figf4 ( a ) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining . Values are means ± s . e . m . of three independent experiments . Bax transfection was used as a positive control . ( b ) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h . Quantitative analysis of these experiments is shown in a . Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining . Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis ( nuclear condensation , cell shrinkage ; approximately 80 % of the cells ) . ( c ) Jurkat cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer . Cell death was observed as a consequence of enforced expression of truncated Atg5 within two days . Morphological analysis ( Diff - Quik ) is also shown and revealed that the type of death was apoptosis ( nuclear condensation , cell shrinkage ) . ( d ) CEM cells that either lacked Bcl - 2 or expressed high levels of Bcl - 2 received truncated Atg5 or GFP by lentiviral gene transfer . In the absence of Bcl - 2 , induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days . Apoptosis was largely blocked in cells expressing high levels of Bcl - 2 . GFP transfection did not affect viability of the cells in this system . ( e ) Staurosporine - induced Atg5 cleavage ( 5 h treatment ) in CEM cells independent of Bcl - 2 expression . Blots were stripped and reprobed with anti - GAPDH antibody . ( f ) Detection of autophagic activity by staining of acidic vesicular organelles ( VAOs , red ) followed by flow cytometry analysis in genetically modified HeLa cells . That 24K Atg5 did not induce autophagy was confirmed by transmission electron microscopy ( data not shown ) . ( g ) Cell death after 24K Atg5 gene transfer in HeLa cells exhibiting different autophagic activities . Values are means ± s . e . m . of three independent experiments ( lentiviral gene transfer for 24 h ) . The asterisks indicate P 0 . 05 . n . s . , no significant difference . The scale bars represent 10 μM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining. Values are means ± s.e.m. of three independent experiments. Bax transfection was used as a positive control. (b) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h. Quantitative analysis of these experiments is shown in a. Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining. Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis (nuclear condensation, cell shrinkage; approximately 80% of the cells).(c) Jurkat cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer. Cell death was observed as a consequence of enforced expression of truncated Atg5 within two days. Morphological analysis (Diff-Quik) is also shown and revealed that the type of death was apoptosis (nuclear condensation, cell shrinkage).(d) CEM cells that either lacked Bcl-2 or expressed high levels of Bcl-2 received truncated Atg5 or GFP by lentiviral gene transfer. In the absence of Bcl-2, induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days. Apoptosis was largely blocked in cells expressing high levels of Bcl-2. GFP transfection did not affect viability of the cells in this system.(e) Staurosporine-induced Atg5 cleavage (5 h treatment) in CEM cells independent of Bcl-2 expression. Blots were stripped and reprobed with anti-GAPDH antibody.(f) Detection of autophagic activity by staining of acidic vesicular organelles (VAOs, red) followed by flow cytometry analysis in genetically modified HeLa cells. That 24K Atg5 did not induce autophagy was confirmed by transmission electron microscopy (data not shown).(g) Cell death after 24K Atg5 gene transfer in HeLa cells exhibiting different autophagic activities. Values are means ± s.e.m. of three independent experiments (lentiviral gene transfer for 24 h). The asterisks indicate P 0.05. n.s., no significant difference. The scale bars represent 10 μM.


figf5 ( a ) Confocal microscopy of HeLa cells were treated with anti - CD95 antibody for 5 h or left untreated . Cells were stained with anti - Atg5 and anti - VDAC antibodies . In untreated cells , there was only little evidence for colocalization of Atg5 and VDAC . In anti - CD95 antibody treated cells some of the Atg5 colocalized with VDAC . The images of colocalization were prepared using Imaris software . Numerical analysis was performed on 10 cells in each group and the correlation coefficient calculated . The error bars represent the s . e . m . ( b ) Subcellular fractionation of blood neutrophils that were left untreated to induce spontaneous apoptosis . After 4 h incubation , cleavage of Atg5 was evident and both full - length and truncated Atg5 were present in the cytosol . After 8 h incubation , some 24K Atg5 but not full - length Atg5 was found in the mitochondrial fraction . Mitochondrial association of truncated Atg5 coincided with the release of cytochrome c into the cytosol . The same data were observed in staurosporine - induced apoptosis of Jurkat cells ( data not shown ) . C , cytosol ; M , mitochondria . ( c ) Cytochrome c release assay . Truncated and full - length recombinant Atg5 were added to isolated mitochondria and incubated at 37 ° C for 1 h . Supernatants and mitochondrial pellets were then analysed for cytochrome c by immunoblotting . Truncated Bid ( tBid ) was used as a positive control for mitochondrial permeabilization . Truncated Atg5 released cytochrome c in a concentration - dependent manner , indicating mitochondrial permeabilization . Full - length Atg5 had only little effect in this system . ( d ) HeLa cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer for 24 h and analysed for caspase - 3 cleavage . GAPDH expression demonstrates equal loading . ( e ) Jurkat cells were treated with staurosporine for 5 h or left untreated . Staurosporine induced Atg5 cleavage and both full - length and truncated Atg5 were present in total lysates . Truncated but not full - length Atg5 was detected in Bcl - xL immunoprecipitates obtained from apoptotic Jurkat cells . No evidence for physical interaction between Atg5 and Bax was obtained . The lower panels demonstrate that both Bax and Bcl - xL were enriched following immunoprecipitation . The asterisk indicates P 0 . 05 . The scale bars represent 10 μM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Confocal microscopy of HeLa cells were treated with anti-CD95 antibody for 5 h or left untreated. Cells were stained with anti-Atg5 and anti-VDAC antibodies. In untreated cells, there was only little evidence for colocalization of Atg5 and VDAC. In anti-CD95 antibody treated cells some of the Atg5 colocalized with VDAC. The images of colocalization were prepared using Imaris software. Numerical analysis was performed on 10 cells in each group and the correlation coefficient calculated. The error bars represent the s.e.m.(b) Subcellular fractionation of blood neutrophils that were left untreated to induce spontaneous apoptosis. After 4 h incubation, cleavage of Atg5 was evident and both full-length and truncated Atg5 were present in the cytosol. After 8 h incubation, some 24K Atg5 but not full-length Atg5 was found in the mitochondrial fraction. Mitochondrial association of truncated Atg5 coincided with the release of cytochrome c into the cytosol. The same data were observed in staurosporine-induced apoptosis of Jurkat cells (data not shown). C, cytosol; M, mitochondria.(c) Cytochrome c release assay. Truncated and full-length recombinant Atg5 were added to isolated mitochondria and incubated at 37 °C for 1 h. Supernatants and mitochondrial pellets were then analysed for cytochrome c by immunoblotting. Truncated Bid (tBid) was used as a positive control for mitochondrial permeabilization. Truncated Atg5 released cytochrome c in a concentration-dependent manner, indicating mitochondrial permeabilization. Full-length Atg5 had only little effect in this system.(d) HeLa cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer for 24 h and analysed for caspase-3 cleavage. GAPDH expression demonstrates equal loading.(e) Jurkat cells were treated with staurosporine for 5 h or left untreated. Staurosporine induced Atg5 cleavage and both full-length and truncated Atg5 were present in total lysates. Truncated but not full-length Atg5 was detected in Bcl-xL immunoprecipitates obtained from apoptotic Jurkat cells. No evidence for physical interaction between Atg5 and Bax was obtained. The lower panels demonstrate that both Bax and Bcl-xL were enriched following immunoprecipitation. The asterisk indicates P 0.05. The scale bars represent 10 μM.


