assembling diverse regulatory factors that are involved in autophagy24. Expression of Beclin 1 contributes to the induction of autophagy25,26. So far, the role and underlying mechanisms of RhoB in regulating Beclin 1 and bacterial clearance are unknown.

In this study, we found that UPEC induced RhoB in human bladder epithelial cells, which subsequently promoted clearance of intracellular bacteria. RhoB upregulation increased Beclin 1 stabilization and LC3 lipidation, both hallmarks of autophagy. Notably, we identified that RhoB physically bound to Beclin 1 and enhanced the association of Hsp90 with Beclin 1, resulting in LC3 lipidation and clearance of intracellular UPEC. The present study reveals the role of RhoB in defending against intracellular UPEC and highlights the importance of the physical association between RhoB, Beclin 1, and Hsp90 to achieve anti-bacterial effects.

Results

RhoB restricts intracellular UPEC

To explore the role of RhoB during UTIs, we first assessed the protein level of RhoB in human bladder epithelial cell line 5637 upon infection with the UPEC strain CFT073. Endogenous RhoB was markedly increased at 2 h post infection (hpi) in a MOI (multiplicity of infection)-dependent manner (Fig. 1a). RhoB was detected to be increased immediately at 0.5 hpi, significantly induced at 1 hpi, reach a peak at 2 hpi, and maintain till 4 hpi (Fig. 1b). To determine if LPS alone is sufficient to induce RhoB, we employed LPS treatment in 5637 bladder epithelial cells and observed that LPS gradually upregulated RhoB expression; whereas, it reached a peak at 2 h post-treatment (Supplementary Fig. 1a and b); which is consistent with an earlier report showing that RhoB expression is induced by LPS19. To explore the underlying functions of RhoB during UTIs, FLAG-tagged RhoB was transfected into 5637 bladder epithelial cells or another human bladder epithelial cell line (J82) for bacterial invasion assays. The number of intracellular bacteria from RhoB-transfected 5637 bladder epithelial cells, compared with that from vector-transfected cells, was reduced by 3-fold at 2 hpi without affecting bacterial entry (Fig. 1c and Supplementary Fig. 1c). Immunofluorescent staining revealed that the number of intracellular bacteria per cell was reduced in RhoB over-expressing cells (Fig. 1d and Supplementary Fig. 1d). Interestingly, a bacterial expulsion assay indicated that the number of extracellular CFT073 in the supernatant from RhoB-overexpressing cells was also decreased, compared with that from control cells (Supplementary Fig. 1e), suggesting that RhoB promotes bacterial clearance post bacterial entry. To further examine this possibility, endogenous RhoB was knocked down using specific siRNAs, which led to a threefold increase of intracellular bacteria, compared with that achieved by siScr control (Fig. 1e). These observations suggest that RhoB is induced by UPEC infection and plays a key role in decreasing intracellular UPEC.

Fig. 1: RhoB restricts intracellular UPEC.
figure1

a RhoB level in 5637 bladder epithelial cells infected by UPEC at indicated MOIs. The RhoB density was normalized to that of β-Actin. The relative density of uninfected cells was set to 100%. (n = 3 independent experiments). MOI 0 vs. 10 P = 0.4998, MOI 0 vs. 50 P = 0.0191. b RhoB level in UPEC-infected 5637 cells at indicated time after infections. The RhoB density was normalized to that of β-Actin. The relative density of uninfected cells was set to 100%. (n = 3 independent experiments). Control vs. 0.5 P = 0.2944, control vs. 1.0 P = 0.0144, control vs. 2.0 P < 1.0e−15, control vs. 4.0 P = 0.0040, control vs. 6.0 P = 0.0909. c RhoB overexpression decreases intracellular UPEC in 5637 bladder epithelial cells or J82 cells at 2 hpi. Left panel: n = 4 independent experiments, P = 0.0286; right panel: n = 6 independent experiments, P = 0.0022. d Representative image of RhoB-overexpressing 5637 bladder epithelial cells infected by CFT073 at 4 hpi. n = 10 random areas assessed from three independent experiments (more than 100 cells for each group). Scale bar, 10 μm. P = 1.1e−5. e Knockdown of RhoB increases intracellular UPEC in 5637 bladder epithelial cells. Knockdown efficiency of RhoB siRNAs in 5637 cells was shown on the right. n = 3 independent experiments. siScr vs. siRhoB #1 P = 0.1299, siScr vs. siRhoB #2 P = 0.0070, siScr vs. siRhoB #3 P = 0.0007. f Representative confocal images and quantification of GFP-LC3-positive UPEC in RhoB-overexpressing 5637 bladder epithelial cells. n = 5 random areas in images of each group from three independent experiments. Scale bars, 8 μm. P = 0.0079. gh Representative confocal images of 5637 cells infected with GFP-expressing CFT073 for 1 h in the presence of BafA1. Scale bars, 8 μm. iQuantification of P62- or Ubiquitin-positive UPEC in RhoB-overexpressing 5637 cells at 1 hpi. n = 3 independent experiments. P62 + P = 0.8627, Ub+ P = 0.5941. j Quantification of GFP-CFT073 colocalization with P62, Ubiquitin and mCherry-LC3 in 5637 cells at 1 hpi. n = 3 independent experiments. P62 + LC3 + P = 0.0121, Ub+ LC3 + P = 0.0006. k Knockdown of ATG7 and P62 restores bacterial survival in RhoB-overexpressing 5637 bladder epithelial cells. Bacterial survival of vector and scramble siRNA-transfected cells was set to 100%. Statistical significance was calculated by comparing the mean of each group with that of RhoB siScr group. n = 3 independent experiments. RhoB siScr vs. Vector siScr P < 1.0e−15, RhoB siScr vs. RhoB siATG7 #1 P = 0.0004, RhoB siScr vs. RhoB siATG7 #2 P = 0.0093, RhoB siScr vs. RhoB siP62 #1 P < 1.0e−15, RhoB siScr vs. RhoB siP62 #2 P= 0.0710, RhoB siScr vs. RhoB siRubicon #1 P = 0.0770, RhoB siScr vs. RhoB siRubicon #2 P = 0.4768. Data are shown as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired Student’s t test (cd, f), one-way ANOVA (ab, e, k), two-way ANOVA (ij).

