Microbiology and Infectious Disease

Salmonella-induced SIRT1 and SIRT3 are crucial for maintaining the metabolic switch in bacteria and host for successful pathogenesis

Dipasree Hajra
Raju S. Rajmani
Ayushi Devendrasingh Chaudhary
Shashi Kumar Gupta
Dipshikha Chakravortty author has email address

Department of Microbiology & Cell Biology, Indian Institute of Science
Centre of Infectious Disease Research, Indian Institute of Science, Bangalore, India
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
Adjunct Faculty, School of Biology, Indian Institute of Science Education and Research, Thiruvananthapuram

https://doi.org/10.7554/eLife.93125.1

Open access
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Figures and data

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 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)

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 5^th 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)

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 5^th 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-1^α (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. 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.
H. Densitometric plot depicting the band intensities of SIRT3 interaction over total input in blot G.
I. 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.
J. Densitometric plot depicting the band intensities of Acetylated PDHA1 over total PDHA1 in blot I.
K . Densitometric plot depicting the band intensities of SIRT3 interaction over total input in blot I.

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-1^α (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. 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.
H. Densitometric plot depicting the band intensities of SIRT3 interaction over total input in blot G.
I. 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.
J. Densitometric plot depicting the band intensities of Acetylated PDHA1 over total PDHA1 in blot I.
K . 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. 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. S8, B. 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 5^th day post S. Typhimurium (expressing GFP) infection (10 ⁸ 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 5^th 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. Representative image of haematoxylin-eosin-stained liver sections to assess the liver tissue architecture upon Salmonella infection at 5^th 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.
N. 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).
O. 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).
P. 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).
Q. 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.
R. Representative image of haematoxylin-eosin-stained liver sections upon Salmonella infection at 5^th 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.

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. 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. S8, B. 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 5^th day post S. Typhimurium (expressing GFP) infection (10 ⁸ 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 5^th 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. Representative image of haematoxylin-eosin-stained liver sections to assess the liver tissue architecture upon Salmonella infection at 5^th 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.
N. 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).
O. 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).
P. 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).
Q. 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.
R. Representative image of haematoxylin-eosin-stained liver sections upon Salmonella infection at 5^th 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 primers

List of Antibodies

List of Antibodies

Salmonella modulates the expression of SIRT1 and SIRT3 along its course of infection
A-B. Expression studies of SIRT1 (A) and SIRT3 (B) through immunoblotting in RAW 264.7 macrophages. Data is representative of N=3, n=1.
C-D. Quantification of the densitometric plot showing the fold intensities of SIRT1 (C) and SIRT3 (D) protein expression. 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- SIRT1 and SIRT3 transcript level expression in M1 polarized RAW264.7 macrophages. Data is representative of N=3, n=3. . Un-paired t-test were performed to obtain p-values. (****p<0.0001,***p < 0.001, ** p<0.01, *p<0.05)
F- Expression profile of SIRT1 and SIRT3 in M2 polarized RAW264.7 macrophages. Data is representative of N=3, n=3. Un-paired t-test were performed to obtain p-values. (****p<0.0001,***p < 0.001, ** p<0.01, *p<0.05)
G- Intracellular fold proliferation of S. Typhimurium within non-polarized, M1 and M2 polarized RAW264.7 macrophages. Data is representative of N=3, n=3. Un-paired t-test were performed to obtain p-values. (****p<0.0001,***p < 0.001, ** p<0.01, *p<0.05)
H- Validation of M1 and M2 polarized status of RAW264.7 macrophages by estimation of IL- 6 and IL-10 production via ELISA. Data is representative of N=3, n=3. Un-paired t-test were performed to obtain p-values. (****p<0.0001,***p < 0.001, ** p<0.01, *p<0.05)

Validation of SIRT1 and SIRT3 knockdown in RAW 264.7 macrophages
A. Knockdown confirmation of SIRT1 at transcript level through qPCR.
B. qPCR expression of SIRT3 in order to validate knockdown efficiency.
C. Immunoblotting of SIRT1 for verification of knockdown. Data is representative of N=3, n=1. (Uns=Un-transfected, Scr=Scrambled, Mix=transfection with equimolar concentration of both SIRT1 shRNA construct C4 and C12)
D. SIRT3 knockdown validation through western blotting. Data is representative of N=3, n=1.(Scr=Scrambled, Mix=transfection with equimolar concentration of both SIRT3 shRNA construct E8 and E12)
E. Densitometric analysis of the immunoblot represented in C.
F. Densitometric analysis of the immunoblot represented in D.