Figure 1A , B Representative images of OG immunohistochemistry on paraffin - embedded colon sections from control , UC and CD patients ( n = 4 ) . Intensities of staining were scored on the right . Scale bars = 50 μm . ( one - way ANOVA , OGT : Healthy vs UC P = 0 . 0002 , Healthy vs CD P = 0 . 003 ; O - GlcNAc : Healthy vs UC P = 0 . 0365 , Healthy vs CD P = 0 . 0125 A , B Representative images o O - GlcNAc ( B ) immunohistochemistry on paraffin - embedded colon sections from control , UC and CD patients ( n = 4 ) . Intensities of staining were scored on the right . Scale bars = 50 μm . ( one - way ANOVA , OGT : Healthy vs UC P = 0 . 0002 , Healthy vs CD P = 0 . 003 ; O - GlcNAc : Healthy vs UC P = 0 . 0365 , Healthy vs CD P = 0 . 0125 C Levels of intestinal epithelial OGT and O - GlcNAcylation in a second cohort of control , UC , and CD patients . ( one - way ANOVA , OGT : Control n = 10 , UC n = 9 , CD n = 6 ; Control vs UC P = 0 . 035 , Control vs CD P = 0 . 003 ; O - GlcNAc : Control n = 10 , UC n = 8 , CD n = 8 ; Control vs UC P = 0 . 0027 , Control vs CD P = 0 . 008 D , E Regression plots of average immune - staining scores of OGT and O - GlcNAcylation against histological scores in UC ( D ) and CD ( E ) ( D : OGT n = 19 , O - GlcNAc n = 18 ; E : OGT n = 15 , O - GlcNAc n = 16 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, B Representative images of OG immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125 A, B Representative images o O-GlcNAc (B) immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125 C Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (one-way ANOVA, OGT: Control n=10, UC n=9, CD n=6; Control vs UC P=0.035, Control vs CD P=0.003; O-GlcNAc: Control n=10, UC n=8, CD n=8; Control vs UC P=0.0027, Control vs CD P=0.008 D, E Regression plots of average immune-staining scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n=19, O-GlcNAc n=18; E: OGT n=15, O-GlcNAc n=16)


Figure 2A Body weight of male wildtype and Vil - Ogt KO mice at 6 weeks of age ( n = 6 , P = 0 . 000015 ) B Incidence of rectal prolapse in male wildtype and Vil - Ogt KO mice ( WT n = 17 , KO n = 9 , P < 0 . 0001 ) C Representative colonoscopy images of male wildtype and Vil - Ogt KO mice D Body weight of female wildtype and Vil - Ogt KO mice at 9 weeks of age ( WT n = 11 , heterozygous KO n = 4 , homozygous KO n = 5 , P < 0 . 0001 ) E Incidence of rectal prolapse in female wildtype and Vil - Ogt KO mice ( WT n = 9 , heterozygous KO n = 8 , homozygous KO n = 6 , P = 0 . 0439 ) F H & E staining of duodenum , jejunum , ileum , colon , and rectum of 10 - week - old male wildtype and Vil - Ogt KO mice . Scale bars = 100 μm

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Body weight of male wildtype and Vil-Ogt KO mice at 6 weeks of age (n = 6, P = 0.000015) B Incidence of rectal prolapse in male wildtype and Vil-Ogt KO mice (WT n = 17, KO n = 9, P < 0.0001)  C Representative colonoscopy images of male wildtype and Vil-Ogt KO mice  D Body weight of female wildtype and Vil-Ogt KO mice at 9 weeks of age (WT n = 11, heterozygous KO n = 4, homozygous KO n = 5, P < 0.0001)  E Incidence of rectal prolapse in female wildtype and Vil-Ogt KO mice (WT n = 9, heterozygous KO n = 8, homozygous KO n = 6, P = 0.0439) F H&E staining of duodenum, jejunum, ileum, colon, and rectum of 10-week-old male wildtype and Vil-Ogt KO mice. Scale bars = 100 μm


Figure 6 A Numbers of Paneth cells in the ileum of wildtype and Vil - Ogt KO mice ( n = 6 , P = 0 . 0048 ) B Representative electron microscopic images of Paneth cells . Scale bars = 1 μm C Expression of AMP genes in ileum tissues of wildtype and Vil - Ogt KO mice ( n = 6 , Pan Defesin P = 0 . 031 , Defa3 P = 0 . 049 , Defa4 P = 0 . 013 , Defa5 P = 0 . 023 , Defa6 P = 0 . 034 , Ang4 P = 0 . 031 ) D Immunoblotting of LC3 in IEC cells isolated from WT and Vil - Ogt KO mice E Representative images of O - GlcNAc staining in Paneth cells of male wildtype and Defa6 - Ogt KO mice . Stars indicate lysozyme - positive Paneth cells F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 , P = 0 . 024 ) . Arrow indicate Paneth cell . Scale bars = 10 μm G Expression of AMP genes in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 Pan Defesin P = 0 . 0291 , Defa3 P = 0 . 0462 ) H Representative images of cultured organoid on Day 3 I Expression of inflammatory markers in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 ) J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis . Daily changes in body weight ( J ) are shown ( n = 4 - 5 ) . ( L : P = 0 . 049 J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis . Daily changes i colitis scores ( K are shown ( n = 4 - 5 ) . ( L : P = 0 . 049 J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis RT - qPCR of inflammatory genes in the ileum ( L ) are shown ( n = 4 - 5 ) . ( L : P = 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 A Numbers of Paneth cells in the ileum of wildtype and Vil-Ogt KO mice (n = 6, P = 0.0048)  B Representative electron microscopic images of Paneth cells. Scale bars = 1 μm  C Expression of AMP genes in ileum tissues of wildtype and Vil-Ogt KO mice (n = 6, Pan Defesin P = 0.031, Defa3 P = 0.049, Defa4 P = 0.013, Defa5 P = 0.023, Defa6 P = 0.034, Ang4 P = 0.031)  D Immunoblotting of LC3 in IEC cells isolated from WT and Vil-Ogt KO mice  E Representative images of O-GlcNAc staining in Paneth cells of male wildtype and Defa6-Ogt KO mice. Stars indicate lysozyme-positive Paneth cells  F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6-Ogt KO mice (n = 5, P = 0.024). Arrow indicate Paneth cell. Scale bars = 10 μm  G Expression of AMP genes in the ileum of wildtype and Defa6-Ogt KO mice (n = 5 Pan Defesin P = 0.0291, Defa3 P = 0.0462)  H Representative images of cultured organoid on Day 3  I Expression of inflammatory markers in the ileum of wildtype and Defa6-Ogt KO mice (n = 5)  J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes in body weight (J) are shown (n = 4-5). (L: P = 0.049 J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes i colitis scores (K are shown (n = 4-5). (L: P = 0.049 J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049


Figure 7 A Expression of phosphorylated and total STAT3 and NF - κB p65 in the colon of wildtype and Vil - Ogt KO mice B Ileum and colon IECs from 10 - week - old wildtype and Vil - Ogt KO mice were isolated for RNA - sequencing analyses . The heatmaps of the sample - to - sample distances are shown C Volcano plots of gene expression changes . The x - axis specifies the fold - changes ( FC ) and the y - axis specifies the negative logarithm to the base 10 of the p - values adjusted for multiple testing with the Benjamini - Hochberg procedure . FC > 2 and p < 0 . 05 genes are shown in solid dots F Levels of STAT1 protein in IECs determined by immunoblotting and O - GlcNAc modification of STAT1 determined by immunoprecipitation using the O - GlcNAc antibody followed by Immunoblotting G , H Caco - 2 cells were treated with 100 uM Ac45S - GlcNAc for 24 hours , levels of STAT1 and global O - GlcNAcylatio were determined G , H Caco - 2 cells were treated with 100 uM Ac45S - GlcNAc for 24 hours , levels of STAT1 an STAT1 O - GlcNAcylation ( H ) were determined I siRNAs against scrambled sequence or human OGT were transfected in 293T cells and STAT1 levels were determined 48 hours later J The pGAS - luc Cis - Reporter plasmid was co - transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 ( n = 4 , P = 0 . 0003 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 A Expression of phosphorylated and total STAT3 and NF-κB p65 in the colon of wildtype and Vil-Ogt KO mice  B Ileum and colon IECs from 10-week-old wildtype and Vil-Ogt KO mice were isolated for RNA-sequencing analyses. The heatmaps of the sample-to-sample distances are shown  C Volcano plots of gene expression changes. The x-axis specifies the fold-changes (FC) and the y-axis specifies the negative logarithm to the base 10 of the p-values adjusted for multiple testing with the Benjamini-Hochberg procedure. FC > 2 and p < 0.05 genes are shown in solid dots  F Levels of STAT1 protein in IECs determined by immunoblotting and O-GlcNAc modification of STAT1 determined by immunoprecipitation using the O-GlcNAc antibody followed by Immunoblotting  G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 and global O-GlcNAcylatio were determined G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 an STAT1 O-GlcNAcylation (H) were determined I siRNAs against scrambled sequence or human OGT were transfected in 293T cells and STAT1 levels were determined 48 hours later  J The pGAS-luc Cis-Reporter plasmid was co-transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 (n = 4, P=0.0003)