Since autophagy plays an essential role in cell defense against bacterial infection14,27,28, we hypothesized that autophagy is involved in RhoB-mediated clearance of intracellular UPEC. We found that the level of LC3-II was increased in 5637 bladder epithelial cells infected with CFT073 in a MOI-dependent manner (Supplementary Fig. 1f), and significantly increased at 1 to 2 hpi (Supplementary Fig. 1g). Accumulation of LC3-II was also observed in LPS-treated cells (Supplementary Fig. 1h). To further verify this result, we transfected tandem-tagged mCherry-GFP-LC3 into 5637 bladder epithelial cells and treated them with CFT073 or LPS. As expected, a higher number of autophagosomes (Yellow puncta) and autolysosomes (Red puncta) were detected in CFT073 or LPS-treated cells, compared with that detected in control cells (Supplementary Fig. 1i–j).

Interestingly, we observed that intracellular CFT073 decorated by GFP-LC3 was increased in 5637 bladder epithelial cells when RhoB was overexpressed (Fig. 1f and Supplementary Fig. 2a). In addition, both P62-LC3 and Ubiquitin-LC3 double-positive CFT073 were detected to be increased in RhoB overexpressing cells (Fig. 1g–j and Supplementary Fig. 2b–c), and colocalization of CFT073 with P62 or Ubiquitin alone was not affected by RhoB (Fig. 1i). In addition, inhibitors of autophagic flux, such as ammonium chloride (NH4Cl) and bafilomycinA1 (BafA1), were applied in RhoB-overexpressing cells infected by CFT073. Of note, both inhibitors efficiently restored the number of intracellular bacteria reduced by overexpression of RhoB (Supplementary Fig. 2d). We also knocked down ATG7, P62, or Rubicon using siRNAs, to specifically block biogenesis of autophagosomes or LC3-associated phagocytosis (LAP) and found that efficient knockdown of ATG7 or P62 abolished RhoB-mediated clearance of intracellular UPEC, and Rubicon knockdown did not attenuate the effect of RhoB (Fig. 1k and Supplementary Fig. 2e–g). It is likely that RhoB promotes intracellular UPEC clearance through autophagy-related pathways.

RhoB induces LC3 lipidation and UPEC clearance through Beclin1

We detected changes of autophagic proteins in HEK293 cells that overexpressed RhoB. Interestingly, RhoB dosage-dependently accelerated the processing of LC3 lipidation, shown as an accumulation of LC3-II (Fig. 2a), which was also observed in human prostate epithelial PC-3 cells (Supplementary Fig. 3a). The level of P62, a marker protein of autophagic degradation29, was conversely reduced by RhoB overexpression (Fig. 2a). Transmission electron microscope (TEM) revealed more autophagic vacuoles in the cytosol of RhoB-overexpressing cells, in stark contrast to those in control cells (Fig. 2b). The number of LC3 puncta (both yellow and red) in RhoB-overexpressing cells was also threefold higher than that in control cells (Fig. 2c). In line with these results, knockdown of RhoB in 5637 bladder epithelial cells mitigated LC3 lipidation and led to an accumulation of P62 (Fig. 2d). Interestingly, treatment with BafA1 also led to an increase of LC3-II in RhoB-overexpressing cells (Supplementary Fig. 3b), reflecting that RhoB likely promotes autophagic flux rather than blocking lysosomal degradation. Notably, the decrease of Beclin 1, the core adaptor of PI3K complex that mediates autophagosome initiation, was observed when RhoB was knocked down (Fig. 2d). This reduction was validated in CFT073-infected 5637 bladder epithelial cells (Fig. 2e), and increased Beclin 1 was observed in both RhoB-transfected T24 bladder epithelial cells and LPS-pretreated 5637 bladder epithelial cells (Supplementary Fig. 3c and d). Moreover, knockdown of Beclin 1 in RhoB-overepxressing cells restored LC3-II protein level and intracellular bacteria survival (Fig. 2f–g and Supplementary Fig. 3e), suggesting that RhoB affects autophagic protein LC3 lipidation and UPEC clearance through Beclin 1.

