– Salmonella modulates the expression of SIRT1 and SIRT3 along its course of infection

A-B. Expression studies of SIRT1 and SIRT3 through qPCR in RAW 264.7 macrophages. Data is representative of N=4, n=2. Unpaired two tailed Student’s t test was performed to obtain the p values. (****p<0.0001,***p < 0.001, ** p<0.01, *p<0.05)

C-D-Expression studies of SIRT1 and SIRT3 through qPCR in peritoneal macrophages derived from C57BL/6. Data is representative of N=3, n=2. Unpaired two tailed Student’s t test was performed to obtain the p values. (****p<0.0001,***p < 0.001, ** p<0.01, *p<0.05)

E. Representative confocal images of RAW264.7 macrophages exhibiting SIRT1 expression upon S. Typhimurium infection at indicated time points post infection. Data is representative of N=3, n=80 (microscopic field).

F. Quantitative representation of the expression profile as depicted in the confocal images (E) in terms of Mean Fluorescence Intensity (MFI). Unpaired two tailed Student’s t test was performed to obtain the p values. (****p<0.0001,***p < 0.001, ** p<0.01)

G. Representative confocal images of RAW264.7 macrophages exhibiting SIRT3 expression upon S. Typhimurium infection at indicated time points post infection. Data is representative of N=3, n=80 (microscopic field).

H. Quantitative representation of the expression profile as depicted in the confocal images (G) in terms of Mean Fluorescence Intensity (MFI). Unpaired two tailed Student’s t test was performed to obtain the p values. (****p<0.0001, *** p < 0.001, ** p<0.01)

I. qPCR mediated expression of SIRT1 in RAW264.7 macrophages upon infection with wildtype S. Typhimurium or SPI-1 (ΔinvC) or SPI-2 (ΔssaV and ΔsteE) mutants of S. Typhimurium. Data is representative of N=3,n=3. Unpaired two tailed Student’s t test was performed to obtain the p values. (****p<0.0001,***p < 0.001, ** p<0.01)

J. qPCR mediated expression of SIRT3 in RAW264.7 macrophages upon infection with wildtype S. Typhimurium or SPI-1 (ΔinvC) or SPI-2 (ΔssaV and ΔsteE) mutants of S. Typhimurium. Data is representative of N=3, n=3. Unpaired two tailed Student’s t test was performed to obtain the p values. (****p<0.0001,***p < 0.001, ** p<0.01)

– Effect of SIRT1 and SIRT3 knockdown in intracellular bacterial proliferation within RAW 264.7 and primary murine macrophages.

A. Fold Proliferation of Salmonella Typhimurium within RAW 264.7 macrophages in transfected and un-transfected conditions. Data is representative of N=3, n=3. Unpaired two tailed Student’s t test was performed to obtain the p values. (** p<0.01, * p<0.05)

B. Fold Proliferation of Salmonella Typhimurium within infected peritoneal macrophages isolated from adult male C57BL/6 mice upon SIRT1 (EX-527) or SIRT3 (3TYP) inhibitor treatment. Unpaired two tailed Student’s t test was performed to obtain the p values. (** p<0.01, * p<0.05)

– Salmonella Typhimurium skews the polarization state of the macrophage toward an immunosuppressive M2 state along the course of infection

A. nanoString gene expression profiling data of S. Typhimurium infected RAW 264.7 macrophages versus uninfected control data sets at 2hr, 6hr and 16hr time points of infection. Data is representative of N=2,n=2.

B. Quantitative representation of flow cytometric analysis of alteration in M1 CD86 positive population in S. Typhimurium (STM) infected samples in comparison to uninfected (UI) and Paraformaldehyde Fixed (PFA) bacteria at the indicated time post-infection. Data is representative of N=2, n=3. Two-way ANOVA and Bonferroni post-t-test was used to obtain p values. (*** p < 0.001)

C. Quantitative representation of flow cytometric analysis of M2 surface marker CD206 in S. Typhimurium (STM) infected SIRT1 or SIRT3 knockdown RAW264.7 macrophages in comparison to the scrambled control at the indicated time post-infection. UI-Uninfected sample. Data is representative of N=3, n=3. Two-way ANOVA and Bonferroni post-t-test was used to obtain p values. (*** p < 0.001)

– SIRT1 mediates modulation of immune functions via deacetylation of p65 subunit of NF-κB in S.Typhimurium infected macrophages.