Figure 8 A , B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS . Daily changes in body weight ( A ) and colitis score ( B ) ( n = 5 ) . ( B : day6 P = 0 . 0171 , day7 P = 0 . 0445 C Intestinal barrier functional assays measuring serum FITC - dextran before DSS treatment ( n = 5 , P = 0 . 0117 ) D - G Albumin levels in feces ( D on day 4 of recovery ( n = 5 ) colon length ( E ) on day 4 of recovery ( n = 5 ) H & E staining of colon tissues ( F on day 4 of recovery ( n = 5 ) . Scale bars = 100 μmRT - qPCR of inflammatory genes in the colon ( G ) on day 4 of recovery ( n = 5 ) . Scale bars = 100 μm . ( E : P = 0 . 0474 ; G : Tnfa P = 0 . 0190 , Il1b P = 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 A, B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS. Daily changes in body weight (A) and colitis score (B) (n= 5). (B: day6 P = 0.0171, day7 P= 0.0445  C Intestinal barrier functional assays measuring serum FITC-dextran before DSS treatment (n = 5, P = 0.0117) D-G Albumin levels in feces (D on day 4 of recovery (n = 5)colon length (E) on day 4 of recovery (n = 5)H&E staining of colon tissues (F on day 4 of recovery (n = 5). Scale bars = 100 μmRT-qPCR of inflammatory genes in the colon (G) on day 4 of recovery (n = 5). Scale bars = 100 μm. (E: P = 0.0474; G: Tnfa P = 0.0190, Il1b P = 0.0301


f1Dose - dependent increase of C . albicans ( SC5314 ) aggregation after 4 - h exposure ( a ) and filamentation after 24 - h exposure ( b ) to different concentrations of IL - 17A in RPMI - 1640 liquid medium . Cells aliquots were imaged by microscopy and magnified in the insets . Also note the increased turbidity of cell suspension at 24 h . Scale bars , 100 μm . ( c ) Percentages of germination of C . albicans untreated ( vehicle - treated , open circle ) or treated with IL - 17A at 1 ( black triangle ) or 10 ng ml − 1 ( black square ) for 4 or 24 h . Data are mean ± s . d . P 0 . 05 , IL - 17A - treated versus vehicle - treated cells , at 4 h ; P 0 . 01 ( 1 ng ) and P 0 . 001 ( 10 ng ) at 24 h ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) . ( d , e ) IL - 17A promotes biofilm formation . SEM images of C . albicans SC5314 untreated ( d ) or exposed to 10 ng ml − 1 IL - 17A ( e ) for 4 h and subsequently cultured onto plastic discs for 24 h . Scale bars , 10 μm . ( f ) IL - 17A increases resistance to nitrogen starvation . Cells were first grown in YPD medium overnight and then switched to nitrogen - free SLAD medium in the presence of 10 ng ml − 1 IL - 17A . Viability , expressed as a percentage of colony - forming units at zero time , was measured over a 15 - day period . Cells were exposed to 0 . 1 ( black circle ) , 1 ( black triangle ) or 10 ng ml − 1 ( black square ) IL - 17A or drug vehicle ( open circle ) at the beginning of the cultures or , in the case of 1 ng ml − 1 ( open triangle ) , also added every two other days . Data are mean ± s . d . P - values ranged from 0 . 05 to 0 . 001 for all samples treated with 10 ng IL - 17A ; P0 . 05 , on days 3 and 5 for samples treated 1 ng IL - 17A every two other days ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) . ( g , l ) C . albicans cells are metabolically active . Untreated ( g - i ) or IL - 17A - treated ( 10 ng ml − 1 in RPMI - 1640 liquid media for 24 h ) ( j - l ) cells were red after staining with FUN - 1 ( g , j ) , green with Con A ( h , k ) and yellow in the merged images ( i , l ) . Metabolically active cells are shown in red , and cell wall polysaccharides are shown in green . Scale bars , 5 μm . Images from one representative experiment out of two are shown .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1Dose-dependent increase of C. albicans (SC5314) aggregation after 4-h exposure (a) and filamentation after 24-h exposure (b) to different concentrations of IL-17A in RPMI-1640 liquid medium. Cells aliquots were imaged by microscopy and magnified in the insets. Also note the increased turbidity of cell suspension at 24 h. Scale bars, 100 μm.(c) Percentages of germination of C. albicans untreated (vehicle-treated, open circle) or treated with IL-17A at 1 (black triangle) or 10 ng ml−1 (black square) for 4 or 24 h. Data are mean±s.d. P 0.05, IL-17A-treated versus vehicle-treated cells, at 4 h; P 0.01 (1 ng) and P 0.001 (10 ng) at 24 h (one-way analysis of variance Bonferroni post-test) (n=3).(d,e) IL-17A promotes biofilm formation. SEM images of C. albicans SC5314 untreated (d) or exposed to 10 ng ml−1 IL-17A (e) for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 10 μm.(f) IL-17A increases resistance to nitrogen starvation. Cells were first grown in YPD medium overnight and then switched to nitrogen-free SLAD medium in the presence of 10 ng ml−1 IL-17A. Viability, expressed as a percentage of colony-forming units at zero time, was measured over a 15-day period. Cells were exposed to 0.1 (black circle), 1 (black triangle) or 10 ng ml−1 (black square) IL-17A or drug vehicle (open circle) at the beginning of the cultures or, in the case of 1 ng ml−1 (open triangle), also added every two other days. Data are mean±s.d. P-values ranged from 0.05 to 0.001 for all samples treated with 10 ng IL-17A; P0.05, on days 3 and 5 for samples treated 1 ng IL-17A every two other days (one-way analysis of variance Bonferroni post-test) (n=3).(g,l) C. albicans cells are metabolically active. Untreated (g-i) or IL-17A-treated (10 ng ml−1 in RPMI-1640 liquid media for 24 h) (j-l) cells were red after staining with FUN-1 (g,j), green with Con A (h,k) and yellow in the merged images (i,l). Metabolically active cells are shown in red, and cell wall polysaccharides are shown in green. Scale bars, 5 μm. Images from one representative experiment out of two are shown.


f2 ( g , h ) TOR inhibition in cells treated with 10 ng ml − 1 IL - 17A , 15 nM rapamycin or vehicle ( none ) by western blotting after 4 - h ( g ) or 24 - h ( h ) exposure . i - n ) Immunofluorescence of ATG8 in untreated ( i - k ) or IL - 17A - treated ( 10 ng ml − 1 for 4 h ) ( l - n ) cells . Cells were red after TRITC - ATG8 staining ( i , l ) , green after ConA staining ( j , m ) , merged images ( k , n ) . Mean values ( % ATG8 + cells ( merge ) ; mean ± s . d . ) : ATG8 / none , 8 . 2 ± 1 . 6 ; ATG8 / IL - 17A , 58 . 0 ± 11 . 3 ( P = 0 . 002 ) ( n = 3 ) . Scale bars , 10 μm . ( o - r ) Monodansilcadaverine ( MDC ) labelling performed on cells unexposed ( o ) or exposed to 1 ( p ) or 10 ng ml − 1 ( q ) IL - 17A for 4 h . Cells were magnified in the inset ( r ) . Representative results from three independent experiments . The mean values ( % hyphal cells with MDC - stained organelles ( merge ) ; mean ± s . d . ) were as follows : none , 9 . 5 ± 3 . 2 ; 1 ng ml − 1 IL - 17A , 14 . 0 ± 4 . 7 ; 10 ng ml − 1 IL - 17A , 56 . 2 ± 18 . 71 ( P = 0 . 004 , IL - 17A 10 ng ml − 1 versus none ) . Scale bars , 10 μm . ( s , t ) Quantitative real - time PCR of orfs . 19 . 4325 , 19 . 130 , RIC1 on cDNA from Candida SC5314 ( s ) or the TOR1 - 1 / TOR1 mutant ( t ) strains exposed to vehicle ( open column ) , 10 ng ml − 1 IL - 17A ( black column ) or 15 nM rapamycin ( grey column ) for 4 h . The actual P - values are indicated ( two - tailed t - test ) ( n = 3 ) . ( u , v ) Intracellular ( u ) or extracellular ( v ) cAMP levels , vehicle ( open circle ) , 10 ng ml − 1 IL - 17A ( black square ) or rapamycin ( grey square ) . P0 . 05 , IL - 17A - treated versus untreated at 1 h and P0 . 01 , IL - 17A - treated versus untreated at 24 h ( u ) , and P0 . 05 , rapamycin - treated versus untreated at 6 h ( v ) ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) . ( w ) orf . 19 . 5258 expression after 24 - h Candida SC5314 exposure as in ( n , o ) . Data are mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2(g,h) TOR inhibition in cells treated with 10 ng ml−1 IL-17A, 15 nM rapamycin or vehicle (none) by western blotting after 4-h (g) or 24-h (h) exposure.i-n) Immunofluorescence of ATG8 in untreated (i-k) or IL-17A-treated (10 ng ml−1 for 4 h) (l-n) cells. Cells were red after TRITC-ATG8 staining (i,l), green after ConA staining (j,m), merged images (k,n). Mean values (% ATG8+ cells (merge); mean±s.d.): ATG8/none, 8.2±1.6; ATG8/IL-17A, 58.0±11.3 (P=0.002) (n=3). Scale bars, 10 μm.(o-r) Monodansilcadaverine (MDC) labelling performed on cells unexposed (o) or exposed to 1 (p) or 10 ng ml−1 (q) IL-17A for 4 h. Cells were magnified in the inset (r). Representative results from three independent experiments. The mean values (% hyphal cells with MDC-stained organelles (merge); mean±s.d.) were as follows: none, 9.5±3.2; 1 ng ml−1 IL-17A, 14.0±4.7; 10 ng ml−1 IL-17A, 56.2±18.71 (P=0.004, IL-17A 10 ng ml−1 versus none). Scale bars, 10 μm.(s,t) Quantitative real-time PCR of orfs. 19.4325, 19.130, RIC1 on cDNA from Candida SC5314 (s) or the TOR1-1/TOR1 mutant (t) strains exposed to vehicle (open column), 10 ng ml−1 IL-17A (black column) or 15 nM rapamycin (grey column) for 4 h. The actual P-values are indicated (two-tailed t-test) (n=3).(u,v) Intracellular (u) or extracellular (v) cAMP levels, vehicle (open circle), 10 ng ml−1 IL-17A (black square) or rapamycin (grey square). P0.05, IL-17A-treated versus untreated at 1 h and P0.01, IL-17A-treated versus untreated at 24 h (u), and P0.05, rapamycin-treated versus untreated at 6 h (v) (one-way analysis of variance Bonferroni post-test) (n=3).(w) orf. 19.5258 expression after 24-h Candida SC5314 exposure as in (n,o). Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).