Fig. 2: RhoB induces LC3 lipidation and upregulates Beclin1 level.
figure2

a Protein levels of LC3 and P62 in HEK293 cells transfected with RhoB or vector. Signal densities of RhoB and P62 were normalized to that of β-Actin. The relative density of vector-transfected cells was set to 100%. n = 3 independent experiments. LC3-II/β-Actin: 0 vs. 0.1 P = 0.1768, 0 vs. 0.5 P = 0.0306, 0 vs. 1.0 P = 0.0001; P62/β-Actin: 0 vs. 0.1 P = 0.2468, 0 vs. 0.5 P = 0.0103, 0 vs. 1.0 P = 0.0031. b TEM images of RhoB overexpressing cells. Quantification on the right shows the number of autophagic vacuoles per cell (n = 12 cells per group from three independent experiments). Arrows show autophagic vacuoles. Scale bars, 2 or 0.5 μm. P = 7.4e−7. c Representative images of HEK 293 cells transfected with mCherry-GFP-LC3 in the presence of RhoB or vector. Autophagosomes, yellow puncta; autolysosomes, red-only puncta. Bar graph shows the quantification of LC3 puncta per cell (n = 10 random areas per group from three independent experiments). Scale bar, 10 μm. Yellow P = 1.1e−5, Red P = 3.2e−8. d, e Western blotting showing the levels of autophagic proteins in RhoB knockdown 5637 bladder epithelial cells with LPS (d) or with CFT073 infection (e). 5637 bladder epithelial cells were transfected with RhoB siRNAs or scramble siRNA for 48 h followed by infection of CFT073 at MOI of 50 for 2 h. Signal densities of LC3-II, P62, and Beclin 1 were normalized to that of β-Actin. The relative density of siScr-transfected cells was set to 100%. n = 3 independent experiments. d LC3-II/β-Actin: siScr vs. siRhoB #1 P = 0.0133, siScr vs. siRhoB #2 P = 0.0011, siScr vs. siRhoB #3 P = 0.0013; P62/β-Actin: siScr vs. siRhoB #1 P = 0.5419, siScr vs. siRhoB #2 P = 0.2053, siScr vs. siRhoB #3 P = 0.0290; Beclin 1/β-Actin: siScr vs. siRhoB #1 P = 0.3483, siScr vs. siRhoB #2 P = 0.0400, siScr vs. siRhoB #3 P = 0.0390. e LC3-II/β-Actin: siScr vs. siRhoB #1 P = 0.1356, siScr vs. siRhoB #2 P = 0.0225, siScr vs. siRhoB #3 P = 0.0030; Beclin 1/β-Actin: siScr vs. siRhoB #1 P = 0.0131, siScr vs. siRhoB #2 P = 0.0004, siScr vs. siRhoB #3 P < 1.0e−15. fKnockdown of Beclin 1 restores LC3 lipidation in RhoB-overexpressing HEK 293 cells. Three independent experiments. g Knockdown of Beclin 1 restores bacterial survival in RhoB-overexpressing 5637 bladder epithelial cells. 5637 cells were co-transfected with RhoB and Beclin 1siRNAs for 48 h followed by bacterial invasion assay. Bacterial survival of vector and siScr-transfected cells was set to 100%. n = 3 independent experiments. RhoB siScr vs. Vector siScr P = 0.0062, RhoB siScr vs. RhoB siBeclin 1 #1 P = 0.0003, RhoB siScr vs. siBeclin 1 #2 P = 0.0004, RhoB siScr vs. siBeclin 1 #3 P = 0.0002. h LC3 lipidation in HEK293 cells transfected with plasmids encoding RhoB-WT, Q63E, Q63L, and T19N. The LC3-II density was normalized to that of β-Actin. The relative density of vector-transfected cells was set to 100%. n = 3 independent experiments. LC3-II/β-Actin: Vector vs. RhoB P = 0.0069, Vector vs. Q63E P = 0.0249, Vector vs. Q63L P = 0.0480, Vector vs. T19N P = 0.0002. i LC3 lipidation and Beclin 1 level in HEK293 cells transfected with plasmids encoding RhoB-WT, S185A, and C193S for 48 h. Signal densities of LC3-II and Beclin 1 were normalized to that of β-Actin. The relative density of vector-transfected cells was set to 100%. n = 3 independent experiments. LC3-II/β-Actin: Vector vs. RhoB P = 0.0459, Vector vs. S185A P = 0.0398, Vector vs. C193S P = 0.7997; Beclin 1/β-Actin: Vector vs. RhoB P = 0.0136, Vector vs. S185A P = 0.0092, Vector vs. C193S P = 0.9421. Data are the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired Student’s t test (b), one-way ANOVA (a, de, gi), or two-way ANOVA (c).

We next investigated whether activation of RhoB affected its activity in LC3 lipidation. Constitutively active (Q63E and Q63L) and dominant-defective (T19N) constructs of RhoB were generated and validated using the Rhotekin pull-down assay (Supplementary Fig. 3f). No significant difference was observed between the effects of Q63L, Q63E, T19N, and wild-type RhoB on LC3 lipidation (Fig. 2h), suggesting that RhoB activation is not important toward its role in LC3 lipidation.

Posttranslational modifications of RhoB at its C-terminal region are known to be functional-related30. Geranylgeranylation and farnesylation of RhoB at cysteine 193 of its C-terminal CAAX motif is critical for its membrane-associated localization (including the plasma membrane, endosomes, and multivesicular bodies) and biological activities18. Phosphorylation of RhoB at serine 185 affects its functions in cytoskeleton organization31. To abrogate its posttranslational modifications, we selectively made two substitutions of RhoB, RhoB-S185A, and RhoB-C193S. Using immunofluorescent staining, we observed that wild-type RhoB and RhoB-S185A were similar, while subcellular location of RhoB-C193S was altered, similar as reported previously30 (Supplementary Fig. 3g). Overexpression of RhoB-C193S in 293 cells impeded LC3 lipidation and upregulation of Beclin 1, whereas RhoB-S185A retained similar effects as the wild-type (Fig. 2i). These observations demonstrate that the activity of RhoB in LC3 lipidation relies on its localization with membrane.

As RhoB was induced by LPS, we examined whether TLR4 (receptor of LPS) is involved in LPS-mediated RhoB upregulation. TLR4 knockdown using siRNA in 5637 bladder epithelial cells greatly reduced RhoB, Beclin 1, and LC3-II levels stimulated by LPS (Supplementary Fig. 3h).