A. Immunoblot depicting p65 NF-κB interaction with SIRT1 post immunoprecipitation of SIRT1 in uninfected (UI) or S. Typhimurium (STM) infected RAW264.7 macrophages at 16hr post-infection. Data is representative of N=3, n=1

B. An immunoblot depicting p65 NF-κB interaction with SIRT1 as well as the p65 NF-κB acetylation status post immunoprecipitation of p65 (IP: p65) or with control IgG (IP: IgG) in uninfected (UI) or S. Typhimurium (STM) infected RAW264.7 macrophages upon knockdown with SIRT1 shRNA or scrambled control.

C. Densitometric plot depicting the band intensities of Acetylated p65 over total p65 in blot B.

D. An immunoblot depicting p65 NF-κB interaction with SIRT1 as well as the p65 NF-κB acetylation status post immunoprecipitation of p65 (IP: p65) or with control IgG (IP: IgG) in uninfected (UI) or S. Typhimurium (STM) infected RAW264.7 macrophages upon SIRT1 inhibitor (EX-527, 1µM) treatment at 16hr post-infection. UT-Untreated.

E. Densitometric plot depicting the band intensities of Acetylated p65 subunit of NF-κ B over total p65 NF-κB in blot D.

– Salmonella Typhimurium drives the metabolism of the infected macrophage toward fatty acid oxidation

A. Metabolic gene expression data of S. Typhimurium infected RAW 264.7 macrophages at 2hr, 6hr and 16hr time points of infection through nanoString. Data is representative of N=2, n=1.

B. PPARδ qPCR expression data in S. Typhimurium infected RAW 264.7 macrophages at the indicated time points of infection. Data is representative of N=3, n=2.

C. Lactate estimation assay of S.Typhimurium infected RAW264.7 macrophages at the initial time point of 2hr and at the late time point of 16hr post infection. Data is representative of N=3, n=3. Unpaired two-tailed Student’s t test was performed to obtain the p values. (** p<0.01, * p<0.05)

D-E-Lactate estimation assay of S.Typhimurium infected RAW264.7 macrophages upon SIRT1 (D) or SIRT3 (E) knockdown condition at16h post-infection. Data is representative of N=3, n=3. Unpaired two-tailed Student’s t test was performed to obtain the p values.

F. Immunoblotting of host glycolytic (PGK), TCA cycle (PDHA1) and fatty acid oxidation (HADHA, ACOX-1) proteins under SIRT1 and SIRT3 knockdown condition of S. Typhimurium infected RAW264.7 macrophages at 16h post-infection.

G. Fatty Acid Oxidation (FAO) Assay of uninfected (UI) and infected (STM) RAW264.7 macrophages under SIRT1 or SIRT3 knockdown or inhibitor treatment. N=2, n=2. (** p<0.01, * p<0.05)

H. Fatty Acid Oxidation (FAO) Assay of uninfected (UI) and infected RAW264.7 macrophages under infection with wildtype S. Typhimurium (STM WT), SPI-1 (ΔinvC) or SPI-2 (ΔssaV and ΔsteE) mutants of S. Typhimurium. Data is representative of N=2,n=2. (** p<0.01, * p<0.05)

– Salmonella Typhimurium infection proceeds with increased glycolysis and glucose uptake inside the infected RAW 264.7 macrophages

A. Salmonella gene expression profiling data of S. Typhimurium infected RAW 264.7 macrophages at 2hr, 6hr and 16hr time points of infection through nanoString. SI-S. Typhimurium infected, PI-PFA fixed S. Typhimurium infected. SPI-1 genes-Inv, PrgH, SPI-2 genes-ssaV,stfF, glk-glucokinase, pfkA-phosphofrucktose kinase A, ptsG-Phosphophenolpyruvate-dependent sugar phosphotransferase system (PTS). Data is representative of N=2, n=2.

B. qRT PCR gene expression profiling of Salmonella metabolic genes within infected RAW264.7 macrophages in knockdown condition of either SIRT1 or SIRT3 at 6hr post infection. Data is representative of N=3, n=3.

C-D. qRT PCR gene expression profiling of Salmonella metabolic genes within infected female C57BL/6 mice spleen (C) and liver (D) under SIRT1 or SIRT3 inhibitor treatment harvested at 5th day post infection of S.Typhimurium (10^7 cfu units/animal). Unpaired two-tailed Student’s t test was performed to obtain the p values. Data is representative of N=3, n=3. (****p<0.0001, *** p < 0.001, ** p<0.01, * p<0.05)

– SIRT1 inhibition triggers hyperacetylation of glycolytic master regulator HIF-1α within S. Typhimurium infected macrophages while SIRT3 skews metabolism of S. Typhimurium-infected macrophages via interaction with PDHA1.