f3 ( a - g ) GFP C . albicans cells were untreated ( a - c ) or treated ( d - g ) for 4 h with 10 ng ml − 1 IL - 17A followed by anti - IL17A - PE and examined by fluorescence microscopy ( scale bars , 5 μm ) . Cells were green in ( a , d ) , red upon staining with anti - IL17A - PE ( b , e ) and double stained in the merged images ( c , f ) . Note the binding of IL - 17A to hyphal tips ( arrows and magnified in g ) ( n = 3 ) . Images are from one representative experiment out of three . ( h , i ) Binding of murine ( h ) or human ( i ) IL - 17A to clinical isolates of C . albicans from human urine ( yellow triangle ) , blood ( green triangle ) , vagina ( red triangle ) and gut ( blue triangle ) . IL - 17A was added to paraformaldehyde - fixed fungal cells followed by biotinylated secondary anti - IL - 17A antibody and streptavidin - horse radish peroxidase . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . P - values of samples exposed to 0 . 1 and 1 ng IL - 17A ranged from 0 . 05 to 0 . 001 ( one - way analysis of variance Bonferroni post - test ) . All samples exposed to 10 ng IL - 17A displayed significant ( P0 . 001 , one - way analysis of variance Bonferroni post - test ) OD values relative to unexposed samples ( 0 ) . ( j - o ) Direct visualization of IL - 17A on Candida freshly harvested from human vagina . Same experiment as in ( a - g ) on cells previously stained with Con A ( j , m , green ) and incubated with PE - anti - human IL - 17A ( k , n , red ) and double stained in the merged images ( l , o ) . Scale bars , 10 μm . Shown is one representative experiment out of three . ( p - r ) Cytospins of vaginal fluids from IL - 17F - deficient ( p ) , IL - 17A - deficient ( q ) and C57BL / 6 ( r ) mice intravaginally infected two days before with C . albicans SC5314 blastospores . Note the presence of hyphae in the vagina of IL - 17F - deficient mice after May - Grünwald - Giemsa staining ( scale bars , 20 μm ) . Shown is one representative experiment out of three . ( s ) Binding of IL - 17A to clinical isolates of C . krusei ( red square ) , C . glabrata ( green square ) and S . cerevisiae ( yellow square ) done as in ( h , i ) . IL - 17A was added to paraformaldehyde - fixed fungal cells followed by biotinylated secondary anti - IL - 17A antibody and streptavidin - horse radish peroxidase . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . P - values of samples exposed to 0 . 1 and 1 ng IL - 17A ranged from 0 . 05 to 0 . 001 ( one - way analysis of variance Bonferroni post - test ) . All samples exposed to 10 ng IL - 17A displayed significant ( P0 . 001 , one - way analysis of variance Bonferroni post - test ) OD values relative to unexposed samples ( 0 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a-g) GFP C. albicans cells were untreated (a-c) or treated (d-g) for 4 h with 10 ng ml−1 IL-17A followed by anti-IL17A-PE and examined by fluorescence microscopy (scale bars, 5 μm). Cells were green in (a,d), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Note the binding of IL-17A to hyphal tips (arrows and magnified in g) (n=3). Images are from one representative experiment out of three.(h,i) Binding of murine (h) or human (i) IL-17A to clinical isolates of C. albicans from human urine (yellow triangle), blood (green triangle), vagina (red triangle) and gut (blue triangle). IL-17A was added to paraformaldehyde-fixed fungal cells followed by biotinylated secondary anti-IL-17A antibody and streptavidin-horse radish peroxidase. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. P-values of samples exposed to 0.1 and 1 ng IL-17A ranged from 0.05 to 0.001 (one-way analysis of variance Bonferroni post-test). All samples exposed to 10 ng IL-17A displayed significant (P0.001, one-way analysis of variance Bonferroni post-test) OD values relative to unexposed samples (0).(j-o) Direct visualization of IL-17A on Candida freshly harvested from human vagina. Same experiment as in (a-g) on cells previously stained with Con A (j,m, green) and incubated with PE-anti-human IL-17A (k,n, red) and double stained in the merged images (l,o). Scale bars, 10 μm. Shown is one representative experiment out of three.(p-r) Cytospins of vaginal fluids from IL-17F-deficient (p), IL-17A-deficient (q) and C57BL/6 (r) mice intravaginally infected two days before with C.albicans SC5314 blastospores. Note the presence of hyphae in the vagina of IL-17F-deficient mice after May-Grünwald-Giemsa staining (scale bars, 20 μm). Shown is one representative experiment out of three.(s) Binding of IL-17A to clinical isolates of C. krusei (red square), C. glabrata (green square) and S. cerevisiae (yellow square) done as in (h,i). IL-17A was added to paraformaldehyde-fixed fungal cells followed by biotinylated secondary anti-IL-17A antibody and streptavidin-horse radish peroxidase. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. P-values of samples exposed to 0.1 and 1 ng IL-17A ranged from 0.05 to 0.001 (one-way analysis of variance Bonferroni post-test). All samples exposed to 10 ng IL-17A displayed significant (P0.001, one-way analysis of variance Bonferroni post-test) OD values relative to unexposed samples (0).