RhoB physically interacts with Beclin1 and prevents its degradation

In order to elucidate the underlying mechanism through which RhoB affects Beclin 1 level, we examined RhoB-induced changes of Beclin 1 mRNA transcription and protein degradation. No significant change was observed in Beclin1 mRNA level when RhoB was ectopically expressed in 5637 bladder epithelial cells (Supplementary Fig. 4a). To evaluate the effect of RhoB on Beclin 1 protein degradation, RhoB-transfected HEK293 cells (or vector control-transfected cells) were treated with cycloheximide (CHX), an inhibitor of protein synthesis. The level of MYC-Beclin 1 was strongly decreased in vector-transfected HEK293 cells in the presence of CHX, whereas the level of MYC-Beclin 1 was still increased in RhoB-overexpressing cells treated by CHX, suggesting that RhoB does not affect Beclin 1 synthesis but may prevent the Beclin 1 degradation (Supplementary Fig. 4b). To confirm this, endogenous RhoB in 5637 bladder epithelial cells was knocked down using siRNAs and the cells were treated with the proteasomal inhibitor MG-132 to block Beclin 1 degradation simultaneously. The level of endogenous Beclin 1 was markedly decreased without MG-132 treatment in RhoB knocked down cells, but not in control cells; however, no decrease of Beclin 1 was found in RhoB knocked down cells treated with MG-132 (Supplementary Fig. 4c). These results indicate that RhoB increases Beclin 1 level via blockage of its degradation, and RhoB has no effect on Beclin 1 transcription and synthesis.

RhoB acts as an intracellular transporter that mediates trafficking events of cellular factors through protein-protein interactions. This activity regulates many cellular functions, such as integrity of endothelial barrier, cell migration, and adhesion, as well as cancer development18,20,32,33,34. Therefore, we hypothesized that RhoB increases Beclin 1 stabilization through directly interacting with Beclin 1. To evaluate this, we carried out co-immunoprecipitation assays using HEK293 cells co-transfected with MYC-tagged Beclin 1, FLAG-tagged RhoB-WT or RhoB-C193S. Intriguingly, Beclin 1 was efficiently co-immunoprecipitated with RhoB-WT, but not with vector or RhoB-C193S, and vice versa (Fig. 3a and b). This RhoB-Beclin 1 association was reciprocally verified by co-immunoprecipitation with endogenous proteins from 5637 bladder epithelial cells (Fig. 3c and d). Purified GST-fused RhoB and Beclin 1 were next applied in immunoprecipitation assays, to determine if RhoB directly binds to Beclin 1. As expected, GST-Beclin 1 with anti-Beclin 1 antibody, but not with the IgG control, was found to efficiently pull down GST-RhoB, and vice versa (Fig. 3e and f). Moreover, colocalization of endogenous RhoB and Beclin 1 was observed with LC3 puncta (Fig. 3g and Supplementary Fig. 4d, Pearson’s coefficients for RhoB-Beclin 1, RhoB-LC3 and Beclin 1-LC3 colocalization were ~0.52, ~0.32, and ~0.18, respectively), reflecting that RhoB and Beclin 1 might form a complex to maintain Beclin 1 for autophagosome formation. Taken together, these data reveal that RhoB binds to Beclin 1 directly and maintains Beclin 1 level by attenuating its degradation. Colocalization of endogenous RhoB and Beclin 1 was also observed in the cytoplasm of 5637 bladder epithelial cell infected by CFT073, and less colocalization of RhoB with CFT073 was detected (Supplementary Fig. 4e, Pearson’s coefficients for RhoB-Beclin 1, RhoB-CFT073 and Beclin 1-CFT073 colocalization were ~0.45, ~0.06, and ~0.19, respectively).

Fig. 3: RhoB physically interacts with Beclin1 and prevents its degradation.
figure3

a, b The interaction of overexpressed RhoB with Beclin 1 in HEK 293 cells. HEK 293 cells were co-transfected with MYC-Beclin 1, FLAG-RhoB WT, FLAG-RhoB-C193S or Vector. At 48 h post transfection, cell lysates were prepared and subjected to immunoprecipitation with anti-FLAG (a) or anti-MYC (b) antibodies followed by western blotting analysis. n = 3 independent experiments. c, d Reciprocal immunoprecipitation of endogenous Beclin 1 and RhoB in 5637 bladder epithelial cells. 5637 cells were treated with LPS for 2 h to upregulate RhoB expression and lysed. Cell lysates were immunoprecipitated with anti-RhoB (c) or anti-Beclin 1 (d) antibodies and analyzed by western blotting. The slanting labels identify the IP antibodies. IgG served as a negative control. Three independent experiments. e, f RhoB directly interacts with Beclin 1. GST-fused RhoB and Beclin 1 were expressed in E. coli and subjected to GST pull-down assay with anti-RhoB (e) or anti-Beclin 1 (f) antibodies. The slanting labels identify the IP antibodies. IgG served as a negative control. n = 3 independent experiments. g Colocalization of endogenous RhoB and Beclin 1 in mCherry-LC3-expressing in CFT073-infected 5637 cells (left). 5637 cells were transfected with mCherry-LC3 and infected with CFT073 for 2 h, followed by immunostaining with Alexa Fluor 488-conjugated anti-RhoB antibody (green), anti-Beclin 1 antibody plus Alexa Fluor 405-conjugated secondary antibody (blue). Quantification of colocalization was shown by calculating the Pearson correlation coefficients (R values) using Image Pro Plus software (right). n = 10 random areas per group from three independent experiments. Scale bar, 10 μm. RhoB-Beclin 1 vs. RhoB-LC3 P < 1.0e−15, RhoB-Beclin 1 vs. Beclin 1-LC3 P < 1.0e−15. Pearson’s coefficients for RhoB-Beclin 1, RhoB-LC3 and Beclin 1-LC3 colocalization were ~0.52, ~0.32, and ~0.18, respectively. Data are the mean ± SD, ****P < 0.0001, one-way ANOVA.