A. An immunoblot depicting HIF-1α interaction with SIRT1 post immunoprecipitation of SIRT1 in uninfected (UI) or S. Typhimurium (STM) infected RAW264.7 macrophages at 16hr post-infection. (Derived from same SIRT1 IP blot as in Fig. 4A)

B. Immunoblotting of SIRT1 in SIRT1 knockdown uninfected (UI) or S. Typhimurium (STM) infected RAW 264.7 cells at 16hr post-infection to assess the acetylation status of HIF-1α.

C. Densitometric plot depicting the band intensities of Acetylated HIF-1α over total HIF-1α in blot B.

D. An immunoblot depicting HIF-1α interaction with SIRT1 as well as the HIF-1α acetylation status post immunoprecipitation of HIF-1α (IP:HIF-1α or with control IgG (IP:IgG) in uninfected (UI) or S. Typhimurium (STM) infected RAW264.7 macrophages upon SIRT1 inhibitor (EX-527, 1µM) treatment at 16hr post-infection. UT-untreated.

E. Densitometric plot depicting the band intensities of Acetylated HIF-1α over total HIF-1α in blot D.

F. Densitometric plot depicting the band intensities of SIRT1 in blot D.

G. Lactate estimation assay of S.Typhimurium infected RAW264.7 macrophages upon SIRT1 and SIRT3 knockdown condition in the presence of HIF-1α inhibitor, chetomine (50nM) at16h post-infection. Data is representative of N=3, n=3. Unpaired two-tailed Student’s t test was performed to obtain the p values. (****p<0.0001, *** p < 0.001, ** p<0.01, * p<0.05)

H. An immunoblot depicting PDHA1 interaction with SIRT1 or SIRT3 post immunoprecipitation of PDHA1 in uninfected (UI) or S. Typhimurium (STM) infected RAW264.7 macrophages in SIRT3 knockdown condition at 16hr post-infection.

I. Densitometric plot depicting the band intensities of SIRT3 interaction over total input in blot

J. An immunoblot depicting PDHA1 interaction with SIRT3 post immunoprecipitation of PDHA1 in uninfected (UI) or S. Typhimurium (STM) infected RAW264.7 macrophages at 16hr post-infection under SIRT3 inhibitor (3-TYP, 1µM) treatment at 16hr post-infection. UT-untreated.

K. Densitometric plot depicting the band intensities of Acetylated PDHA1 over total PDHA1 in blot I.

L. Densitometric plot depicting the band intensities of SIRT3 interaction over total input in blot I.

– Effect of SIRT1 or SIRT3 inhibition on S. Typhimurium infected C57BL/6 mice

A. The schematic representation of the experimental strategy for studying the effect of SIRT1 and SIRT3 on the in vivo pathogenesis of STM (WT).

B. In-vivo organ burden of S. Typhimurium upon SIRT1 or SIRT3 inhibition in C57BL/6 mice at day 5 post-infection under specified dosage of inhibitor treatment. Data is representative of N=4, n=8. Mann-Whitney Test was performed to obtain the p values.(****p<0.0001, *** p < 0.001, ** p<0.01, * p<0.05).

C. Percent survival of S. Typhimurium infected C57BL/6 mice upon SIRT1 or SIRT3 inhibitor treatment at a specific dose of inhibitor treatment. Data is representative of N=4, n=5.

D. Representation of splenic length of S. Typhimurium infected spleen tissue harvested from C57BL/6 mice (males) at day 5 post-infection upon SIRT1 or SIRT3 inhibition at 1mg/kg dosage. Data is representative of N=4, n=8. (** p<0.01, * p<0.05).

E. Bacterial load in blood at different days post-infection upon SIRT1 or SIRT3 inhibition at 1mg/kg dosage in C57BL/6 mice (males). Data is representative of N=3, n>5. (** p<0.01, * p<0.05) Mann-Whitney Test was performed to obtain the p values. (** p<0.01, * p<0.05).

F. Bacterial load in blood at day 5 post-infection upon SIRT1 or SIRT3 inhibition at 1mg/kg dosage in C57BL/6 WT mice (males) and gp91phox-/− (males) mice. Data is representative of N=3, n>5. Mann-Whitney Test was performed to obtain the p values. (** p<0.01, * p<0.05).

G. Serum IL-6 levels of S. Typhimurium infected C57BL/6 WT mice (males) mice treated with SIRT1(EX-527) or SIRT3 (3TYP) inhibitors or SRT1720 (SIRT1 activator) at 1mg/kg dosage at 5th day post-infection. Data is representative of N=3, n>5. Unpaired two-tailed Student’s t test was performed to obtain the p values.(** p<0.01, * p<0.05).

H. Quantitative analysis of percentage population of cells within liver showing DCFDA positive staining shown in Fig. S9, A. Data is representative of N=3, n>5. Unpaired two-tailed Student’s t-test was performed to obtain the p values. (** p < 0.01).

I. Enumeration of GFP positive bacterial cells through flow cytometry in splenic tissues homogenate harvested from adult male 6-8 week old C57BL/6 mice (subjected to different chemical treatment-Vehicle treated or SIRT1 (EX-527) or SIRT3 (3-TYP) inhibitor or SIRT1 activator SRT1720 treated at a dose of 1mg/kg) at 5th day post S. Typhimurium (expressing GFP) infection (10 7 CFU orally). Data is representative of N=3, n>5. Unpaired two-tailed Student’s t test was performed to obtain the p values. (** p < 0.01).

J. Quantitative analysis of the percentage population of splenic cells harboring GFP+ bacterial cells shown in I. Unpaired two-tailed Student’s t test was performed. (** p<0.01, * p<0.05).

K. Enumeration of GFP positive bacterial cells through flow cytometry within F4/80 positive splenic macrophages present within splenic tissues homogenate harvested from adult male 6-8 week old C57BL/6 mice (subjected to different chemical treatment-Vehicle treated or SIRT1 (EX-527) or SIRT3 (3-TYP) inhibitor or SIRT1 activator SRT1720 treated aa t dose of 1mg/kg) at 5th day post S. Typhimurium infection. Data is representative of N=3, n>5.

L. Quantitative analysis of percentage population of F4/80 positive macrophage cells harboring GFP+ bacteria shown in K. Unpaired two-tailed Student’s t test was performed to obtain the p values. Data is representative of N=3, n>5.(* p < 0.05).

M-S. Quantitative analysis of different mCherry-S.Typhimurium-infected splenic populations harboring mCherry+bacteria via flow cytometry depicted in Fig. S8. Unpaired two-tailed Student’s t test was performed. (*** p < 0.001,** p<0.01, * p<0.05).

T. Representative image of haematoxylin-eosin-stained liver sections to assess the liver tissue architecture upon Salmonella infection at 5th days post-infection in different mice cohorts. (UI-Uninfected, STM-S. Typhimurium infected, EX-527-SIRT1 inhibitor, 3TYP-SIRT3 inhibitor, SRT1720-SIRT1 activator, Vehicle Control-(PBS containing 0.1% DMSO). Scale bar-50µm. Scoring system:- according to pathological changes the tissue sections are scored as 0 for normal pathology, 1 for mild/ minor pathology, 2 for moderate pathology, and 3 for severe pathological changes.

U. Graph representing the histopathological scoring of the liver sections depicted in L. Unpaired two-tailed Student’s t test was performed to obtain the p values. (* p < 0.05)(****p<0.0001, *** p < 0.001, ** p<0.01, * p<0.05).

V. In-vivo organ burden of S. Typhimurium upon SIRT1 or SIRT3 adenovirus-mediated in vivo knockdown in C57BL/6 mice at day 5 post-infection. Data is representative of N=3, n>3. Mann-Whitney Test was performed to obtain the p values. (****p<0.0001, *** p < 0.001, ** p<0.01, * p<0.05).

W. Serum IL-6 levels of S. Typhimurium infected C57BL/6 WT mice (males) upon in vivo adenovirus-mediated SIRT1 or SIRT3 knockdown. Data is representative of N=3, n>3. Unpaired two-tailed Student’s t test was performed to obtain the p values. (** p<0.01, * p<0.05).

X. Percent survival of S. Typhimurium infected C57BL/6 mice upon in vivo adenovirus-mediated SIRT1 or SIRT3 knockdown. Data is representative of N=3, n>3.

Y. Representative image of haematoxylin-eosin-stained liver sections upon Salmonella infection at 5th days post-infection in different mice cohorts. (Scrambled STM, shSIRT1 STM, shSIRT3 STM). Scale bar-50µm. Scoring system:- according to pathological changes the tissue sections are scored as 0 for normal pathology, 1 for mild/ minor pathology, 2 for moderate pathology, and 3 for severe pathological changes.

List of strains and plasmids used in this study

List of shRNA used for knockdown

List of primers

List of Antibodies