f4 ( a ) Dose - dependent binding of IL - 17A to fungal Crh11p ( red circle ) and Gas1p ( light blue circle ) proteins , but not to the fungal polysaccharides β - 1 , 3 - D - glucan ( open circle ) , mannan ( blue circle ) and zymosan ( grey circle ) . The lack of binding of IL - 17E to Crh11p ( orange circle ) serves as a negative control . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . Data are mean ± s . d . , n = 3 . P0 . 001 , Crh11p and Gas1p without versus with IL - 17A ( one - way analysis of variance Bonferroni post - test ) . ( b ) Co - immunoprecipitation assay of fungal Crh11p and murine IL - 17A proteins . Immunoprecipitates were probed with antibodies to the corresponding antigens and secondary HRP - conjugated antibody . C , irrelevant antibody . ( c ) Failure of IL - 17A to bind to wild - type ( red square ) or mutant C . albicans strains in which the three genes of the CRH family ( UTR2 , open light blue , CRH1 , open black and CRH12 , open blue square ) had been deleted , and restoration of binding by CRH1 ( black square ) and CRH12 ( blue square ) , but not UTR2 ( light blue square ) , reconstitution . Data are mean ± s . d . ( n = 3 ) . P0 . 001 for red square , open light blue , black square and blue square ( one - way analysis of variance Bonferroni post - test ) . ( d , e ) Cellular localization of bound IL - 17A . Electron micrographs of ultrafine sections of C . albicans MKY 378 wild - type ( d ) or the crh11Δ mutant ( e ) incubated with biotinylated IL - 17A and streptavidin gold - conjugated particles . Arrows indicate immunogold particles . Scale bars , 200 nm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4(a) Dose-dependent binding of IL-17A to fungal Crh11p (red circle) and Gas1p (light blue circle) proteins, but not to the fungal polysaccharides β-1,3-D-glucan (open circle), mannan (blue circle) and zymosan (grey circle). The lack of binding of IL-17E to Crh11p (orange circle) serves as a negative control. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. Data are mean±s.d., n=3. P0.001, Crh11p and Gas1p without versus with IL-17A (one-way analysis of variance Bonferroni post-test).(b) Co-immunoprecipitation assay of fungal Crh11p and murine IL-17A proteins. Immunoprecipitates were probed with antibodies to the corresponding antigens and secondary HRP-conjugated antibody. C, irrelevant antibody.(c) Failure of IL-17A to bind to wild-type (red square) or mutant C. albicans strains in which the three genes of the CRH family (UTR2, open light blue, CRH1, open black and CRH12, open blue square) had been deleted, and restoration of binding by CRH1 (black square) and CRH12 (blue square), but not UTR2 (light blue square), reconstitution. Data are mean±s.d. (n=3). P0.001 for red square, open light blue, black square and blue square (one-way analysis of variance Bonferroni post-test).(d,e) Cellular localization of bound IL-17A. Electron micrographs of ultrafine sections of C. albicans MKY 378 wild-type (d) or the crh11Δ mutant (e) incubated with biotinylated IL-17A and streptavidin gold-conjugated particles. Arrows indicate immunogold particles. Scale bars, 200 nm.


f5Fungi were harvested from the stomachs of C57BL / 6 ( a - c ) , IL - 17F - deficient ( d - f ) or IL - 17A - deficient ( g - i ) mice infected with GFP - Candida for 2 days , and stained using PE - labeled antibody to IL - 17A . Cells were green in ( a , d and g ) , red upon staining with anti - IL17A - PE ( b , e and h ) and double stained in the merged images ( c , f and i ) . Scale bars , 10 μm . Images are from one representative experiment out of two . ( j - l ) SEM images showing fungal germination in the stomachs of C57BL / 6 mice ( j ) , filamentation in IL - 17F - deficient mice ( k ) and neither one in IL - 17A - deficient ( l ) mice intragastrically infected 2 days before . Scale bars , 10 μm . ( a - l ) Images are from one representative experiment out of two . ( m - o ) C57BL / 6 mice were infected intragastrically with C . albicans MKY 378 strain ( m ) or the crh11Δ ( n ) and crh12Δ ( o ) mutants , and were monitored for fungal growth ( log10 colony - forming units ( CFU ) at 2 ( open circle ) or 8 ( black circle ) days post infection ) . Data are mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) for 2 and 8 days ( m ) and for 2 days ( o ) . Fungal cells were untreated ( none ) or exposed to 10 ng ml − 1 IL - 17A for 4 h before injection .

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f5Fungi were harvested from the stomachs of C57BL/6 (a-c), IL-17F-deficient (d-f) or IL-17A-deficient (g-i) mice infected with GFP-Candida for 2 days, and stained using PE-labeled antibody to IL-17A. Cells were green in (a,d and g), red upon staining with anti-IL17A-PE (b,e and h) and double stained in the merged images (c,f and i). Scale bars, 10 μm. Images are from one representative experiment out of two. (j-l) SEM images showing fungal germination in the stomachs of C57BL/6 mice (j), filamentation in IL-17F-deficient mice (k) and neither one in IL-17A-deficient (l) mice intragastrically infected 2 days before. Scale bars, 10 μm. (a-l) Images are from one representative experiment out of two.(m-o) C57BL/6 mice were infected intragastrically with C. albicans MKY 378 strain (m) or the crh11Δ (n) and crh12Δ (o) mutants, and were monitored for fungal growth (log10 colony-forming units (CFU) at 2 (open circle) or 8 (black circle) days post infection). Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test) for 2 and 8 days (m) and for 2 days (o). Fungal cells were untreated (none) or exposed to 10 ng ml−1 IL-17A for 4 h before injection.


f6Candida SC5314 ( a ) , TOR1 - 1 / TOR1 ( b ) and atg9Δ / atg9Δ ( c ) mutant strains were untreated ( open column ) or pre - incubated with IL - 17A ( filled column ) for 4 h before exposure to neutrophils for additional 2 h before the assessment of adhesion , phagocytosis and killing . Error bars , mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) . i - n ) Immunofluorescence of ATG8 in untreated ( i - k ) or IL - 17A - treated ( 10 ng ml − 1 for 4 h ) ( l - n ) cells . Cells were red after TRITC - ATG8 staining ( i , l ) , green after ConA staining ( j , m ) , merged images ( k , n ) . Mean values ( % ATG8 + cells ( merge ) ; mean ± s . d . ) : ATG8 / none , 8 . 2 ± 1 . 6 ; ATG8 / IL - 17A , 58 . 0 ± 11 . 3 ( P = 0 . 002 ) ( n = 3 ) . Scale bars , 10 μm . ( g ) Fungi purified from the stomachs of mice infected as in ( d - f ) were subjected to RT - PCR for autophagy genes expression . C + , fungal cells grown on SPIDER solid medium . C - , no cells . ( h - j ) Log10 colony - forming units ( CFU ) at 2 days post infection with C . albicans SC5314 ( wild - type ) or different mutant strains in C57BL / 6 ( h ) , IL - 17F - deficient ( i ) or IL - 17A - deficient ( j ) mice . Data are mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) .

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f6Candida SC5314 (a), TOR1-1/TOR1(b) and atg9Δ/atg9Δ (c) mutant strains were untreated (open column) or pre-incubated with IL-17A (filled column) for 4 h before exposure to neutrophils for additional 2 h before the assessment of adhesion, phagocytosis and killing. Error bars, mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).i-n) Immunofluorescence of ATG8 in untreated (i-k) or IL-17A-treated (10 ng ml−1 for 4 h) (l-n) cells. Cells were red after TRITC-ATG8 staining (i,l), green after ConA staining (j,m), merged images (k,n). Mean values (% ATG8+ cells (merge); mean±s.d.): ATG8/none, 8.2±1.6; ATG8/IL-17A, 58.0±11.3 (P=0.002) (n=3). Scale bars, 10 μm.(g) Fungi purified from the stomachs of mice infected as in (d-f) were subjected to RT-PCR for autophagy genes expression. C+, fungal cells grown on SPIDER solid medium. C-, no cells.(h-j) Log10 colony-forming units (CFU) at 2 days post infection with C. albicans SC5314 (wild-type) or different mutant strains in C57BL/6 (h), IL-17F-deficient (i) or IL-17A-deficient (j) mice. Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).