Molecular interactions between RhoB and Beclin 1

Three functional domains of Beclin 1, including Bcl-2-homology-3 (BH3)-only domain, central coiled-coiled domain (CCD), and evolutionary conserved domain (ECD) have been identified. In order to map the interface of RhoB-Beclin 1 interaction in detail, a series of truncation mutants of MYC-Beclin1 and FLAG-RhoB were genetically engineered. Co-immunoprecipitation assay with MYC-Beclin 1 constructs and FLAG-RhoB indicated that MYC-Beclin 1 truncation mutants including 1–244, 1–337, and 150–450, but not 1–88 and 1–150, were able to bind to FLAG-RhoB (Fig. 4a and Supplementary Fig. 5a). In addition, GST-Beclin 1 truncation mutants (88–450, 150–450, 244–450, and 337–450) were constructed for GST pull-down analysis, and the interactions between RhoB and two Beclin 1 truncation mutants (88–450 and 150–450) could be detected. These results suggest that the CCD domain (150–244) of Beclin 1 is necessary for RhoB-Beclin 1 interaction (Supplementary Fig. 5b). Reciprocally, co-immunoprecipitation assay with FLAG-RhoB constructs and MYC-Beclin 1 demonstrated that residues (89–140) of RhoB are indispensable for their interaction (Fig. 4b and Supplementary Fig. 5c). These RhoB constructs, such as RhoB-WT and RhoB (89–196), that interacted with Beclin 1 coordinately enhanced Beclin 1 stabilization and LC3 lipidation, supporting the idea that binding of RhoB to Beclin 1 is essential for the activity of RhoB in mediating LC3 lipidation (Supplementary Fig. 5d and e).

Fig. 4: Molecular interactions between RhoB and Beclin 1.
figure4

a, b Mapping the interacting regions between RhoB and Beclin 1. HEK 293 cells were co-transfected with truncation mutants of MYC-Beclin 1 and 3×FLAG-RhoB (a), or reciprocally with truncation mutants of 3×FLAG-RhoB and MYC-Beclin 1 (b), followed by immunoprecipitation and western blotting analysis. The density of MYC-Beclin 1 was normalized to that of FLAG-RhoB in IP group. In Fig. 4a, the percentage of bound MYC-Beclin 1 150–450 was set to 100%. In Fig. 4b, the percentage of bound MYC-Beclin 1 to RhoB WT was set to 100%. n = 3 independent experiments. a MYC-Beclin 1/FLAG-RhoB: 1–88 vs. 1–150 P = 0.9771, 1–88 vs. 1–244 P = 0.0033, 1–88 vs. 1–337 P < 1.0e−15, 1–88 vs. 150–450 P < 1.0e−15; b MYC-Beclin 1/FLAG-RhoB: WT vs. 42–196 P = 0.5048, WT vs. 89–196 P = 0.6684, WT vs. 141–196 P < 1.0e−15, WT vs. 1–180 P < 1.0e−15. c Interaction of Beclin 1 with RhoB truncation mutants at helical region. The density of MYC-Beclin 1 was normalized to that of FLAG-RhoB in IP group. The percentage of bound MYC-Beclin 1 to RhoB WT was set to 100%. n = 3 independent experiments. MYC-Beclin 1/FLAG-RhoB: WT vs. 89–196 P = 0.9445, WT vs. 118–196 P = 0.0680, WT vs. 141–196 P = 0.0001. d GST pull-down assay with GST-fused Beclin 1 and RhoB truncation mutants. The slanting labels identify the IP antibodies. IgG served as a negative control. The density of GST-RhoB was normalized to that of GST-Beclin 1 in IP group. The percentage of bound GST-RhoB WT to GST-Beclin 1 was set to 100%. n = 3 independent experiments. GST-RhoB/GST-Beclin 1: WT vs. 42–196 P = 0.8909, WT vs. 89–196 P = 0.9759, WT vs. 118–196 P = 0.9924, WT vs. 141–196 P = 0.0257. e Mapping the key binding site of RhoB-Beclin 1 interaction at helical region of RhoB. The density of MYC-Beclin 1 was normalized to that of FLAG-RhoB in IP group. The percentage of bound MYC-Beclin 1 to RhoB WT was set to 100%. n = 3 independent experiments. MYC-Beclin 1/FLAG-RhoB: WT vs. T129V P = 0.3089, WT vs. R133A P = 0.0133, WT vs. Q136G P = 0.9961. f GST pull-down assay with GST-fused Beclin 1 and RhoB single mutants at helical region. The slanting labels identify the IP antibodies. IgG served as a negative control. The density of GST-RhoB was normalized to that of GST-Beclin 1 in IP group. The percentage of bound GST-RhoB WT to GST-Beclin 1 was set to 100%. n = 3 independent experiments. GST-RhoB/GST-Beclin 1: WT vs. T129V P = 0.3277, WT vs. R133A P = 0.0305, WT vs. Q136G P = 0.5267. Data are the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA (af).

Based on structural information obtained from the Protein Data Bank (RhoB, 2FV8), we noticed that RhoB contains a helical region at residues (118–134) that might be crucial for its interaction with Beclin 1 (Supplementary Fig. 5f). Therefore, we additionally generated truncation mutant RhoB (118–196) to find out the exact region of RhoB-Beclin 1 interaction. Co-immunoprecipitation and GST pull-down results revealed that the helical region located in residues (118–140) is required for RhoB-Beclin 1 interaction (Fig. 4c and d). Based on RhoB structure, we hypothesized two amino acids (T129, R133) in this helical region, which contain side-chain groups that may form hydrogen bonds with other proteins, are involved in RhoB-Beclin 1 interaction. Therefore, substitutions of RhoB, T129V, R133A, and Q136G (located in the adjacent loop region, used as the negative control), were constructed for co-immunoprecipitation and GST pull-down analysis. Notably, R133A attenuated the interaction between RhoB and Beclin 1 compared with RhoB-WT, Q136G and T129V (Fig. 4e and f). This disruption of Beclin 1 interaction by RhoB-R133A led to a decrease of Beclin 1 level and coordinately reduced LC3 lipidation (Supplementary Fig. 5g and h). In addition, R133A did not significantly decrease intracellular bacterial survival compared with RhoB-WT, Q136G, and T129V (Supplementary Fig. 5i)

Collectively, these results suggest that RhoB physically interacts with the CCD domain of Beclin 1 by its residues (118–140), whereas Arg133 of RhoB might be the key binding site for RhoB-Beclin 1 interaction (Supplementary Fig. 5j).

