Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin–mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI

Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin–mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI

Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications

Rab35 is required for the survival of UPEC within BEC 5637.

<p><b>A.</b> BEC5637 cells were transfected with 100nM each of si Rab35 or non-targeting siRNA (si NT). 48h following knockdown, the cells were infected with UPEC at MOI 500. Intracellular bacterial load at different time points {4 h (invasion), 24 h and 48 h, was determined by lysing the cells in 0.1% Triton X-100 and plating on LB-agar as described in Materials and Methods. Results are expressed as bacterial load/ 2x10⁵ cells. Immunoblotting was done to confirm the knock down efficiency of Rab35 siRNA (inset). <b>B.</b> Rab35 silencing does not enhance the efflux rate of UPEC from BEC-5637. BEC-5637 cells were transfected with 100nM each of si Rab35 or non-targeting siRNA (si NT). 48 h following knockdown, the cells were infected with UPEC at MOI 500. After gentamycin (100μg/ml) treatment, cells were washed in left in fresh culture medium containing 100mM methyl-D-mannopyranoside. At 24 h post infection, the culture medium was collected and plated for CFU counts as described in Materials and Methods. Results are expressed % exocytosis relative to siNT cells. <b>C.</b> Over expression of Rab35 protein leads to increase in bacterial load at 48 h post infection. BEC cells were transfected with GFP-Rab35 or control GFP vector for 24 h followed by UPEC infection (MOI 500) for another 48 h. Intracellular bacterial load was calculated as mentioned above. Results are expressed as bacterial load/ 2x10⁵ cells. Immunoblotting was done to confirm the expression levels of Rab35 (inset). * represents <i>p</i> < .05; ** represents <i>p</i><0.01. Values shown represent mean ± standard deviation of results of three independent experiments.</p

Rab35 protein localizes to UPEC-containing vacuole and is up regulated during UPEC infection.

<p><b>A.</b> BEC cells were transfected with GFP-Rab35. At 24 h post transfection the cells were either left uninfected or infected (with RFP-UPEC (MOI 500) and the localization of Rab35 with UPEC-containing vacuole (UCV) was analyzed at 4, 24 and 48 h post-infection by confocal microscopy. UCV with complete rim-like demarcation of Rab35 (category 1, marked as 1 in the merged image) and UCV where Rab35 may not form a complete rim-like structure, but enriched at the periphery of UCV membrane (category 2, marked as 2 in the merged image) by visual determination were counted as the positive population for Rab35 positive UCV. Number of Rab35 positive UCV was divided by the total number of UCVs and is represented in the graph as % of Rab35 positive UCV. Representative z sections of the images at 24 h are shown. Also shown at the bottom right side are the orthogonal sections of bacteria in category 1 and category 2 UCVs in XZ and YZ plane. White lines represent regions in which XYZ sections were taken. Scale bar denotes 2μm. DAPI (blue), Rab35 (green), and UPEC (red). At least 100 UCVs were counted for each experiment. Experiments were repeated three times with similar results. <b>B</b>. BEC cells were transfected with Rab35 ∆Q67L or Rab35 ∆S22N as mentioned above. The cells were infected with RFP-UPEC and the localization of mutant Rab35 with UCV was analyzed at 24 h post infection by confocal microscopy and represented in the graph as % of Rab35 positive UCV (category 1 and 2) as mentioned above. A representative image at 24 h is shown in the right panel. <b>C</b> and <b>D</b>. BEC cells were infected with UPEC and Rab35 mRNA and protein expression was analyzed at 4, 24 and 48 h post infection by qRT-PCR (Fig <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005083#ppat.1005083.g001" target="_blank">1C</a></b>) and Western blot (Fig <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005083#ppat.1005083.g001" target="_blank">1D</a></b>) respectively. The qRT-PCR values are normalized with GAPDH. * represents p < .05, ** represents p < .01. All the values shown represent mean ± standard deviation of results from three independent experiments. Quantitation of the Western blots using the software ImageJ is shown (Fig <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005083#ppat.1005083.g001" target="_blank">1D</a></b>, bottom panel). Relative densitometric data (Rab35 protein normalized with GAPDH) is shown. Values shown represent means ± standard deviations of results of three experiments. (* <i>p</i><0.05; ** represents <i>p</i><0.01 <i>vs</i> values at 0 h time point). <b>E</b>. C57BL/6 mice were infected transurethrally with UPEC (UTI89 strain). Mouse bladders were removed 24 h (top panels) and 2 weeks (bottom panel) post infection and the tissue sections were processed for immunofluorescence. The magnified image of association of Rab35 with UPEC is shown in the zoomed panel {(optical magnification (63x) and electronic zoom (2x)}. Green (Rab35) UPEC (red) and DAPI (blue). n = 4 sections/mouse bladder, n = 3 mice per experiment.</p