f7 ( a - f ) GFPAspergillus conidia , incubated overnight at room temperature , were untreated ( a - c ) or exposed ( d - f ) to 10 ng ml − 1 IL - 17A for 4 h followed by anti - IL - 17A - PE . Cells were green in ( a , b ) , red upon staining with anti - IL17A - PE ( b , e ) and double stained in the merged images ( c , f ) . Scale bars , 10 μm . ( g , h ) IL - 17A promotes biofilm formation in A . fumigatus AF293 strain . SEM images refer to cells untreated ( g ) or pretreated ( h ) with 10 ng ml − 1 IL - 17A for 4 h and subsequently cultured onto plastic discs for 24 h . Scale bars , 20 μm . ( i - k ) IL - 17A induces autophagy in Aspergillus . Representative electron micrographs of vacuoles ( v ) in A . fumigatus AF293 unexposed ( k ) or exposed to 10 ng ml − 1 IL - 17A ( j ) or 15 nM rapamycin ( i ) for 4 h before TEM . Scale bars , 500 nm . ( l ) IL - 17A binds the Aspergillus Crf1p protein . Dose - dependent binding of IL - 17A to Crf1p ( red circle ) , Gel1p ( light blue circle ) or the Aspergillus - derived polysaccharides β - 1 , 3 - D - glucan ( blue circle ) , α - 1 , 3 - D - glucan ( open circle ) and galactomannan ( grey circle ) . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay ( ELISA ) on microtitre - coated plates . Data are mean ± s . d . ( n = 5 ) . P - values ranged from 0 . 01 to 0 . 001 for IL - 17A - treated versus untreated ( 0 ) Crf1p ( one - way analysis of variance , Bonferroni post - test ) . ( m ) ELISA assay showing failure of IL - 17A to bind the Aspergillus mutant crf1Δ ( blue square ) as opposed to the wild - type strain AF293 ( red square ) . Data are mean ± s . d . ( n = 5 ) . P - values ranged from 0 . 05 to 0 . 01 , wild - type versus mutant strain exposed to 1 or 10 ng ml − 1 IL - 17A ( one - way analysis of variance , Bonferroni post - test ) . ( n - q ) IL - 17A promotes filamentation in the mutant crf1Δ ( o ) but not the wild - type AF293 ( q ) strain after 24 - h exposure in liquid culture . Scale bars , 500 μm . ( r , s ) C57BL / 6 mice were infected intranasally with the Aspergillus crf1Δ mutant ( r ) or the AF293 ( s ) strain and monitored for fungal growth ( log10 colony - forming units ( CFU ) ± s . d . ) in the lungs 2 days later . Fungal cells were exposed or not ( none ) to 10 ng ml − 1 IL - 17A for 4 h , extensively washed and injected . Data are mean ± s . d . ( n = 4 ) . The actual P - values are indicated ( two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f7(a-f) GFPAspergillus conidia, incubated overnight at room temperature, were untreated (a-c) or exposed (d-f) to 10 ng ml−1 IL-17A for 4 h followed by anti-IL-17A-PE. Cells were green in (a,b), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Scale bars, 10 μm.(g,h) IL-17A promotes biofilm formation in A. fumigatus AF293 strain. SEM images refer to cells untreated (g) or pretreated (h) with 10 ng ml−1 IL-17A for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 20 μm.(i-k) IL-17A induces autophagy in Aspergillus. Representative electron micrographs of vacuoles (v) in A. fumigatus AF293 unexposed (k) or exposed to 10 ng ml−1 IL-17A (j) or 15 nM rapamycin (i) for 4 h before TEM. Scale bars, 500 nm.(l) IL-17A binds the Aspergillus Crf1p protein. Dose-dependent binding of IL-17A to Crf1p (red circle), Gel1p (light blue circle) or the Aspergillus-derived polysaccharides β-1,3-D-glucan (blue circle), α-1,3-D-glucan (open circle) and galactomannan (grey circle). The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay (ELISA) on microtitre-coated plates. Data are mean±s.d. (n=5). P-values ranged from 0.01 to 0.001 for IL-17A-treated versus untreated (0) Crf1p (one-way analysis of variance, Bonferroni post-test).(m) ELISA assay showing failure of IL-17A to bind the Aspergillus mutant crf1Δ (blue square) as opposed to the wild-type strain AF293 (red square). Data are mean±s.d. (n =5). P-values ranged from 0.05 to 0.01, wild-type versus mutant strain exposed to 1 or 10 ng ml−1 IL-17A (one-way analysis of variance, Bonferroni post-test).(n-q) IL-17A promotes filamentation in the mutant crf1Δ (o) but not the wild-type AF293 (q) strain after 24-h exposure in liquid culture. Scale bars, 500 μm.(r,s) C57BL/6 mice were infected intranasally with the Aspergillus crf1Δ mutant (r) or the AF293 (s) strain and monitored for fungal growth (log10 colony-forming units (CFU)±s.d.) in the lungs 2 days later. Fungal cells were exposed or not (none) to 10 ng ml−1 IL-17A for 4 h, extensively washed and injected. Data are mean±s.d. (n=4). The actual P-values are indicated (two-tailed t-test).


Figure 1Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L + W133V DHFR strains after being grown at 30 ° C , 37 ° C , or 42 ° C ( I91L + W133V was grown at 40 ° C ) in M9 medium supplemented with amino acids for 4 hours ( see Methods ) . Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42 ° C ( for WT , W133V and V75H + I155A strains ) or at 40 ° C ( for I91L + W133V and V75H + I91L + I155A ) in M9 medium supplemented with amino acids . For the boxplots in panels , F the central band represents the median of the distribution , the box ends represent the 25th and 75th percentile , the whiskers represent the 10th and 90th percentile , while the dots represent the 5th and 95th percentile . Data was usually obtained from 2 - 3 biological replicates . The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 ( please refer to Figure 1 source data for exact number of cells for each dataset ) . See related Figure EV1B .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L+W133V DHFR strains after being grown at 30°C, 37°C, or 42°C (I91L+W133V was grown at 40°C) in M9 medium supplemented with amino acids for 4 hours (see Methods).Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42°C (for WT, W133V and V75H+I155A strains) or at 40°C (for I91L+W133V and V75H+I91L+I155A) in M9 medium supplemented with amino acids. For the boxplots in panels , F the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B.


Figure 2 ( A ) and ( B ) shows abundance of selected nucleotides and amino acids for mutants I91L + W133V and W133V as well as WT strain treated with 0 . 5μg / ml of Tmp ( Trimethoprim ) after 4 hours of growth at 40 / 42 ° C in M9 minimal medium ( condition of no filamentation ) , Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions . In minimal medium ( B ) Methionine levels are extremely low Levels of purines ( IMP , AMP ) are also largely rescued with amino acid supplementation , however dTMP , dTDP and dTTP levels remain extremely low . Error bars represent SEM of at least three biological replicates ( see Methods ) . The dashed line in each plot represents value of 1 for WT .

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Figure 2(A) and (B) shows abundance of selected nucleotides and amino acids for mutants I91L+W133V and W133V as well as WT strain treated with 0.5μg/ml of Tmp (Trimethoprim) after 4 hours of growth at 40/42°C in M9 minimal medium (condition of no filamentation), Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions. In minimal medium (B) Methionine levels are extremely low Levels of purines (IMP, AMP) are also largely rescued with amino acid supplementation, however dTMP, dTDP and dTTP levels remain extremely low. Error bars represent SEM of at least three biological replicates (see Methods). The dashed line in each plot represents value of 1 for WT.


Figure 3 ( A ) recA ( B ) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP . WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37 ° C , while WT treated with different concentrations of Tmp were grown for 4 hours at 42 ° C . Brown bars ( M9 + AA ) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes . On the other hand , conditions with loss of filamentation ( with dTMP or no supplementation ) show much less expression . Error bars represent SD of 2 - 3 biological replicates ( see Methods ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) recA (B) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP. WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37°C, while WT treated with different concentrations of Tmp were grown for 4 hours at 42°C. Brown bars (M9+AA) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes. On the other hand, conditions with loss of filamentation (with dTMP or no supplementation) show much less expression. Error bars represent SD of 2-3 biological replicates (see Methods).


Figure 4WT cells were treated with different concentrations of Tmp at 42 ° C for varying amounts of time in amino acid supplemented M9 medium ( panel B ) or in M9 media supplemented with both amino acids and GMP ( panel C ) , following which they were spotted on M9 + AA plates and allowed to grow at 30 ° C . Colonies were counted next day . In the presence of only amino acids , there was no loss in viability for any concentration of Tmp ( panel B ) , despite extensive filamentation ( Figure EV1E ) . In contrast , in the presence of amino acids and GMP , the cells showed sharp loss in viability when grown at high Tmp concentrations . In both panels , error bars represent SD of two biological replicates . The dashed line represents cfu at one - hour timepoint . Expression of SOS response genes recA , recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids ( black bars ) or in the presence of both amino acids and GMP ( gray bars ) at 42 ° C ( I91L + W133V was grown at 40 ° C ) . Error bars represent SD of 2 - 3 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4WT cells were treated with different concentrations of Tmp at 42°C for varying amounts of time in amino acid supplemented M9 medium (panel B) or in M9 media supplemented with both amino acids and GMP (panel C), following which they were spotted on M9+AA plates and allowed to grow at 30°C. Colonies were counted next day. In the presence of only amino acids, there was no loss in viability for any concentration of Tmp (panel B), despite extensive filamentation (Figure EV1E). In contrast, in the presence of amino acids and GMP, the cells showed sharp loss in viability when grown at high Tmp concentrations. In both panels, error bars represent SD of two biological replicates. The dashed line represents cfu at one-hour timepoint.Expression of SOS response genes recA, recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids (black bars) or in the presence of both amino acids and GMP (gray bars) at 42°C (I91L+W133V was grown at 40°C). Error bars represent SD of 2-3 biological replicates.