RhoB-mediated Beclin 1 stabilization and LC3 lipidation is governed by Hsp90

Given that varieties of binding partners of Beclin 1 manipulate its degradation through ubiquitin machinery, we next examined the effect of RhoB on Beclin 1 ubiquitination. We found that both K11- and K48-linked ubiquitination of Beclin 1 were similar in RhoB-overexpressing and control cells (Supplementary Fig. 6a), indicating that there is another mechanism through which RhoB inhibits Beclin 1 degradation. Beclin 1 has been shown to be maintained by binding of Hsp9035. To determine whether RhoB-mediated Beclin 1 stabilization was associated with the stabilizing activity of Hsp90, we employed the specific inhibitor of Hsp90, Geldanamycin (GA), in our following experiments. Results showed that GA treatment, attenuated RhoB-induced LC3 lipidation and MYC-Beclin 1 stabilization (Fig. 5a). Similar results were obtained by Hsp90 knocked down with siRNAs, which abrogated the upregulation of MYC-Beclin 1 induced by RhoB (Fig. 5b). In line with this, both knockdown of Hsp90 and GA inhibition restored the diminished survival of intracellular CFT073 in RhoB-overexpressing 5637 bladder epithelial cells (Fig. 5c). No difference for Beclin 1 ubiquitination in RhoB overexpressing and control cells treated by GA was detected (Supplementary Fig. 6a). Taken together, these results support that Hsp90 is indispensable in increasing Beclin 1, LC3 lipidation, and bacterial clearance induced by RhoB.

Fig. 5: RhoB-mediated Beclin 1 stabilization is governed by Hsp90.

a Protein level of Beclin 1 and LC3 in RhoB-overexpressing HEK293 cells in the presence or absence of Geldanamycin (GA, 1 μM). HEK 293 cells were transfected with MYC-Beclin 1 and FLAG-RhoB for 48 h, followed by treatment of GA overnight. n = 3 independent experiments. b Effect of Hsp90knocked down by siRNAs on the protein level of Beclin 1 in RhoB-overexpressing cells. n = 3 independent experiments. c Effects of Hsp90 knocked down by siRNAs (left) or GA (right) on survival of intracellular CFT073 in RhoB-overexpressing cells. 5637 cells were co-transfected with FLAG-RhoB and Hsp90 siRNAs followed by bacterial invasion assay. For the effect of GA, 5637 cells were transfected with FLAG-RhoB or vector in the presence or absence of GA, followed by bacterial invasion assay. Bacterial survival in Vector siScr or Vector DMSO groups was set to 100%. n = 6 independent experiments. Data are the mean ± SD, *P < 0.05. Left panel: Vector siScr vs. RhoB siScr P= 0.0100, Vector siHsp90 vs. RhoB siHsp90 P = 0.7789; right panel: Vector DMSO vs. RhoB DMSO P = 0.0069, Vector GA vs. RhoB GA P = 0.9936. d The interaction of endogenous Hsp90 with Beclin 1 in RhoB-overexpressing cells. n = 3 independent experiments. e, f The interaction of Hsp90 with Beclin 1 in HEK 293 cells transfected with RhoB truncation mutants (e) and single mutants at helical region (f). HEK 293 cells were co-transfected with MYC-Beclin 1, HA-Hsp90 and indicated 3×FLAG-RhoB mutants followed by immunoprecipitation assay with anti-MYC antibody and western blotting analysis. The density of HA-Hsp90 was normalized to that of MYC-Beclin 1 in IP group. The percentage of bound HA-Hsp90 to MYC-Beclin 1 in RhoB WT group was set to 100%. n = 3 independent experiments. e HA-Hsp90/MYC-Beclin 1: Vector vs. WT P = 0.0257, Vector vs. 42–196 P= 0.4885, Vector vs. 89–196 P = 0.0304, Vector vs. 141–196 P = 0.8694, Vector vs. 1–180 P = 0.6351. fHA-Hsp90/MYC-Beclin 1: Vector vs. WT P = 0.0067, Vector vs. T129V P = 0.6605, Vector vs. R133A P = 0.9938, Vector vs. Q136G P = 0.0248. g GST pull-down assay with bacterially expressed GST-fused Beclin 1, Hsp90, and RhoB. Immunoprecipitation was performed with anti-Beclin 1 antibody or IgG. n = 3 independent experiments. h GST pull-down assay with bacterial expressed GST-fused Hsp90 and truncated RhoB proteins. Immunoprecipitation was performed with an anti-RhoB antibody or IgG. The density of GST-Hsp90 was normalized to that of GST-RhoB in IP group. The percentage of bound GST-Hsp90 to GST-RhoB WT was set to 100%. n = 3 independent experiments. GST-Hsp90/GST-RhoB: WT vs. 42–196 P = 0.0509, WT vs. 89–196 P < 1.0e−15, WT vs. 118–196 P < 1.0e−15, WT vs. 141–196 P < 1.0e−15. Data are the mean ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA (c, ef, h).

We further examined if RhoB contributed to Beclin 1-Hsp90 interaction, resulting in decreased Beclin 1 degradation. Co-immunoprecipitation assay in HEK293 cells transfected with MYC-Beclin 1 and FLAG-RhoB showed that RhoB-WT, but not RhoB-C193S, enhanced the binding of endogenous Hsp90 to MYC-Beclin 1 (Fig. 5d). Immunofluorescent assays indicated increased colocalization of Hsp90 with Beclin 1 in RhoB-overexpressing cells (Pearson’s coefficient, ~0.85), compared with that in control cells (Pearson’s coefficient, ~0.69, Supplementary Fig. 6b). Co-immunoprecipitation assay in HEK293 cells transfected with MYC-Beclin 1, HA-Hsp90 and truncation mutants of FLAG-RhoB showed that RhoB-WT and RhoB (89–196) promoted the association of HA-Hsp90 with MYC-Beclin 1; yet, RhoB (42–196) did not obviously enhance the Beclin 1-Hsp90 interaction, in accordance with the above results showing that RhoB (42–196) did not promote LC3 lipidation (Fig. 5e, Supplementary Fig. 5d and e). RhoB-R133A, which attenuates the binding of RhoB to Beclin 1, was also observed to disrupt the Beclin 1-Hsp90 interaction (Fig. 5f). In GST pull-down assays, Beclin 1-Hsp90 interaction was enhanced in the presence of RhoB-WT (Fig. 5g). Collectively, these observations suggest that binding of RhoB to Beclin 1 enhances the Beclin 1-Hsp90 interaction, resulting in the blockage of Beclin 1 degradation and enhancement of LC3 lipidation.

Interestingly, endogenous RhoB was observed to be decreased in Hsp90 knockdown cells, and overexpression of Hsp90 significantly increased the protein level of endogenous RhoB (Supplementary Fig. 6c), suggesting that RhoB may also be a client protein of Hsp90. Co-immunoprecipitation assay with overexpressed truncation mutants of FLAG-RhoB and HA-Hsp90 indicated that RhoB interacted with Hsp90 through RhoB residues 42–89 (Supplementary Fig. 6d), which is different from the interaction between RhoB and Beclin 1. This association was further validated by GST pull-down assay (Fig. 5h). Consistently, colocalization of RhoB with Hsp90 was observed in transfected HEK293 cells (Pearson’s coefficient, ~0.73, Supplementary Fig. 6e). In addition, we examined the interactions between RhoB, Beclin 1, and Hsp90 in 5637 bladder epithelial cells infected by CFT073. Induced RhoB and Beclin 1 were observed when cells were infected by CFT073, and more Beclin 1 was obtained by co-immunoprecipitation, binding with more Hsp90 and RhoB (Supplementary Fig. 6f). Overall, these data indicate that RhoB interacts with Beclin 1 and Hsp90 to form a RhoB-Beclin 1-Hsp90 complex (Supplementary Fig. 6g).

RhoB promotes bacterial clearance in vivo

To determine the effects of UPEC infection on RhoB and autophagic proteins in vivo, we performed immunohistofluorescence (IHF) staining using bladder tissue sections from CFT073-infected mice as a model of acute UTI. Wild-type female C57BL/6J mice were transurethrally injected with 108 CFU of CFT073 for 12 h before analysis. Stronger staining intensity of RhoB was detected at the urothelium beside the bladder lumen of infected mice, compared with that of control mice, as was higher staining intensity of LC3 and Beclin 1 (Fig. 6a). These coordinate enrichments of RhoB and autophagic proteins were also identified in bladder homogenates from infected mice using western blotting (Fig. 6b).

Fig. 6: RhoB promotes bacterial clearance in vivo.
figure6

a, b Protein level of RhoB, Beclin 1 and LC3 in CFT073-infected wild-type mice. Female C57BL/6J mice were transurethrally inoculated with 108 CFU of CFT073 cultured overnight. aImmunohistofluorescence analysis. Levels of indicated proteins were quantified by measurement of fluorescent intensity using Image Pro Plus software. Expression of each indicated protein in control mice was set to 100% (n = 6 images assessed from 3 mice per group) (left). Representative confocal images were shown (right). Indicated proteins were stained with appropriate primary antibodies plus Alexa Fluor-488-conjugated secondary antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. RhoB P = 0.0087, Beclin 1 P = 0.0022, LC3 P = 0.0022. b Western blotting analysis of indicated proteins in CFT073-infected mice. n = 3 mice. cProtein level of RhoB, Beclin 1 and LC3 in RhoB+/− and RhoB−/− mice infected by CFT073 through immunohistofluorescence analysis. Quantification of indicated proteins was shown, and expression of each indicated protein in WT mice was set to 100% (n = 6 images assessed from 3 mice per group) (left). Representative confocal images were shown (right). Scale bar, 25 μm. RhoB: WT vs. RhoB+/− P < 1.0e−15, WT vs. RhoB−/− P < 1.0e−15; Beclin 1: WT vs. RhoB+/− P < 1.0e−15, WT vs. RhoB−/− P < 1.0e−15; LC3: WT vs. RhoB+/− P < 1.0e−15, WT vs. RhoB−/− P < 1.0e−15. d Western blotting analysis of indicated proteins in CFT073-infected RhoB−/− mice. n = 3 mice. e Bacterial titers in bladders of WT, RhoB+/−and RhoB−/− mice infected by CFT073 (n = 5 mice per group). WT vs. RhoB+/− P = 0.0492, WT vs. RhoB−/− P < 1.0e−15. f Schematic diagram illustrating RhoB-induced UPEC clearance. During UPEC infections, RhoB is rapidly induced upon LPS stimulation through TLR4-mediated signaling, possibly through the activated NF-κB pathway. RhoB forms a complex with Beclin 1 and Hsp90, which leads to increased stability of Beclin 1. As a result, more autophagosomes are formed, resulting in enhanced clearance of intracellular UPEC. Data are the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired Mann−Whitney test (a), two-way ANOVA (c), or one-way ANOVA (e).

RhoB heterozygous (RhoB+/−) and knockout (RhoB−/−) mice were next employed in a model of acute UTI. Both IHF staining and western blotting assays indicated that the protein levels of RhoB, Beclin 1, and LC3 at 12 hpi in RhoB+/− and RhoB−/− mice were reduced compared with those of the wild-type mice (Fig. 6c, d and Supplementary Fig. 7a). As a result, bacterial titers in bladder homogenate from RhoB+/− and RhoB−/− mice infected by CFT073 were strongly increased compared with that from wild-type mice (Fig. 6e). However, under normal conditions, no significant physiological changes were observed in bladders of RhoB+/− and RhoB−/− mice, compared with that of wild-type mice based on H&E staining (Supplementary Fig. 7b).