Rab35 is required for the survival of UPEC within BEC 5637.

<p><b>A.</b> BEC5637 cells were transfected with 100nM each of si Rab35 or non-targeting siRNA (si NT). 48h following knockdown, the cells were infected with UPEC at MOI 500. Intracellular bacterial load at different time points {4 h (invasion), 24 h and 48 h, was determined by lysing the cells in 0.1% Triton X-100 and plating on LB-agar as described in Materials and Methods. Results are expressed as bacterial load/ 2x10⁵ cells. Immunoblotting was done to confirm the knock down efficiency of Rab35 siRNA (inset). <b>B.</b> Rab35 silencing does not enhance the efflux rate of UPEC from BEC-5637. BEC-5637 cells were transfected with 100nM each of si Rab35 or non-targeting siRNA (si NT). 48 h following knockdown, the cells were infected with UPEC at MOI 500. After gentamycin (100μg/ml) treatment, cells were washed in left in fresh culture medium containing 100mM methyl-D-mannopyranoside. At 24 h post infection, the culture medium was collected and plated for CFU counts as described in Materials and Methods. Results are expressed % exocytosis relative to siNT cells. <b>C.</b> Over expression of Rab35 protein leads to increase in bacterial load at 48 h post infection. BEC cells were transfected with GFP-Rab35 or control GFP vector for 24 h followed by UPEC infection (MOI 500) for another 48 h. Intracellular bacterial load was calculated as mentioned above. Results are expressed as bacterial load/ 2x10⁵ cells. Immunoblotting was done to confirm the expression levels of Rab35 (inset). * represents <i>p</i> < .05; ** represents <i>p</i><0.01. Values shown represent mean ± standard deviation of results of three independent experiments.</p

Loss of Rab35 targets UPEC to late lysosomal compartment.

<p><b>A.</b> BEC-5637 cells were transfected with Rab35 expressing plasmid. 24 h later the cells were infected with UPEC. The cells were fixed at different stages of infection, permeabilised and stained for LAMP1 and were then analyzed by confocal microscopy. The results (panel a) are represented as % of UCV positive for both Rab35 and LAMP1. At least 100 UCV were counted for each experiment. A representative image is shown in panel b. DAPI {blue, host nuclei (N) or bacteria (UPEC)}, Rab35 (green), and LAMP1 (red). Arrows in the DAPI panel indicates UPEC. Zoomed panel (panel c) shows the magnified image of UCV (marked by arrows in the merged image) {(optical magnification (63x) and electronic zoom (2.5x)}. The graph (panel d) shows the quantification of the fluorescence intensity along the white line shown in the zoomed panel. Both LAMP1 and Rab35 co-localized at the UCV. <b>B.</b> si NT and si Rab35 cells were infected with GFP-UPEC. At 24 h post infection, the cells were stained with LysoTracker Red for 2 h; fixed and analyzed by confocal microscopy. Graph (panel a) is depicted as % bacteria co-localizing with LysoTracker Red, At least 100 bacteria were counted. Representative images are shown in panel b (si NT) and (si Rab35). *** represents <i>p</i><0.001, Values shown represent mean ± standard deviation of results of three independent experiments. The graphs (panel c) show the quantification of the fluorescence intensity along the white line shown in the merged image. UPEC from si NT sample did not co-localize with LysoTracker Red; while UPEC from si Rab35 sample co-localized with LysoTracker Red. <b>C.</b> BEC cells were transfected with si NT or si Rab35. 24 h later the cells were transfected with RFP-Cathepsin D expressing plasmid. After another 24 h the cells were infected with GFP-UPEC. The colocalization between UPEC containing vacuole (UCV) and Cathepsin D was observed 24 h post infection by confocal microscopy. Results are represented as % of cathepsin D positive UCV (% of UCV that is positive for cathepsin D). At least 100 bacteria were counted. ** represents <i>p</i> < .01, Values shown represent mean ± standard deviation of results of three independent experiments. <b>D.</b> Cathepsin D protein expression was analyzed in si NT/ si Rab35 cells left uninfected or infected with UPEC for 4, 24 and 48 h by Western blotting. The experiment was repeated three times with similar results and a representative blot is shown (top panel). Quantitation of the Western blots using the software ImageJ is shown (bottom panel). Relative densitometric data (mature cathepsin D/GAPDH) is shown. Values shown represent means ± standard deviations of results of three experiments. (* p<0.05 vs siNT values at the corresponding time points).</p