SIRT1 and SIRT3 modulates cytokine production in Salmonella infection scenario
A. Estimation of pro-inflammatory IL-6 cytokine production upon S. Typhimurium infection in SIRT1 and SIRT3 knockdown RAW 264.7 macrophages through ELISA. Data is representative of N=2, n=2. Unpaired two tailed Student’s t test was performed to obtain the p values. (** p<0.01, * p<0.05).
B. Estimation of anti-inflammatory IL-10 cytokine production upon S. Typhimurium infection in SIRT1 and SIRT3 knockdown RAW 264.7 macrophages through ELISA. Data is representative of N=2, n=2. Unpaired two tailed Student’s t test was performed to obtain the p values. (*** p < 0.001, * p<0.05)
C. Estimation of pro-inflammatory IL-6 cytokine production within uninfected or S. Typhimurium infected peritoneal macrophages in presence or absence of SIRT1 or SIRT3 inhibitor treatment . Data is representative of N=2, n=2. 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- Flow cytometric analysis of alteration in M1 CD86 (PE) positive population in S. Typhimurium (STM) infected samples in comparison to uninfected (UI) and Paraformaldehyde Fixed (PFA) bacteria at the indicated time post-infection.
B- Flow cytometric analysis of M2 surface marker CD206 (APC) 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.

Salmonella Typhimurium skews the polarization state of the macrophage toward an immunosuppressive M2 state along the course of infection
A- Flow cytometric analysis of alteration in M1 CD86 (PE) positive population in S. Typhimurium (STM) infected samples in comparison to uninfected (UI) and Paraformaldehyde Fixed (PFA) bacteria at the indicated time post-infection.
B- Flow cytometric analysis of M2 surface marker CD206 (APC) 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.

SIRT1 and SIRT3 play a role in maintaining ROS balance within Salmonella infected macrophages.
A. DCFDA fluorescence measured through Flow cytometry in shSIRT1, shSIRT3 or scrambled shRNA transfected and un-transfected RAW 264.7 macrophages at 16hr post S. Typhimurium infection. Data is representative of N=3, n=2.
B. Quantitative analysis of percentage population of DCFDA positive cells shown in Fig.6, A. Unpaired two-tailed Student’s t test was performed. Data is statistically non-significant. (** p<0.01, * p<0.05)
C. Detection of extracellular H2O2 production through Phenol Red Hydrogen peroxidase assay at 610nm absorbance. Two-way ANOVA and Bonferroni post-t-test was used to obtain p values. (** p < 0.01, * p < 0.05, ns-not significant) Data is representative of N=2, n=2. (** p<0.01, * p<0.05)

Effect of SIRT1 and SIRT3 knockdown or inhibition in intracellular bacterial proliferation within RAW 264.7 macrophages or peritoneal macrophages in presence of ROS scavenger N-Acetyl Cysteine (NAC).
A. Fold Proliferation of Salmonella Typhimurium within RAW 264.7 macrophages in transfected and un-transfected conditions in presence or absence of NAC (1mM). Data are 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)
B. Fold Proliferation of Salmonella Typhimurium within peritoneal macrophages isolated from 6-8 week old adult male C57BL/6 mice upon SIRT1 (EX-527, 1µM) or SIRT3 (3TYP, 1µM) inhibitor treatment in presence or absence of NAC (1mM). Data are 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)