Collectively, we conclude that UPEC induces RhoB level, which modulates Beclin 1 degradation and LC3 lipidation to promote intracellular UPEC clearance through the autophagy-related pathway (Fig. 6f).

Discussion

The most studied autophagy protein involved in UPEC infection is ATG16L1, which is reported to enhance UPEC infection9. However, the mechanism through which ATG16L1 deficiency decreases UPEC infection is independent of autophagy but depends on superficial urothelial cell architectural alterations and enhanced IL-1β-mediated hyperinflammatory response in macrophages10,11,12,13. On the other hand, Atg16L1 is required to protect against S. Typhimurium infection through autophagy28,36. We examined the role of ATG16L1 in RhoB-mediated LC3 lipidation and UPEC clearance, and found that RhoB knockdown had no effect on ATG16L1 protein level with or without CFT073 infection, and vice versa (Supplementary Fig. 7c and d). Notably, RhoB knockdown-induced increase of intracellular UPEC is not restored by ATG16L1 knockdown (Supplementary Fig. 7e). These results indicate that RhoB-mediated UPEC clearance may have no direct relationship with ATG16L1. Of note, a recent study from Xu et al. found that virulence factor SopF of Salmonella counteracted ATG16L1-mediated autophagy, which promoted intracellular bacterial survival28. It is possible that UPEC (generically similar to Salmonella) could also inhibit ATG16L1-mediated autophagy, as we observed that ATG16L1 knockdown did not affect intracellular UPEC in vitro (Supplementary Fig. 7e), and similar results were also shown in previous studies37,38. In addition, as ATG16L1 mutation influence urothelial cell architecture to decrease intracellular UPEC, its effect on UPEC clearance in vitro may be affected by many factors. However, our work suggests that RhoB-mediated clearance of CFT073 is through the autophagy-related pathway, as the effects are inhibited by Atg7 and P62 knockdown, and RhoB promotes colocalization of UPEC with LC3. Thus, the effect of RhoB on UPEC clearance could be achieved through other autophagic mechanisms rather than ATG16L1 related pathways, which is complicated and need to be further studied.

It is also reported that ATG3 deficiency in mouse bladder superficial epithelium significantly increases UPEC bacterial load, and an autophagy-inducing peptide markedly reduces the bacterial burden of infected mouse bladders, which supports that autophagy enhances UPEC clearance14. In addition, mice with urothelial-cell-specific knockout of ATG16L1 (involved in phagophore elongation), ATG7 (involved in phagophore elongation), ATG14 (involved in phagophore initiation) or Epg5 (involved in autophagosome-lysosome fusion) have no effect on bacterial load in urine at an early stage (24 h post infection), while ATG16L1 and ATG7 (but not Atg14 and Epg5) deficient mice harbor fewer UPEC QIRs (quiescent intracellular reservoirs) in the bladder at 14 days post infection13. Taken together, different results of UPEC clearance in autophagy-deficient mice may be due to other effects except for autophagy in gene-deficient mice.

RhoB is a member of Rho GTPases family, which shares a high identity with RhoA and RhoC. Unlike constitutively expressed RhoA and RhoC, RhoB maintains low expression in resting cells and is upregulated to exhibit distinct cellular functions upon stimulations18,39. It has been demonstrated that RhoB can be induced in cells treated with LPS19,21 or bacterial toxins;40 however, the possible role of RhoB in bacterial infections is still unclear. Like most Rho GTPases, RhoB activity is mediated by recycling of GTP/GDP loading to regulate the dynamics of cytoskeleton organization41, inflammation19,21, and cancer progression39. RhoB differs from RhoA and RhoC mostly at the hypervariable region of its C-terminus, which is highly posttranslational modified and crucial for its subcellular localization30. Intriguingly, LC3 lipidation enhanced by RhoB is not due to its enzymatic function as Rho GTPases, but correlates with its posttranslational modification that determines its membrane-associated localization.

It has been shown that binding partners of Beclin 1 control its level by manipulating its ubiquitination24. Here, we showed RhoB enhanced Beclin 1 stability in an Hsp90 but not ubiquitination-dependent fashion. Intriguingly, RhoB also seemed to be a client protein of Hsp90 to maintain RhoB homeostasis. RhoB binds to Hsp90 through residues of 42–89, different from RhoB-Beclin 1 interaction, which supports the formation of RhoB-Beclin 1-Hsp90 complex. Obvious colocalization of RhoB and Beclin 1 was observed, and less colocalization of RhoB and CFT073 was detected, and RhoB overexpression has no effect on UPEC uptake or P62/Ubiquitin recruited to UPEC, which suggests that RhoB has not involved in bacteria phagocytosis and intracellular recognition. Taken together, RhoB has mainly involved in Beclin 1 stabilization, resulting in enhanced membrane nucleation, and may leave autophagosome and was recycled during the late phase of autophagosome including bacteria, which should be further studied.

S-guanylated Group A Streptocccus by 8-nitro-cGMP, induced by nitric oxide, is reported to be selected for autophagic degradation42. We found that suppression of 8-nitro-cGMP by NaHS, or a nitric oxide synthase inhibitor L-NMMA, did not significantly affect RhoB effect on intracellular CFT073 survival, indicating that S-guanylation is not related to RhoB-mediated UPEC clearance (Supplementary Fig. 7f and g).

In summary, we identified the interaction between RhoB-Beclin-1-Hsp90 that promoted LC3 lipidation and intracellular UPEC clearance, providing the potential target to develop treatment strategies for UTIs. Whether RhoB has an effect on the clearance of other intracellular bacteria through the same mechanism should be examined in further studies.


Article classification: Biological abstract
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