Iron supplementation is unable to support intracellular UPEC survival in the absence of Rab35 or TfR.

<p><b>A. si</b>NT, siRab35 or siTfR treated BEC cells were maintained in serum-free conditions 4 h prior to infection and were subsequently left in serum free medium for the rest of the duration of the experiment. Cells were infected with UPEC at MOI 500 for 2 h. Following infection, and gentamycin treatment, the cells were washed and left in RPMI with gentamycin (10μg/ml) and treated with 50μg/ml holotransferrin or left untreated for further 24 h. The cells were then lysed and plated for enumeration of intracellular bacteria by cfu assay. Results are expressed as % change in bacterial load/ 2x10⁵ cells. Values shown represent means ± standard deviations of results of three experiments. (** p<0.01 vs untreated siNT values). <b>B.</b> UPEC infection upregulates TfR at the protein level. BEC cells were infected with UPEC and TfR protein expression was analyzed at 4, 24 and 48 h post infection by Western blot analysis. Quantitation of the Western blots using the software ImageJ is shown (bottom panel). Relative densitometric data (TfR/GAPDH) is shown. Values shown represent means ± standard deviations of results of three experiments. (* p<0.05 vs 0 h time point values).</p

Iron acquisition <i>via</i> Rab35/transferrin receptor 1 pathway is critical for the intracellular survival of UPEC within BEC.

<p><b>(A)</b> UPEC-Infected BEC cells were either left untreated (UT) or treated with holotransferrin (iron), deferoxamine (DFO) or iron+DFO in serum-free conditions. The intracellular bacterial load was determined 24 h post-infection by lysing the cells in 0.1% Triton X-100 and plating on LB-agar. <b>B</b> BEC cells transfected with si NT, si Rab35 or si TfR were left either uninfected or infected with UPEC. Intracellular iron levels at 24 h post infection were determined by calcein-AM fluorescence as described in materials and methods. Immunoblotting was done to confirm the knock down efficiency of Rab35 and TfR siRNA (inset panel). <b>C.</b> BEC cells were transfected with si TfR, si Rab35 or siNT for 48 h followed by infection with UPEC. Intracellular bacterial load at different time points was determined as described before. <b>D and E.</b> BEC-5637 cells were transfected with 100nM each of 3’ UTR si Rab35 (<b>D</b>) 3’ UTR si TfR (<b>E</b>). 24 h following knockdown, the cells were transfected with either empty vector control or plasmids encoding Rab35 (D) or TfR (E) for 24 h. Cells were subsequently serum starved for 4 h and infected with UPEC at MOI 500. The infected cells were finally left in Gentamycin (10 μg/ml)—RPMI with 3% serum. Intracellular bacterial load was determined at 24 h. Immunoblotting was done to confirm the knockdown and over expression efficiency (inset panels). <b>F.</b> BEC cells were transfected with GFP-Rab35 for 24 h followed by UPEC infection for 24 h; fixed, permeabilised, stained for intracellular Transferrin Receptor and analyzed by confocal microscopy. Rab35 (green), DAPI {blue, host nuclei or bacteria (UPEC)}, and Transferrin receptor (red). Arrows in the DAPI panel indicate UPEC colonies. Panel b shows the magnified image of the UCV marked by arrows in the merged image {(optical magnification (63x) and electronic zoom (3.7x)}. The graph (panel c) shows the quantification of the fluorescence intensity along the white line shown in panel b. N, host nucleus. Both Rab35 and TfR colocalized at the UCV. The experiment was repeated three times with similar results and a representative image is shown. <b>G.</b> si NT and si Rab35 cells were either left uninfected or infected with UPEC for 24 h. Subsequently, the cells were loaded with Alexa 568 labelled transferrin for 20’ at 37°C. Unbound Transferrin was removed and the cells were incubated with RPMI containing unlabelled holotransferrin for the given time points. The cells were fixed and analyzed for intracellular Transferrin by confocal microscopy. Transferrin receptor recycling (Transferrin release) was measured as % of intracellular Transferrin at 0h - intracellular Transferrin at the given time point. <b>H</b> si NT and si Rab35 cells were either left uninfected or infected with UPEC. 24 h later the cells were fixed and stained for cell surface Transferrin Receptor and represented as Mean fluorescence intensity (MFI) and analyzed by confocal microscopy. <b>I and J.</b> si NT and si Rab35 BEC cells were either left uninfected or infected with UPEC. 24 h later the cells were loaded with Alexa 568 labelled transferrin for 20’ at 37°C. After removing the unbound Transferrin the cells were fixed and analyzed for intracellular Transferrin by confocal microscopy. Mean fluorescence intensity (MFI) is shown in the graph (<b>I</b>). The confocal microscopy experiments were repeated three times with similar results and a representative Fig for the infected samples are shown in (<b>J</b>). Transferrin (red). <b>K.</b> Infected si NT and si Rab35 cells were loaded with Bodipy 493/503 at 24 h post infection. The cells were subsequently fixed and analyzed for lipid staining by confocal microscopy and represented as MFI. Data represent mean ± standard deviation of results of three independent experiments (A, B, C, D, E, G, H, I and K). * represents p<0.05, **represents p<0.01. *** represent <i>p</i><0.001. Results of (A), (C), (D) and (E) are expressed as bacterial load/ 2x10⁵ cells. si = siRNA, si NT = negative control siRNA.</p

RNAi screening and bioinformatics analysis.

<p>(A) Screen methodology. See methods for detailed description of screening methodology. The results of stage specific assays are shown in the brown box on the right side of panel A. Numbers and arrows shown in red and green colour indicate steps of the primary and secondary screens, respectively. (B) Network analysis involving all ‘hits’ in the siRNA screen that have been experimentally validated in the literature to interact with one or more components of the RIG-I pathway. Putative positive regulators of RIG-I are indicated in green, and putative negative regulators in red. Empty circles are previously known genes of the RIG-I pathway that were not defined as hits in our RNAi screen. (C) Gene ontology canonical pathway enrichment analysis among the novel RIG-I regulators identified in this screen. Most enriched categories are shown. RLR, RIG-I Like Receptors. (D) Schematic showing inositol pyrophosphate synthesis pathway. IPPK, inositol 1,3,4,5,6-pentakisphosphate 2-kinase; PPIP5K1/2, diphosphoinositol pentakisphosphate kinase 1/2; IP6K1-3, inositol hexakisphosphate kinase 1–3; IP5, inositol pentakisphosphate; IP6, inositol hexakisphosphate; IP7, diphosphoinositol-pentakisphosphate; IP8, bisdiphosphoinositol-tetrakisphosphate.</p