SIRT1 and SIRT3 knockdown or inhibitor treatment led to decrease in fatty acid oxidation and promoted host glycolysis in S. Typhimurium infected peritoneal macrophages
A-A-qRT PCR mediated gene expression analysis of several host metabolic genes in SIRT1 or SIRT3 knockdown RAW264.7 macrophages at 6 hr 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<0.05)
B- qRT PCR mediated gene expression analysis of several host metabolic genes in SIRT1 or SIRT3 inhibitor-treated (1µM) peritoneal macrophages isolated from 6-8 week old C57BL/6 male mice liver harvested at 5th day post thioglycolate injection. 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)
C-Lactate estimation assay of S.Typhimurium infected peritoneal macrophages upon SIRT1 or SIRT3 catalytic inhibition 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)
D-Immunoblotting of host glycolytic (PGK, PFK) and fatty acid oxidation (HADHA, ACOX- 1) proteins under SIRT1 (EX-527) and SIRT3 (3TYP) catalytic inhibitor treated condition in S. Typhimurium infected peritoneal macrophages (isolated from thioglycolate treated adult male C57BL/6 mice) at 16h post-infection. Data is representative of N=2, n=1.
E- qRT PCR mediated gene expression profiling of several host metabolic genes in SIRT1 or SIRT3 inhibitor treated 6-8 week old C57BL/6 male mice liver harvested at 5-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)
F-qRT PCR mediated gene expression profiling of several host metabolic genes in SIRT1 or SIRT3 inhibitor treated 6-8 week old C57BL/6 male mice spleen harvested at 5 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 and SIRT3 knockdown or inhibitor treatment led to decrease in fatty acid oxidation and promoted host glycolysis in S. Typhimurium infected peritoneal macrophages
A-A-qRT PCR mediated gene expression analysis of several host metabolic genes in SIRT1 or SIRT3 knockdown RAW264.7 macrophages at 6 hr 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<0.05)
B- qRT PCR mediated gene expression analysis of several host metabolic genes in SIRT1 or SIRT3 inhibitor-treated (1µM) peritoneal macrophages isolated from 6-8 week old C57BL/6 male mice liver harvested at 5th day post thioglycolate injection. 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)
C-Lactate estimation assay of S.Typhimurium infected peritoneal macrophages upon SIRT1 or SIRT3 catalytic inhibition 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)
D-Immunoblotting of host glycolytic (PGK, PFK) and fatty acid oxidation (HADHA, ACOX- 1) proteins under SIRT1 (EX-527) and SIRT3 (3TYP) catalytic inhibitor treated condition in S. Typhimurium infected peritoneal macrophages (isolated from thioglycolate treated adult male C57BL/6 mice) at 16h post-infection. Data is representative of N=2, n=1.
E- qRT PCR mediated gene expression profiling of several host metabolic genes in SIRT1 or SIRT3 inhibitor treated 6-8 week old C57BL/6 male mice liver harvested at 5-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)
F-qRT PCR mediated gene expression profiling of several host metabolic genes in SIRT1 or SIRT3 inhibitor treated 6-8 week old C57BL/6 male mice spleen harvested at 5 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 and SIRT3 inhibition led to enhanced ROS production within Salmonella-infected mice liver and spleen tissues.
A-DCFDA fluorescence measured through flow cytometry in liver 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 dose of 1mg/kg) at 5^th day post S. Typhimurium infection (10⁸ CFU orally). Data is representative of N=3, n=2.
B- DCFDA fluorescence measured 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 dose of 1mg/kg) at 5^th day post S. Typhimurium infection. Data is representative of N=3, n=2.
C- Quantitative analysis of percentage population of splenocytes showing DCFDA positive staining shown in C. Unpaired two-tailed Student’s t test was performed to obtain the p values. (** p < 0.01)

SIRT1 and SIRT3 inhibition led to enhanced ROS production within Salmonella-infected mice liver and spleen tissues.
A-DCFDA fluorescence measured through flow cytometry in liver 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 dose of 1mg/kg) at 5^th day post S. Typhimurium infection (10⁸ CFU orally). Data is representative of N=3, n=2.
B- DCFDA fluorescence measured 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 dose of 1mg/kg) at 5^th day post S. Typhimurium infection. Data is representative of N=3, n=2.
C- Quantitative analysis of percentage population of splenocytes showing DCFDA positive staining shown in C. Unpaired two-tailed Student’s t test was performed to obtain the p values. (** p < 0.01)

In vivo knockdown validation by Western blotting and qPCR in liver
A-Immunoblotting of SIRT1 and SIRT3 in harvested liver samples upon AAV- mediated in vivo knockdown in mice.
B- qPCR mediated gene expression of SIRT1 in harvested liver samples upon in vivo AAV- mediated gene inhibition in mice. 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)
C-qPCR mediated gene expression of SIRT1 in harvested liver samples upon in vivo AAV- mediated gene inhibition in mice. 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)

Schematic Diagram
In murine macrophages, Salmonella infection drives an immuno-metabolic shift toward an immunosuppressive state with an increase in host fatty acid oxidation rate by modulating SIRT1 and SIRT3. SIRT1 and SIRT3 regulate the acetylation status of NFκB p65 subunit, HIF-1α, PDHA1 thereby influencing host metabolism which in turn impacts the intracellular Salmonella metabolism. Inhibition or knockdown of SIRT1 or SIRT3 triggers increased ROS, pro-inflammatory cytokine burst with a concomitant decline in host fatty acid oxidation and a corresponding increase in bacterial fatty acid oxidation together amounting to decreased intracellular bacterial replication. Contrastingly, in in vivo mice model of infection, SIRT1 and SIRT3 cause increased bacterial dissemination owing to increased ROS and IL-6 production.

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