Figure 5Comparison of growth rates of WT and mutant DHFR strains at 42 ° C ( 40 ° C for I91L + W133V and V75H + I91L + I155A ) in minimal medium that is supplemented with amino acids and / or 1mM dTMP . Except for W133V and to a lesser extent for V75H + I155A , the effect of dTMP on growth rates is only modest . Error bars represent SEM of 3 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Comparison of growth rates of WT and mutant DHFR strains at 42°C (40°C for I91L+W133V and V75H+I91L+I155A) in minimal medium that is supplemented with amino acids and/or 1mM dTMP. Except for W133V and to a lesser extent for V75H+I155A, the effect of dTMP on growth rates is only modest. Error bars represent SEM of 3 biological replicates.


Figure 6in vitro activity of purified Thymidylate Kinase ( Tmk ) as a function of dTMP concentration shows Michaelis - Menten ( MM ) like kinetics . ATP concentration is saturating at 1mM . The KM of dTMP is 13μM . The error bars represent SD of three technical replicates . The green points were acquired during growth of WT , W133V and I91L + W133V mutants in the presence of different concentrations of thymidine , which follow the red curve . The inset plot shows the same graph with selected datapoints for I91L + W133V mutant . The cyan circle shows I91L + W133V mutant in the absence of any metabolite supplementation , while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6in vitro activity of purified Thymidylate Kinase (Tmk) as a function of dTMP concentration shows Michaelis-Menten (MM) like kinetics. ATP concentration is saturating at 1mM. The KM of dTMP is 13μM. The error bars represent SD of three technical replicates.The green points were acquired during growth of WT, W133V and I91L+W133V mutants in the presence of different concentrations of thymidine, which follow the red curve. The inset plot shows the same graph with selected datapoints for I91L+W133V mutant. The cyan circle shows I91L+W133V mutant in the absence of any metabolite supplementation, while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively.


Figure 7Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for ( A ) Adenylate Kinase ( AK ) ( B ) Uridylate Kinase ( PyrH ) ( C ) Adenylosuccinate Lyase ( PurB ) and ( D ) GAR Transformylase ( PurN ) . AK , PyrH and PurB appear to follow hyperbolic MM kinetics in vivo , while PurN shows Hill - like dependence with cooperativity . In panel ( A ) , the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK ( Adkar et al . , 2019 ) as well as DHFR mutants used in the present study , while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP ( Adkar et al . , 2019 ) . Data points for all other panels ( B - D ) are derived from mutant DHFR strains in the present study . Since SAICAR , AICAR , GAR and FGAR levels were undetectable in WT , all data points in ( C - D ) are relative to those observed in I91L + W133V mutant following 4 hours of growth at 40 ° C . Data points in all panels represent metabolite levels for individual biological replicates without averaging .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for (A) Adenylate Kinase (AK) (B) Uridylate Kinase (PyrH) (C) Adenylosuccinate Lyase (PurB) and (D) GAR Transformylase (PurN). AK, PyrH and PurB appear to follow hyperbolic MM kinetics in vivo, while PurN shows Hill-like dependence with cooperativity. In panel (A), the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK (Adkar et al., 2019) as well as DHFR mutants used in the present study, while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP (Adkar et al., 2019). Data points for all other panels (B-D) are derived from mutant DHFR strains in the present study. Since SAICAR, AICAR, GAR and FGAR levels were undetectable in WT, all data points in (C-D) are relative to those observed in I91L+W133V mutant following 4 hours of growth at 40°C. Data points in all panels represent metabolite levels for individual biological replicates without averaging.


Figure 2 ( A ) Motility assay of C . c . ΔpseI cells expressing predicted Leg or Pse synthases ( LegI or PseI , in yellow or red , respectively ) from Plac on pSRK - Gm . Overnight cultures were spotted on PYE soft ( 0 . 3 % ) agar plates with gentamycin and IPTG ( 0 . 5 mM ) and incubated for 3 days at 30 ° C . Only predicted PseI - like synthase coding sequences ( CDSs ) restore motility of the C . c . ΔpseI cells . Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C . jejuni . Motility assays are representative of three independent experiments . ( B ) Immunoblot of cell extracts from cells in ( A ) probed with antibodies to C . crescentus FljK ( FljKCc ) . The migration of flagellins in ΔpseI cells is shifted towards lower molecular mass suggesting that post - translational modification of flagellin is defective in ΔpseI mutant . Molecular sizes are indicated by the blues lines ( in kDa ) . Bs : Brevundimonas subvibrioides , Bl : Brevundimonas lutea , Bv : Brevundimonas viscosa , Dv : Dermabacter vaginalis , Li : Leptospira interrogans , Tp : Treponema pallidum , Td : Treponema denticola , AbACICU : Acinetobacter baumannii ACICU , AbLUH : Acinetobacter baumannii LUH , AbLAC : Acinetobacter baumannii LAC - 4 , Lp : Legionella pneumophila , PaY82 : Pseudomonas aeruginosa Y82 , PaPAPS475 : Pseudomonas aeruginosa PAPS475 , Sj : Shewanella japonicum , Basu : Bacillus subtilis , Ks : Kurthia sibirica , Cb : Clostridium botulinum , Gk : Geobacillus kaustophilus , Mh : Moorella humiferrea , Myco : Mycobacterium sp . KS0706 , HrrPV6 : Halorubrum sp . PV6 , Ms : Methanobrevibacter smithii . All the genes come from synthetic fragments codon optimized for E . coli ( except the synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Yellow boxes indicate PseI functional orthologs . Immunoblots are representative of at least two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Motility assay of C.c. ΔpseI cells expressing predicted Leg or Pse synthases (LegI or PseI, in yellow or red, respectively) from Plac on pSRK-Gm. Overnight cultures were spotted on PYE soft (0.3%) agar plates with gentamycin and IPTG (0.5 mM) and incubated for 3 days at 30°C. Only predicted PseI-like synthase coding sequences (CDSs) restore motility of the C.c. ΔpseI cells. Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C. jejuni. Motility assays are representative of three independent experiments.(B) Immunoblot of cell extracts from cells in (A) probed with antibodies to C. crescentus FljK (FljKCc). The migration of flagellins in ΔpseI cells is shifted towards lower molecular mass suggesting that post-translational modification of flagellin is defective in ΔpseI mutant. Molecular sizes are indicated by the blues lines (in kDa). Bs: Brevundimonas subvibrioides, Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Yellow boxes indicate PseI functional orthologs. Immunoblots are representative of at least two independent experiments.


Figure 3 ( A ) and ( B ) Motility assays of WT , ΔlegI ( A ) and ΔflmG ( B ) B . s . cells . Overnight cultures of WT and mutants harboring the empty pSRK - Gm vector ( + Plac ) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG ( 0 . 5 mM ) and incubated for 7 days at 30 ° C . Motility assays are representative of three independent experiments . The same original picture was used to generate Figs 3B and EV2C . ( C ) and ( D ) Immunoblots of cell extracts ( cells ) and supernatants ( SN ) of cultures from WT and mutant cells probed with antibodies to FljKCc , that detect the B . s . flagellins . The estimated molecular mass ( in kDa ) are indicated by the blue lines on the left . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of at least two independent experiments . ( F ) Flagellum length measurements determined by TEM of WT and mutants B . s . cells : WT , ΔlegX ( ΔBresu _ 3267 ) , ΔlegB ( ΔBresu _ 3266 ) expressing legX in trans from Plac on a plasmid , ΔlegH ( ΔBresu _ 0506 ) , ΔlegI ( ΔBresu _ 0507 ) , ΔflmG ( ΔBresu _ 2406 ) and ΔlegI ΔflmG . Box plots represent the distribution of the flagellum lengths . The cross indicates the average length , the central band indicates the median , the upper and lower sides of the box indicate the upper and lower quartiles , respectively , and the whiskers indicate the minimum and the maximum length measured . Twenty - five independent flagella were measured different cells ( biological replicates ) for each strain from TEM images .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) and (B) Motility assays of WT, ΔlegI (A) and ΔflmG (B) B.s. cells. Overnight cultures of WT and mutants harboring the empty pSRK-Gm vector (+Plac) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG (0.5 mM) and incubated for 7 days at 30°C. Motility assays are representative of three independent experiments. The same original picture was used to generate Figs 3B and EV2C.(C) and (D) Immunoblots of cell extracts (cells) and supernatants (SN) of cultures from WT and mutant cells probed with antibodies to FljKCc, that detect the B.s. flagellins. The estimated molecular mass (in kDa) are indicated by the blue lines on the left. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.(F) Flagellum length measurements determined by TEM of WT and mutants B.s. cells: WT, ΔlegX (ΔBresu_3267), ΔlegB (ΔBresu_3266) expressing legX in trans from Plac on a plasmid, ΔlegH (ΔBresu_0506), ΔlegI (ΔBresu_0507), ΔflmG (ΔBresu_2406) and ΔlegI ΔflmG. Box plots represent the distribution of the flagellum lengths. The cross indicates the average length, the central band indicates the median, the upper and lower sides of the box indicate the upper and lower quartiles, respectively, and the whiskers indicate the minimum and the maximum length measured. Twenty-five independent flagella were measured different cells (biological replicates) for each strain from TEM images.


Figure 4 ( A ) Motility assays of B . s . ΔlegI cells complemented with LegI - type ( yellow ) or PseI - type ( red ) synthases expressed from Plac on pSRK - Gm . Motility assays are representative of three independent experiments . ( B ) Immunoblots probed with antibodies to FljKCc , revealing the cell extracts levels of flagellin in B . s . ΔlegI derivatives shown in ( A ) . The blue lines represent the migration of the molecular size standards ( in kDa ) . Bl : Brevundimonas lutea , Bv : Brevundimonas viscosa , Dv : Dermabacter vaginalis , Li : Leptospira interrogans , Tp : Treponema pallidum , Td : Treponema denticola , AbACICU : Acinetobacter baumannii ACICU , AbLUH : Acinetobacter baumannii LUH , AbLAC : Acinetobacter baumannii LAC - 4 , Lp : Legionella pneumophila , PaY82 : Pseudomonas aeruginosa Y82 , PaPAPS475 : Pseudomonas aeruginosa PAPS475 , Sj : Shewanella japonicum , Basu : Bacillus subtilis , Ks : Kurthia sibirica , Cb : Clostridium botulinum , Gk : Geobacillus kaustophilus , Mh : Moorella humiferrea , Myco : Mycobacterium sp . KS0706 , HrrPV6 : Halorubrum sp . PV6 , Ms : Methanobrevibacter smithii . All the genes come from synthetic fragments codon optimized for E . coli ( except the synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Red boxes indicate LegI functional orthologs . Immunoblots are representative of at least two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Motility assays of B.s. ΔlegI cells complemented with LegI-type (yellow) or PseI-type (red) synthases expressed from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.(B) Immunoblots probed with antibodies to FljKCc, revealing the cell extracts levels of flagellin in B.s. ΔlegI derivatives shown in (A). The blue lines represent the migration of the molecular size standards (in kDa). Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Red boxes indicate LegI functional orthologs. Immunoblots are representative of at least two independent experiments.


Figure 5 ( A ) Motility assays of WT B . s . and ΔBresu _ 3266 ( ΔlegB ) cells expressing Bresu _ 3266 ( LegB ) , Bresu _ 3267 ( LegX ) or Bresu _ 3266 - Bresu _ 3267 ( LegB - LegX , Bresu _ 3266 - 67 ) from Plac on plasmid pSRK - Gm . Motility assays are representative of three independent experiments . ( D ) Motility assay of WT B . s . and ΔBresu _ 0506 ( ΔlegH ) cells complemented with a plasmid expressing Bresu _ 0506 ( LegH ) from Plac on pSRK - Gm . Motility assays are representative of three independent experiments . ( B ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in cell extracts ( cells ) and supernatants ( SN ) of the strains described in ( A ) . ( E ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in cell extracts ( cells ) and supernatants ( SN ) of the strains described in ( D ) . Immunoblots are representative of two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Motility assays of WT B.s. and ΔBresu_3266 (ΔlegB) cells expressing Bresu_3266 (LegB), Bresu_3267 (LegX) or Bresu_3266-Bresu_3267 (LegB-LegX, Bresu_3266-67) from Plac on plasmid pSRK-Gm. Motility assays are representative of three independent experiments. (D) Motility assay of WT B.s. and ΔBresu_0506 (ΔlegH) cells complemented with a plasmid expressing Bresu_0506 (LegH) from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.(B) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (A). (E) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (D). Immunoblots are representative of two independent experiments.


Figure 6 ( A ) Motility assay of ΔBresu _ 3267 ( ΔlegX ) B . s . cells compared to WT B . s . cells harbouring the empty pSRK - Gm vector ( + Plac ) or a complementing derivative with PtmE / LegX orthologs from C . jejuni ( Cj ) , Tanerella forsythia ( Tf ) or P . aeruginosa NMI1897 ( PaNMI ) . Motility assays are representative of three independent experiments . ( B ) Immunoblots probed with anti - FljKCc antibodies from cell lysates ( cells ) and supernatants ( SN ) of WT B . s . and ΔBresu _ 3267 ( ΔlegX ) cultures harbouring either the empty pSRK - Gm vector ( + Plac ) or a complementing plasmid expressing different PtmE / LegX orthologs from C . jejuni ( Cj ) , Tanerella forsythia ( Tf ) or P . aeruginosa NMI1897 ( PaNMI ) . Molecular size standards are indicated by the blue lines with the corresponding value in kDa . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Motility assay of ΔBresu_3267 (ΔlegX) B.s. cells compared to WT B.s. cells harbouring the empty pSRK-Gm vector (+Plac) or a complementing derivative with PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Motility assays are representative of three independent experiments.(B) Immunoblots probed with anti-FljKCc antibodies from cell lysates (cells) and supernatants (SN) of WT B.s. and ΔBresu_3267 (ΔlegX) cultures harbouring either the empty pSRK-Gm vector (+Plac) or a complementing plasmid expressing different PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Molecular size standards are indicated by the blue lines with the corresponding value in kDa. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of two independent experiments.


Figure 7 ( B ) Anti - FljKCc immunoblot analyses of whole cell lysates ( cells ) from C . crescentus ( C . c . ) mutant cultures expressing fljKBssyn ( codon optimized for E . coli ) and flmGBs from the replicative pMT463 plasmid , in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . In the presence of pXGFP4 - legBs ( integrated at the xylX locus ) and pMT375 - Bresu _ 3267 , FljKBssyn migration is shifted toward higher molecular mass , indicative of glycosylation . Note that in the C . c . Δfljx6 background , the six flagellin - encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn . The legBs synthetic operon ( in red ) is composed of Bresu _ 3266 , Bresu _ 0765 , Bresu _ 0506 , Bresu _ 3264 , Bresu _ 0507 ( legI ) and Bresu _ 3265 . Molecular masses are indicated in kDa by the blue lines . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg . Immunoblots are representative of at least two independent experiments . ( C ) Anti - FljKCc immunoblot analyses of whole cell lysates from C . crescentus mutant cultures expressing FlmGCc , FlmGBs and FlmGCc - Bschim chimera from the replicative pSRK - Gm plasmid ( Plac ) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . Molecular masses are indicated in kDa by the blue lines . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of at least two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B) Anti-FljKCc immunoblot analyses of whole cell lysates (cells) from C. crescentus (C.c.) mutant cultures expressing fljKBssyn (codon optimized for E. coli) and flmGBs from the replicative pMT463 plasmid, in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). In the presence of pXGFP4-legBs (integrated at the xylX locus) and pMT375-Bresu_3267, FljKBssyn migration is shifted toward higher molecular mass, indicative of glycosylation. Note that in the C.c. Δfljx6 background, the six flagellin-encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn. The legBs synthetic operon (in red) is composed of Bresu_3266, Bresu_0765, Bresu_0506, Bresu_3264, Bresu_0507 (legI) and Bresu_3265. Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg. Immunoblots are representative of at least two independent experiments.(C) Anti-FljKCc immunoblot analyses of whole cell lysates from C. crescentus mutant cultures expressing FlmGCc, FlmGBs and FlmGCc-Bschim chimera from the replicative pSRK-Gm plasmid (Plac) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.


Figure 1Three independent samples ( Exp 1 to 3 ) of EV secreted by labeled 3T3 - F442A cells were analyzed by mass spectrometry ( in duplicate injections , Inj1 / 2 ) . The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Three independent samples (Exp 1 to 3) of EV secreted by labeled 3T3-F442A cells were analyzed by mass spectrometry (in duplicate injections, Inj1/2). The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated.