Introduction

Sirtuins are NAD+-dependent deacetylases that are present in all forms of life. Sirtuins comprise a conserved core catalytic domain that removes acetyl moiety from the lysine residues of proteins in presence of NAD+ as a cofactor [1] giving rise to 2’O-acetyl-ADP-ribose and free nicotinamide as products[2][3]. Free nicotinamide acts as a non-competitive inhibitor of sirtuins[4]. They possess variable N terminal and C terminal domains that confer different subcellular localization, substrate specificity, and functions[5]. Mammals have seven sirtuins that are responsible for regulating various biological functions such as cell survival, apoptosis, oxidative stress, metabolism, and inflammation[6][7]. SIRT1,6 and 7 have nuclear localization, SIRT2 is cytoplasmic and SIRT 3,4 and 5 comprise the mitochondrial SIRTs. In addition to their deacetylase activity, they possess ADP ribosylation (SIRT1, SIRT4, and SIRT6), desuccinylation and demalonylation (SIRT5), delipoylation (SIRT4), and demyristoylation and depalmitoylation (SIRT6) enzymatic activities [8]. Previous studies have shown that SIRT1 gets activated in response to acute immune response and it deacetylates RelA/p65 component of NFκB thereby mediating its proteasomal degradation[9]. On the other hand, it activates RelB component of NFκB pathway. RelB causes heterochromatinization of pro-inflammatory genes like TNF-α and IL-β[10]. SIRT1 activates Peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) mediating a metabolic switch from glycolysis toward fatty acid oxidation. SIRT1-mediated RelB activation, in turn, activates SIRT3 causing the promotion of mitochondrial bioenergetics[11]. PGC-1α, a major player in mitochondrial biogenesis, activates SIRT3 [12] which in turn causes activation of PGC-1α, thereby fuelling a positive feedback loop. SIRT3 accounts for the major mitochondrial deacetylase orchestrating several metabolic processes such as fatty acid oxidation, promotion of the TCA cycle, and inhibition of ROS production[13].

Salmonella enterica serovar Typhimurium is a facultative intracellular Gram-negative enteric pathogen, causing a wide array of infections ranging from self-limiting gastroenteritis to diarrhea in humans [14]. Salmonella enterica serovar Typhi cause systemic infection in humans with typhoidal symptoms. Recent reports reported incidences of 21 million [15] typhoid cases and 93 million of non-typhoidal [16] cases round the year. The virulence of Salmonella is majorly regulated by two pathogenicity islands, namely, SPI-1 and SPI-2. It uses SPI-1 encoded T3SS and the effector proteins to invade host cells [17]. Inside the macrophages, they harbour within the Salmonella containing vacuoles (SCV) by virtue of its SPI-2 effectors [18]. Macrophages, dendritic cells and neutrophils are responsible for successful dissemination throughout the body through the reticulo-endothelial system (RES) [17].

Macrophages, serving as an intracellular niche for Salmonella, exhibit several continua of polarization states. At the two extreme ends of the spectrum lie the classically polarized M1 macrophages and alternatively activated M2 macrophages. M1 macrophages comprise of the proinflammatory antimicrobial state producing IL-1β, IL-6, TNF-α,IL-12, IFN-γ cytokines and exhibit enhanced expression of CD80, CD86 surface markers. The anti-inflammatory M2 macrophages promote bacterial persistence by producing anti-inflammatory cytokines like IL-10,TGF-β and show increased expression of Arg-1, CD206 surface markers[19][20]. To sustain the continuous production of proinflammatory cytokines M1 macrophages rely on glycolysis for their energy requirements. On the other hand, M2 macrophages are fuelled by enhanced oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO)[21].It has been previously reported that sirtuins mediated attuning of metabolism impact polarization of macrophages in vivo. SIRT1 has the ability to promote the polarization of M2 macrophages and inhibit inflammation in macrophages of adipose tissue[22][23][24]. SIRT3 suppresses ROS by deacetylating and activating MnSOD[25].

Several bacteria are known to subvert the host immune system toward an immunosuppressive state. Salmonella or Mycobacterium have evolved mechanisms to counteract the M1 state of the host macrophage. Salmonella Typhimurium uses its SPI-2 effectors to inhibit the recruitment of NADPH oxidase to the SCV, thereby preventing oxidative burst mediated microbicidal activity[26]. Similarly, Mycobacterium bovis bacillus Calmette-Guérin prevents NOS2 recruitment to phagosomes[27]. Salmonella Dublin causes inhibition of the production of pro-inflammatory cytokine like IL-18 and IL-12p70[28]. Moreover, Mycobacteria inhibits NFκB signalling and IFN-γ mediated downstream pathways[29]. Furthermore, Yersinia enterocolitica elicits an M2 response by inducing Arginase-1 expression and TGFβ1 and IL-4production[30]. Yersinia TTSS effector LcrV induces an M2 phenotype supposedly by IL-10 production[31].

Since SIRT1 and SIRT3 are the major modulators of the immuno-metabolic paradigm, we intend to decipher the role of SIRT1 and SIRT3 in influencing host and Salmonella metabolism. This study highlights the role of SIRT1 and SIRT3 in intracellular pathogen survival by promoting Salmonella glycolysis and concomitantly driving host metabolism towards fatty acid oxidation. Additionally, Salmonella trigger an immunosuppressive M2 environment conducive to its intravacuolar proliferation by modulating SIRT1 and SIRT3 levels. Here, we have shown that SIRT1 and SIRT3 knockdown cause decreased M2 surface marker expression such as CD206 along with increased production of pro-inflammatory cytokines and ROS generation, together amounting to attenuated bacterial intracellular proliferation within the infected macrophages. Moreover, SIRT1 mediated p65 NF-κB deacetylation played a vital role in immune function regulation within the Salmonella infected macrophages with increased interaction of SIRT1 with p65 NF-κB. SIRT1 knockdown or inhibition resulted in hyperacetylation of p65 NF-κB thereby leading to enhanced pro-inflammatory response in S. Typhimurium infected macrophages. Further, SIRT1 and SIRT3 knockdown or inhibition skewed the Salmonella-induced host metabolic shift by regulating acetylation status of HIF-1α and PDHA. This caused increased host glycolysis and reduced fatty acid oxidation. However, the Salmonella shows the opposing metabolic profile with increased fatty acid oxidation and reduced glycolysis upon SIRT1 or SIRT3 inhibition. In the contrary to the macrophages, in in vivo mice-model of infection, SIRT1 and SIRT3 inhibition resulted in increased pathogen loads in organs and triggered enhanced bacterial dissemination, together leading to increased susceptibility of the mice to S. Typhimurium infection owing to increased ROS and IL-6 production. To the best of our knowledge, this is the first report implying the ability of host Sirtuins in impacting intracellular bacterial metabolism crucial for successful pathogenesis.

Results

Salmonella modulates SIRT1 and SIRT3 expression along its course of infection in macrophages

Upon infection of RAW 264.7 murine macrophages with wildtype Salmonella Typhimurium strain 14028S, we observed an increased expression level of SIRT 1 and SIRT3 at initial and middle phases of infection, precisely at 2hr and 6hr post infection through qPCR (Fig. 1, A and B). The SIRT1 expression level declined at later phases of infection. On the other hand, the SIRT3 transcript levels remained elevated at all time points with respect to uninfected control with a marked increment at 16hr time point post-infection which subsided at 20hr post-infection. We even monitored the expression profile of SIRT1 and SIRT3 in primary macrophages like peritoneal macrophages of C57BL/6 mice and observed a similar trend of elevated expression at initial (2hr), middle (6hr), and late (16hr) time points post-infection (Fig. 1, C and D). In Confocal Laser Scanning Microscopy (CSLM) studies, we observed a similar increase in SIRT1 and SIRT3 expression at 6hr post infection within the infected macrophages RAW264.7 macrophages. (Fig. 1, E-H). Immunoblotting revealed increased protein expression of both SIRT1 and SIRT3 at 2hr post-infection in comparison to the uninfected control (Fig. S1). However, SIRT1 expression exhibits a gradual decline at the late phase of infection (Fig. S1A, C). In line with the confocal microscopy data, SIRT3 immunoblotting data shows an increased protein expression profile at 6hr and 16hr post-infection (Fig. S1, B-D). Subsequently, to ascertain whether indeed Salmonella could modulate SIRT1 or SIRT3 expression levels, we evaluated the SIRT1 and SIRT3 mRNA and SIRT1 and SIRT3 protein expression profile within RAW264.7 macrophages upon infection with wildtype S. Typhimurium and SPI-1 (ΔinvC) (InvC-protein export apparatus) or SPI-2 (ΔssaV and ΔsteE) (SsaV-Structural component of SPI-2 needle apparatus, SteE-SPI-2 effector protein involved in driving M2 polarization) mutants of S. Typhimurium. Our results depict the ability of wildtype S. Typhimurium to induce the expression of both SIRT1 and SIRT3 within the infected RAW264.7 macrophages. However, infection with either SPI-1 or SPI-2 mutant abrogates the induction of SIRT1 whereas only SPI-2 mutants (ΔssaV and ΔsteE) and not SPI-1 mutant infection caused reduction in SIRT3 transcript level expression in the infected macrophages, implicating the role of SPI-1 and SPI-2 genes in triggering SIRT1 and SIRT3 in the infected macrophages (Fig. 1, I-J). However, at the protein level, only SPI-2 mutant infection resulted in a predominant decline in SIRT3 expression and a mild reduction in SIRT1 expression (Fig. S1B). Further, we examined the transcript level profile of SIRT1 and SIRT3 in M1 or M2 polarized RAW264.7 macrophages at 16hr post-infection and we observed 20-fold and 5-fold increase in SIRT1 and SIRT3 expression in M2 polarized infected macrophages as opposed to 0.5-fold and 0.4 fold downregulation in M1 polarized infected macrophages (Fig. S1C-D). Thus, an increase in expression profile both at transcript and protein levels indicates their role in Salmonella pathogenesis.

Fig. 1

SIRT1 and SIRT3 play crucial role in intracellular bacterial proliferation in infected murine macrophages

As our previous SIRT1 and SIRT3 expression data in the polarized macrophages, hinted at the role of SIRT1 and SIRT3 in driving polarization of macrophages in the infected macrophages. We validated the intracellular replication of S. Typhimurium within the infected polarized macrophages. S. Typhimurium exhibited increased intracellular fold proliferation within anti-inflammatory M2 polarized macrophages in comparison to the pro-inflammatory M1 polarized RAW264.7 macrophages (Fig. S1 E-F). To evaluate the role of SIRT 1 and SIRT3 in the intracellular proliferation of the bacteria within murine macrophages, we have undertaken knockdown of SIRT1 and SIRT3 in RAW 264.7 (Fig. S2) macrophages through PEI-mediated transfection of shRNA plasmids directed against SIRT1 and SIRT3. Post 48hr of transfection, the transfected cells were infected with MOI=10 of wildtype S. Typhimurium and a gentamicin protection assay was performed. Intracellular proliferation of the bacteria was quantified by plating the cell lysate at 2hr and 16hr post-infection. Salmonella exhibits compromised intracellular survival in SIRT1 and SIRT3 knock-down RAW264.7 macrophages in comparison to the un-transfected and scrambled controls (Fig. 2, A). Further, we have assessed the intracellular proliferation in peritoneal macrophages isolated from thioglycolate-treated adult C57BL/6 mice post SIRT1 (EX-527) and SIRT3 (3TYP) inhibitor treatment. SIRT1 or SIRT3 inhibitor-treated macrophages exhibited attenuated intracellular replication in comparison to the untreated peritoneal macrophages (Fig,2, B). Together, our results depict the role of SIRT1 and SIRT3 in controlling the intracellular proliferation of S. Typhimurium.

Fig. 2

SIRT1 and SIRT3 inhibition contribute to skewed inflammatory host responses upon Salmonella infection

Several reports indicate the role of SIRT1 and SIRT3 in the modulation of host immune responses pertaining to infection scenarios [35][36][37][38]. Therefore, we intend to check whether SIRT1 or SIRT3 regulates immune functions in Salmonella-infected macrophages. To delineate the role of SIRT1 and SIRT3 in the modulation of immune responses, we wished to investigate the production of pro-inflammatory and anti-inflammatory cytokines in knockdown (KD) RAW 264.7 macrophages upon S. Typhimurium infection. Post 48hr transfection, cells were subjected to wildtype S. Typhimurium infection at an MOI of 10. At the indicated time points, cell-free supernatant was harvested and evaluated for pro-inflammatory and anti-inflammatory cytokine production by ELISA. Inhibition of both SIRT1 and SIRT3 increased production of pro-inflammatory cytokine IL-6 significantly at 2hr and 20hr post-infection (Fig.S3, A). Moreover, there was only a significant reduction in anti-inflammatory IL-10 production upon SIRT1 and SIRT3 KD at 2hr and 20hr post-infection (Fig.S3, B). Further, we estimated the production of another pro-inflammatory cytokine, IL-1β at 20hr post-infection under the knockdown condition of SIRT1 and SIRT3 (Fig.S3, C) and we observed heightened IL-1β production under knockdown of SIRT1 and SIRT3 in comparison to the scrambled infected control. In peritoneal macrophages upon SIRT1(EX-527-1µM) or SIRT3(3-TYP-1µM) chemical inhibitor treatment, an increase in IL-6 and IL-1β cytokine levels was observed at 6hr post-infection (Fig.S3, D-E). This indicates the possible role of SIRT1 and SIRT3 in the regulation of cytokine production upon Salmonella infection.

Immune functions are an important determinant of macrophage polarization. Since SIRT1 and SIRT3 played an immune-modulatory role in Salmonella infection, we investigated whether Salmonella infection is associated with a shift in macrophage polarization status. To assess the ability of the pathogen to alter the polarization state of the macrophage, we have undertaken gene expression profiling of various M1 and M2 markers using nanoString nCounter technology along the course of Salmonella Typhimurium infection at the indicated time points in RAW 264.7 macrophages. A gradual shift from pro-inflammatory M1 toward the anti-inflammatory M2 state was observed with the progression of Salmonella infection. Along the course of infection, there was a reduction in the expression of M1 markers like NOS2, CD40,CD86, TNF-α, Nfkb2,IL-6 and a corresponding increase in the expression of the M2 markers such as Arg-1, CCL-17, CD 206, IL4ra with an exception of Tgf-β (Fig. 3, A).In order to validate the polarization potency of the pathogen, FACS was performed using a pro-inflammatory M1 surface marker, CD86 tagged with PE. The data suggests a distinct decrease in CD86 positive population in the infected sets in comparison to the uninfected and the fixed dead bacteria control along the course of S. Typhimurium infection (Fig. 3, B, Fig. S4, A). Thus, the live pathogen has a propensity to skew the polarization state of the macrophage toward an anti-inflammatory M2 state to subvert the initial acute inflammatory response mounted by the host immune system.

Fig. 3

To assess the role of SIRT1 and SIRT3 in macrophage polarization, we determined anti-inflammatory CD206 surface marker profiling of the infected macrophages through flow cytometry. Knockdown of SIRT1 or SIRT3 in infected RAW 264.7 macrophages resulted in a reduction in anti-inflammatory CD206 surface marker expression at 16hr post-infection (Fig. 3, C, Fig. S4, B). Moreover, SIRT1 or SIRT3 knockdown led to enhanced intracellular ROS generation within the infected macrophages in comparison to the scrambled or the untransfected control (Fig.S5). Further, the Haematoxylin and Eosin staining of the S. Typhimurium infected mice liver tissue sections depicted exacerbated signs of inflammation in the SIRT1 (EX-527) or SIRT3 (3TYP) inhibitor-treated cohorts in comparison to the untreated controls with multiple necrotic foci and increased influx of inflammatory cell inflates. Moreover, these acute inflammatory signs of the liver sections get ameliorated in the SIRT1 (SRT1720) activator-treated infected cohort (Fig. 8, T-U) (Data described later). Together, these data suggest the role of SIRT1 and SIRT3 in the modulation of host inflammatory response. Previous literature reports have shown that SIRT1 physically interacts with p65 subunit of NF-κB and inhibits transcription by deacetylating p65 at lysine 310 [39]. Moreover, SIRT1-mediated deacetylation of the p65 subunit of the master regulator of the inflammatory response, NF-κB, results in the reduction of the inflammatory responses mediated by this transcription factor [40]. To evaluate the immune regulatory mechanism of SIRT1 in the S. Typhimurium (STM) infection scenario, we undertook SIRT1 immunoprecipitation in the infected RAW264.7 macrophages at 16h post-infection alongside the uninfected macrophages and probed for NF-κB p65 interaction. We observed an increased interaction of SIRT1 with NF-κB p65 in the infected macrophages in comparison to the uninfected control (Fig. 4, A). Further, the knockdown of SIRT1 resulted in increased acetylation status of the NF-κB p65 upon infection in comparison to the scrambled, infected control (Fig. 4, B, C). To understand whether the enzymatic domain of SIRT1 possess any role in mediating this interaction, we carried out NF-κB p65 immunoprecipitation in infected RAW264.7 macrophages in presence or absence of SIRT1 catalytic chemical inhibitor, EX-527(1µM) treatment at 16h post-infection. We observed an increased interaction of NF-κB p65 with SIRT1 in the infected untreated macrophages when compared to the untreated uninfected control (Fig. 4, D). However, the interaction of NF-κB p65 with SIRT1 gets abrogated under the EX-527 inhibitor treatment in the infected macrophages thereby implying the function of the catalytic domain in mediating the interaction (Fig. 4, D). Moreover, an increased acetylation status of NF-κB p65 was observed in the EX-527 treated infected macrophages in comparison to the untreated infected macrophages (Fig. 4 E).

Fig. 4

SIRT1 and SIRT3 relieve oxidative stress in infected macrophages and alleviation of the intracellular ROS generation restores intracellular survival of S. Typhimurium within the SIRT1 or SIRT3 knockdown macrophages

Previous reports have suggested the role of SIRT1 and SIRT3 in oxidative stress conditions. They are known to act in concert as anti-oxidants by reducing ROS production[41][42][43]. Moreover, enhanced ROS production is also a prototype of the classically activated macrophages[44][45]. Here, we examined the effect of SIRT1 and SIRT3 knockdown in intracellular ROS generation in S. Typhimurium infected RAW264.7 macrophages through DCFDA staining in FACS. Results depicted significant enhancement in the production of ROS at 16hr post infection upon knockdown of SIRT1 or SIRT3 in comparison to untransfected or scrambled control (Fig.S5, A,B). Upon detection of extracellular ROS generation through Phenol Red Hydrogen Peroxidase assay, we detected higher ROS generation upon SIRT3 KD at 6hr post infection and greater ROS production at 16hr and 20hr time points in SIRT1 knockdown macrophages (Fig.S5,C). Therefore, SIRT1 and SIRT3 play an important role in mitigating the oxidative burst in Salmonella infected macrophages. Our previous findings depicted decreased intracellular burden of S. Typhimurium within the SIRT1 or SIRT3 knockdown macrophages along with increase in intracellular ROS generation. Therefore, we speculated that the decreased intracellular proliferation within the SIRT1 or SIRT3 knockdown macrophages might be on account of increased intracellular ROS production which might lead to increased killing of the intracellular bacteria. This hypothesis led us to evaluate the intracellular bacterial burden within the infected SIRT1 or SIRT3 knockdown RAW264.7 macrophages or SIRT1 or SIRT3 inhibitor treated peritoneal macrophages (isolated from C57BL/6 adult mice post 5^th day of thioglycolate injection) in presence of a ROS inhibitor named N-Acetyl Cysteine (NAC). NAC acts as a scavenger of ROS by antagonizing the activity of proteasome inhibitors [46]. The attenuated intracellular proliferation of Salmonella Typhimurium within the knockdown or the chemical inhibitor treated macrophages got restored upon 1mM treatment of ROS scavenger, NAC (Fig.S6). Therefore, intracellular ROS production within the knockdown murine macrophages is one of the reasons for the attenuated survival of the bacteria.

SIRT1 and SIRT3 play crucial role in mediating metabolic switch in infected macrophages

Macrophage polarization is not only governed by immunological changes but also contributed by metabolic reprogramming[47][24][48]. Since previous data suggested progression of Salmonella infection with the shift in polarization state of the macrophage, we decided to investigate alteration of the metabolic state of the macrophages as macrophage polarization is governed by immune-metabolic shift. In pursuit of fulfilling such requirement, we performed gene expression studies of various metabolic genes through nanoString nCounter technology in S. Typhimurium infected RAW 264.7 macrophages. Analysis of the gene profile revealed upregulation of genes involved in fatty acid oxidization and tricarboxylic acid cycle and corresponding downregulation of genes involved in glycolysis (Fig. 5, A). To validate the findings, we carried out qRTPCR to quantitatively measure the expression of a fatty acid oxidation gene, PPARδ in infected RAW 264.7 macrophages. We found that mRNA level was elevated to 2-fold at 2hr and 6hr post-infection. At the late phase of infection, 16hr post infection around 6-fold upregulation in mRNA transcript was noted (Fig. 5, B). 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 revealed decline in lactate (glycolysis end product) production at 16hr in comparison to 2hr post-infection timepoint (Fig. 5C). Together, these results suggest the capability of the pathogen to drive a shift in the metabolic status of the infected macrophages toward fatty acid oxidation. Next, we evaluated the function of SIRT1 and SIRT3 in influencing the metabolic switch in the infected macrophages through qRT PCR with several host fatty acid oxidizing genes (Acox, Hadha, Pdha, and AcadL) and glycolytic gene (PfkL) in SIRT1 and SIRT3 knockdown macrophages (Fig. S7, A) and via lactate production assay (Fig. 5, D-E). Lactate estimation assay in SIRT1 and SIRT3 knockdown condition revealed enhanced lactate production at 16hr post infection in comparison to the scrambled control which further authenticates the increased host glycolysis upon SIRT1 and SIRT3 knockdown scenario (Fig. 5, D-E). SIRT1 and SIRT3 knockdown RAW 264.7 infected macrophages showed decreased expression of fatty acid oxidizing genes and increased expression of glycolytic PfkL gene in comparison to the scrambled infected control (Fig. S7, A). Similar results were obtained in the infected peritoneal macrophages under the SIRT1 or SIRT3 catalytic inhibitor treatment (Fig. S7, B-D). Moreover, qRT PCR-mediated metabolic gene profiling of liver and spleen isolated from wildtype S. Typhimurium infected macrophages revealed decreased transcript level expression of murine fatty acid oxidation genes upon treatment with SIRT1 (EX-527) or SIRT3 (3TYP) catalytic inhibitors which got reversed upon SIRT1 activator (SRT1720) treatment (Fig. S7, E-F). Moreover, SIRT1 and SIRT3 knockdown or catalytic inhibition in peritoneal macrophages resulted in increased protein expression of host glycolytic genes such as Phosphoglycerate kinase (Pgk), Phosphofructokinase (Pfk) with concomitant reduction in protein expression of TCA cycle gene like Pyruvate dehydrogenase (Pdha1) and fatty acid oxidation genes such as Hadha and Acox1 (Fig. 5, F, Fig. S7D). Fatty acid oxidation assay in the RAW264.7 macrophages under SIRT1 or SIRT3 knockdown condition or inhibition treatment revealed significant decrease in fatty acid β oxidation activity of the infected macrophages in comparison to the scrambled or the untreated control (Fig. 5, G). To investigate whether the host metabolic switch toward increased fatty acid oxidation is being driven by the pathogen, we performed fatty acid β oxidation activity assay under wildtype S. Typhimurium (STM WT), SPI-1 (ΔinvC) or SPI-2 (ΔssaV and ΔsteE) mutants of S. Typhimurium infection condition. We found that the wildtype bacteria with its intact SPI-2 effector secretion apparatus could promote increased host fatty acid β oxidation. In the contrary, the SPI-2 (ΔssaV and ΔsteE) mutants of S. Typhimurium failed to drive host metabolic shift towards increased fatty acid oxidation (Fig. 5, H).

Fig. 5

Collectively, these data suggest the role of SIRT1 and SIRT3 in mediating the Salmonella induced host metabolic shift in the infected macrophages.

SIRT1 and SIRT3 concomitantly influences Salmonella metabolism

Our previous data indicated a shift in host metabolism toward increased fatty acid oxidation along the course of Salmonella Typhimurium infection in murine RAW 264.7 macrophages. Salmonella Typhimurium drives the metabolism of the infected macrophage toward fatty acid oxidation. This observation led us to investigate the influence of host metabolic shift on the metabolic status of the pathogen harboring inside the infected macrophages[49]. We were intrigued whether increased glucose availability within the fatty-acid oxidizing macrophages is utilized by the bacteria. Thus, we undertook simultaneous gene expression studies of various Salmonella genes involved in their pathogenesis and metabolism through nanoString nCounter technology in S. Typhimurium infected RAW 264.7 macrophages. The nanoString gene profile revealed enhanced expression of genes involved in Salmonella glycolysis and glucose uptake such as pfkA and ptsG, respectively (Fig. 6, A). This finding indicates the ability of the pathogen to drive the metabolic state of the host toward fatty acid oxidation with corresponding increased glucose utilization by the bacteria favoring their survival inside the host. qRT PCR results with several bacterial fatty acid oxidizing genes (fadA, fadB,fadL,aceA, aceB) and glycolytic genes (ptsG) in knockdown condition of SIRT1 and SIRT3 in RAW 264.7 macrophages further validated the nanoString gene expression profiles (Fig. 6, B). In scrambled control, Salmonella infection progresses with increased Salmonella glycolysis and reduced bacterial fatty acid oxidation. However, knockdown of SIRT1 and SIRT3 abrogate this bacterial metabolic shift by reducing its glycolysis and by exhibiting enhanced fatty acid oxidation thereby attenuating pathogen intracellular survival. Similar observations were obtained from the qPCR data in the infected mice liver and spleen samples with increased transcript level expression of bacterial fatty acid oxidation genes and decreased expression of bacterial glycolytic genes upon SIRT1 or SIRT3 inhibitor treatment (Fig. 6 C, D). Therefore, SIRT1 and SIRT3 driven host metabolic switch potentially influence the metabolic profile of the intracellular pathogen.

Fig. 6

Mechanism behind SIRT1 or SIRT3 mediated metabolic switch

As per our previous findings, SIRT1 or SIRT3 inhibition led to increased host glycolysis and decline in fatty oxidation in the infected macrophages. HIF1α is a master regulator of glycolysis in host during stress conditions [50]. Previous reports have suggested HIF1α to be a target of deacetylation by SIRT1 at Lys 674 which contribute to metabolic reprogramming in cancer cells. During hypoxia, downregulation of SIRT1 leads to increased acetylation and activation of HIF1α [51]. Additionally, in CD4+ T cells, ectopic expression of SIRT1 inhibited IL-9 production and glycolysis by negatively regulating HIF1α [52]. To delve into the mechanism behind SIRT1 mediated modulation of metabolic responses, we assessed the interaction of SIRT1 with HIF-1α in infected RAW264.7 macrophages. The immunoprecipitation studies of SIRT1 showed increased interaction of the SIRT1 with HIF1α in the S. Typhimurium infection scenario with respect to the uninfected control (Fig. 7, A). Further, we evaluated the acetylation status of HIF1α in the SIRT1 knockdown status of the infected macrophages. We found that SIRT1 knockdown showed increased acetylation of HIF1α in the infected macrophages in comparison to the scrambled infected control at 16hr post-infection (Fig. 7, B,C). Immunoprecipitation studies under SIRT1 (EX-527) inhibitor treatment in RAW264.7 macrophages revealed increased acetylation of HIF1-α along with reduced interaction of HIF-1α with SIRT1, thereby indicating the probable role of the catalytic domain in influencing the interaction (Fig. 7 D-F). Further, to ascertain the role of HIF-1α in mediating the glycolytic switch in the infected macrophages, we estimated the lactate production under SIRT1 and SIRT3 knockdown conditions in the presence or absence of HIF-1α inhibitor (chetomin)[53]. Our results depicted a decline in lactate production upon chetomine treatment including under SIRT1 and SIRT3 knockdown conditions (Fig. 7G). Together, our results implicate the role of SIRT1 in governing glycolytic shift in the infected macrophages by deacetylating HIF1α. Upon SIRT1 knockdown or inhibition, HIF1α gets hyperacetylated which causes activation of the downstream glycolytic genes. Alternatively, several key literatures suggest the role of SIRT3 in modulating metabolic programming by deacetylating several proteins involved in fatty acid oxidation, the tricarboxylic acid cycle and oxidative phosphorylation [54][55]. PDHA1 (Pyruvate Dehydrogenase E1 subunit alpha) is a key enzyme linking glycolysis to TCA cycle and oxidative phosphorylation. SIRT3 regulates PDHA1 acetylation by deacetylating PDHA1 at lysine 385 residue, thereby playing a key role in metabolic reprogramming [55]. PDHA1 acetylation coincides with PDH activity and increased PDHA1 phosphorylation [55]. Therefore, we investigated the role of SIRT3 in the modulation of host fatty acid oxidation upon S. Typhimurium infection in RAW264.7 macrophages. To do so, we immunoprecipitated PDHA1 and checked for its interaction with SIRT3 or SIRT1 under the knockdown condition of SIRT3 or upon SIRT3 inhibitor treatment (Fig. 7 I-J). We observed an increased interaction of PDHA1 with SIRT3 in the infection scenario in comparison to the uninfected control which gets eventually abolished under the knockdown condition (Fig. 7 I-J) and under the chemical inhibitor treatment of SIRT3 (Fig. 7, K-L) suggesting the role of the SIRT3 in mediating the interaction with PDHA1. Alongside the decreased interaction of PDHA1 with SIRT3, increased acetylation of PDHA1 was detected upon SIRT3 inhibitor treatment in infected macrophages (Fig. 7, K-L).

Fig. 7

SIRT1 or SIRT3 inhibition enhances bacterial burden in mice in vivo

6-8 weeks old adult male C57BL/6 mice were treated with SIRT1 inhibitor EX-527, SIRT3 inhibitor 3TYP and SIRT1 activator SRT1720 at a dose of 1mg/kg each via intraperitoneal injection (every alternate Day) (Fig. 8A). Following the inhibitor treatment, the mice were orally gavaged with 10⁷ CFU of S. Typhimurium 14028S for organ burden evaluation or with 10⁸ CFU of wildtype S. Typhimurium for survival studies. On day 5^th post-infection, mice were sacrificed, and the liver, spleen and Mesenteric Lymph Node (MLN) were harvested for enumeration of the organ burden. The SIRT1 inhibitor, EX-527 and SIRT3 inhibitor, 3TYP-treated mice cohorts exhibited increased organ loads in liver, spleen and MLN in comparison to the vehicle control. On the contrary, the SRT1720 treated mice group showed organ burden comparable to that of the vehicle control (Fig. 8, B). Moreover, the SIRT1 and the SIRT3 inhibitor-treated mice cohorts died earlier than the vehicle-treated control mice group or the SIRT1 activator-treated group (Fig. 8, C). Further, the SIRT1 and the SIRT3 inhibitor-treated mice cohorts showed increased splenic length in comparison to the vehicle-treated mice group and the SIRT1 activator-treated mice cohort (Fig. 8, D). The increased organ burden in the EX-527 or 3TYP treated group might be due to increased bacterial dissemination in blood. To assess bacterial dissemination, blood was collected from infected mice post-inhibitor treatment at specific days post-infection retro-orbitally and plated onto Salmonella Shigella (SS) agar plates for bacterial enumeration. Indeed, increased bacterial dissemination was observed in the blood of mice treated with SIRT1 inhibitor, EX-527 or SIRT3 inhibitor, 3TYP at day 1-, 2-, 3-, 4-post-infection in comparison to the vehicle-treated mice (Fig. 8, E). Further, we wanted to examine whether the increased bacterial dissemination was due to increased ROS production or due to the presence of elevated inflammatory cytokine levels like IL-6 and IL-1β. In the wildtype C57BL/6 mice treated with SIRT3 inhibitor 3TYP showed heightened bacterial burden in blood at 5^th day post-infection in comparison to the vehicle control. Nevertheless, the gp91phox-/− mice group lacking the catalytic subunit of NADPH oxidase did not depict significant variation in the bacterial load among the different mice treatment cohorts (Fig. 8, F). Further, the EX-527 (SIRT1 inhibitor) and the 3TYP (SIRT3 inhibitor) treated mice possessed elevated levels of serum IL-6 (Fig. 8G), IL-1β (Fig. S3F) and showed increased intracellular ROS burden in infected liver tissues in comparison to the vehicle-treated control and the SRT1720 (SIRT1 activator) treated mice group (Fig. 8H). The increased mouse serum IL-6 and IL-1β production was in a similar line with the increased IL-6 or IL-1β cytokine generation in EX-527 or 3TYP treated peritoneal macrophages under the infection scenario (Fig. S3, D-E). Moreover, estimation of IL-1β within the infected intestinal ileal sections of the mice revealed increased pro-inflammatory IL-1β generation in the SIRT1 and SIRT3 inhibitor-treated mice groups in comparison to the untreated or the SIRT1-activator treated mice cohorts (Fig. S3G). However, contrary to the in vitro studies wherein SIRT1 or SIRT3 knockdown or inhibition resulted in attenuated intracellular proliferation, here in in vivo mouse model of infection, we observed increased bacterial organ loads owing to increased bacterial dissemination. To delineate this observation further, we evaluated the bacterial load within splenocytes isolated from control or inhibitor-treated C57BL/6 mice infected with GFP expressing S. Typhimurium at 5^th day post-infection via flow cytometry. We observed heightened bacterial load in the EX-527 or the 3TYP treated mice cohorts (Fig. 8, I-J). However, when we enumerated the bacterial count within the F4/80+ macrophage population of the infected splenocytes, we noticed decreased bacterial loads in the EX-527 or 3TYP –treated mice group in comparison to the vehicle-treated control group or the SRT-1720 activator-treated group (Fig. 8, K-L). Further, we evaluated additional splenic populations including CD45+, Ly6C+, and CD11c+ populations. Our results show that the CD45+ splenic population depicts increased bacterial loads like that of the total splenic population within the SIRT1 or SIRT3 inhibitor-treated cohorts. However, CD45+ monocytes and Ly6C positive splenic population exhibit compromised burden within the SIRT1 and SIRT3 inhibitor-treated cohorts. Moreover, CD11c+ population, CD45+ granulocytes, or lymphocytes show comparable organ loads to that of the vehicle control or SIRT1 activator-treated mice group (Fig. 8M-S, Fig. S8). Overall, our data suggest heterogeneous bacterial burden in diverse splenic populations. This opposing phenotype could be attributed to the increased IL-6 and IL-1β cytokine storm and elevated ROS production upon the SIRT1 or SIRT3 inhibitor treatment which in turn resulted in bacterial dissemination in vivo and concomitantly restricted the in vitro intracellular proliferation within macrophages. To validate this observation, we estimated the ROS levels within the liver and spleen tissues harvested from S. Typhimurium infected C57BL/6 mice, treated with specific catalytic inhibitor, activator or vehicle via DCFDA staining using flow cytometry at 5^th day post-infection. We detected escalated levels of ROS within both the infected liver and spleen tissues of the EX-527 or 3TYP-treated mice groups in comparison to the vehicle-treated or the SRT1720 treated mice cohorts (Fig. 8, H, S9). Haematoxylin and eosin staining of the liver sections (harvested at 5^th day post-infection) revealed increased inflammation with multiple areas of severe acute hepatic necrosis with complete loss of hepatic architecture in the EX-527 and 3-TYP treated liver samples in comparison to the vehicle-treated control and SRT-1720 treated liver samples (Fig. 8, T-U). In line with the inhibitor-treated studies, the increased organ loads, and systemic dissemination driven heightened susceptibility of mice toward S. Typhimurium infection were replicated in in vivo SIRT1 and SIRT3 adeno-associated virus serotype 6 (AAV6) mediated knockdown mice model which showed elevated IL-6 production in comparison to the scrambled control treated mice cohort (Fig. 8, V-X, Fig. S10). Simultaneously, the haematoxylin-eosin-stained sections of the liver tissues harvested from the shSIRT1 or shSIRT3 mice cohorts depicted increased pathological scoring with multiple necrotic areas and severely damaged liver tissue architecture in comparison to the scrambled mice control (Fig. 8, Y). Altogether, our results implicate the role of SIRT1 and SIRT3 in controlling S. Typhimurium infection in vivo.

Fig. 8

Discussion

Several studies have confirmed the propensity of Salmonella to skew the polarization state of the infected macrophages toward an immunosuppressive anti-inflammatory state[56][57][58]. We have validated such findings and further elaborated it by depicting the role of SIRT1 and SIRT3 in the modulation of host immune responses as well as host-bacterial metabolism. Salmonella Typhimurium infection modulates the expression profile of both SIRT1 and SIRT3 in the infected macrophages at both mRNA and protein level via its SPI-2 effector. Downregulation of SIRT1 and SIRT3 through shRNA mediated knockdown resulted in heightened pro-inflammatory immune responses with increased production of IL-6 cytokine and decreased surface expression of anti-inflammatory CD206. SIRT1 and SIRT3 downregulation also resulted in increased intracellular ROS production in the infected macrophages. SIRT1 and SIRT3 knockdown macrophages not only show altered host immune status but also depicted shift in the metabolic state with increased glycolytic shift. This altered host metabolism upon SIRT1 and SIRT3 knockdown condition influences the outcome of infection by regulating the intracellular bacterial metabolism which shows reduced bacterial glycolysis and increased fatty acid oxidation. All these outcomes account for attenuated intracellular bacterial proliferation in the SIRT1 and SIRT3 knockdown macrophages. However, in murine model of infection, SIRT1 or SIRT3 inhibitor treatment led to increased organ burden and triggered bacterial dissemination (Fig. S11). Overall, our study highlights the crucial role of SIRT1 and SIRT3 in governing the host immune-metabolic shift during Salmonella infection which in turn is vital for maintaining Salmonella metabolism.

Previous reports have elucidated the role of SIRT1 and SIRT2 pertaining to Salmonella infection. Ganesan et al., have depicted the role of SIRT1 in autophagy in Salmonella infection scenario[59]. Gogoi et al., have demonstrated SIRT2 mediated modulation of immune responses in dendritic cells[60]. Till date, there is no report highlighting the role of SIRT3 governing the Salmonella pathogenesis. The function of SIRT3 in infection scenario has been explored quite recently. In Mycobacterium tuberculosis infection condition, SIRT3 control mitochondrial function and autophagy[38]. SIRT3 downregulation in Mycobacterium tuberculosis infected macrophages is associated with dysregulated mitochondrial metabolism and increased cell death[61]. In this study we have explored the role of SIRT1 and SIRT3 in mediating host immune-metabolic switch in Salmonella Typhimurium infected macrophages which further govern intracellular bacterial metabolism and pathogenesis.

Our findings suggest the role of SIRT1 and SIRT3 in mediating polarization of the Salmonella infected macrophages toward an anti-inflammatory state. Upon knockdown of SIRT1 and SIRT3 in the infected macrophages we detect robust pro-inflammatory response and oxidative burst. This is in line with the findings by S.Elsela et al., wherein SIRT1 knockout RSV(Respiratory Syncytial Virus) – infected BMDCs showed significant increase in Il1β, Il6 and Il23 expression and ROS generation in comparison to the wild type RSV-infected BMDCs[35]. Also, Kim et al., showed presence of aggravated inflammatory responses in Mycobacterium tuberculosis infected SIRT3-/− BMDMs[38]. This heightened pro-inflammatory cytokine and oxidative burst restrict the intracellular survival of the pathogen as detected by the lower intracellular bacterial burden in the SIRT1 and SIRT3 knockdown murine macrophages. Salmonella showed enhanced proliferation in the M2 macrophages owing to the reduced arsenals in terms of pro-inflammatory cytokines and ROS production. Moreover, the M2 macrophages are fuelled by increased fatty acid oxidation and reduced glycolysis[62]. This might facilitate enhanced bacterial proliferation as the host un-utilized intracellular glucose can be readily up taken by the pathogen and used to support its own glycolysis. Similarly, M1 or pro-inflammatory macrophages resort to glycolysis to meet their energy demands[63] thereby limiting the glucose availability for the intracellular pathogen[63][64]. In such condition, bacteria show enhanced fatty acid metabolism to sustain their energy demand[65]. In our study, we found that wild type S. Typhimurium infection drive host metabolism toward increased fatty acid oxidation via its SPI-2 effector protein with concomitant increase in the bacterial glycolysis. SIRT1 and SIRT3 inhibition abrogates the metabolic switch and triggers increase in host glycolysis which in turn skew the bacterial metabolism from increased glycolysis to enhanced fatty acid oxidation and reduced glycolysis. Together, these findings implicate the role of SIRT1 and SIRT3 in reprograming the host metabolism which in turn affect the intracellular nutrient niche of the pathogen thereby influencing intracellular Salmonella proliferation. However, our in vivo findings in the murine model of infection show increased bacterial burden upon SIRT1 or SIRT3 inhibition. This increased burden could be attributed to increased dissemination from the macrophages into the bloodstream owing to the increased level of serum IL-6 levels. This is in concert with previous findings in Klebsiella pneumoniae infection in mice wherein increased inflammatory response upon HIF-1α activation induces bacterial dissemination [66]. Further correlation analysis of immune responses to Salmonella infection revealed that increased innate immune “cassette” opposes the adaptive immune arm leading to increased bacterial load [67]. Future studies might explore the host and bacterial interacting partners of SIRT1 and SIRT3 through mass spectrometry analyses in Salmonella infected macrophages which might hint at the underlying mechanism of their action and regulation. Together, this study highlights the complex and multifaceted nature of host-pathogen interactions, and the need for further research to fully understand the role of SIRT1 and SIRT3 in the context of Salmonella infection.

Materials and methods

Bacterial Strains, and culture conditions

Salmonella enterica serovar Typhimurium (STM) strain ATCC 14028S or ATCC 14028S constitutively expressing green fluorescent protein (eGFP) or mCherry (RFP) through pFPV plasmid were used. 4% paraformaldehyde fixed STM (PFA) was used as the killed fixed bacteria control. The above-mentioned live bacterial strain was grown overnight in LB broth in 37 ⁰C at 160 rpm shaking condition in presence or absence of appropriate antibiotic (Ampiciilin-50µg/ml) (Table-1) after revival of the bacterial strains from glycerol stock (stored at –80 ⁰C).

Table 1:

Table 2:

Cell Culture

RAW 264.7 murine macrophages were cultured in DMEM (Lonza) containing 10% FBS (Gibco) at 37 ⁰C in a humified incubator with 5% CO_2. Prior to each experiment, cells were seeded into 24 well or 6 well plate as per requirement at a confluency of 60%.

For macrophage polarization experiments, the seeded macrophages were subjected to 100ng/ml LPS + 20ng/ml IFN-_γ treatment for M1 polarization and 20ng/ml IL-4 treatment for M2 polarization for 24 hrs. Post-polarization, the cell supernatant were collected for validation of polarization status by ELISA and was further subjected to infection protocol.

Peritoneal macrophages were collected in PBS from the peritoneal cavity of C57BL/6 mice aseptically post thioglycolate treatment using 20G needle and 5ml syringe. Following centrifugation, cell pellet was resuspended in RPMI-1640 (Lonza) containing 10% heat-inactivated FBS (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin and seeded into 6 well-plate. 6hr prior to infection, antibiotic-containing media was replaced with Penicillin-Streptomycin free RPMI-1640 (Lonza) containing 10% heat-inactivated FBS (Gibco).

Transfection

shRNA mediated knockdown was carried out by PEI mediated transfection protocol. Plasmid harbouring shRNA in pLKO.2 vector backbone specific to SIRT1 and SIRT3 were used for transfection. Plasmid harbouring scrambled sequence of shRNA, served as a control, was also used for transfection. Plasmid DNA was used at a concentration of 500ng and 1µg per well of a 24-well plate and 6-well plate respectively. Plasmid and PEI were added in 1:2 ratio in serum free DMEM media and incubated for 20 mins at room temperature. Post incubation, the DNA: PEI cocktail was added to the seeded RAW 264.7 macrophages. After 6-8hrs of incubation, serum-free media was replaced with complete media containing 10% FBS. Post 48hr of transfection, transfected cells were either harvested for knockdown confirmation studies or subjected to infection with STM.

Infection Protocol

Macrophages were infected with stationary-phase bacterial culture with MOI of 10. For synchronization of the infection, tissue culture plates were subjected to centrifugation at 600xg for 5 min and incubated at 37 ⁰C humified incubator with 5% CO₂ for 25 min. Cells were washed with PBS and were treated with DMEM (Sigma) + 10% FBS (Gibco) containing 100 μg/ml gentamicin for 1 hr. Subsequently, the gentamicin concentration was reduced to 25 μg/ml and maintained until the cells were harvested. For the inhibitor treatment studies, along with 25 μg/ml containing complete media 1µM of SIRT1 (EX-527) inhibitor or SIRT3 (3TYP) or 10mM of N-Acetyl Cysteine (NAC, Sigma) or 50nM of chetomin (Sigma) were added to the cells.

Immunofluorescence confocal microscopic studies

At the specified time points post infection with GFP tagged STM, cells were fixed with 3.5% paraformaldehyde for 15 min. Primary antibody staining was performed with specific primary antibody in the presence of a permeabilizing agent, 0.01% saponin (Sigma) dissolved in 2.5% BSA containing PBS at 4°C for overnight or for 6hr at room temperature (RT). Following this, cells were washed with PBS stained with appropriate secondary antibody tagged with fluorochrome for 1 hr at RT. This was followed by DAPI staining and mounting of the coverslip onto a clean glass slide using the mounting media containing the anti-fade agent. The coverslip sides were sealed with a transparent nail paint. All immunofluorescence images were obtained using Zeiss LSM 710 or Zeiss LSM 880 and were analyzed using ZEN black 2012 software.

Quantitative Real Time PCR

Total RNA was isolated at specific time points post infection by using TRIzol (Takara) as per manufacturer’s protocol. Quantification of RNA was performed in Nano Drop (Thermo-Fischer scientific). Quality of isolated RNA was detected by performing 2% agarose gel electrophoresis. 2 µg of RNA was subjected to DNaseI (Thermo Fischer Scientific) treatment at 37°C for 1 hr followed by addition of 0.5M EDTA (final concentration 5mM) and heat inactivation at 65°C for 10 mins. The mRNA was reverse transcribed to cDNA using oligo (dT)₁₈ primer, buffer, dNTPs and reverse transcriptase (Takara) as per manufacturer’s protocol. The expression profile of target gene was evaluated using specific primers (Table-3) by using SYBR green RT-PCR master mix (Takara) in BioRad Real time PCR instrument. β-actin was used as an internal control for mammalian genes and for bacterial genes 16S rRNA was used. All the reaction was setup in 384 well plate with two replicates for each sample.

Table 3:

Intracellular proliferation or gentamicin protection assay

Following infection of the transfected cells with STM at an MOI of 10, cells were treated with DMEM (Sigma) + 10% FBS (Gibco) containing 100 μg/ml gentamicin for 1 hr. Subsequently, the gentamicin concentration was reduced to 25 μg/ml and maintained until the specified time point. Post 2hr and 16hr post-infection, cells were lysed in 0.1% triton-X-100. Lysed cells were serially diluted and plated on Salmonella-Shigella (SS) agar to obtain colony-forming units (cfu). Fold proliferation was calculated as cfu at 16hr divided by cfu at 2hr.

Fold Proliferation= [CFU at 16h]/[CFU at 2h]

Western Blotting

Post appropriate time points of infection, the cells were washed in PBS and subsequently harvested in PBS. The cell pellets were obtained after centrifugation at 300g for 7 minutes at 4°C. Cells were lysed in 1X RIPA (10X-0.5M NaCl, 0.5M EDTA pH-8.0, 1M Tris, NP-40, 10% sodium deoxycholate, 10% SDS) buffer containing 10% protease inhibitor cocktail (Roche) for 30 min on ice. Total protein was estimated using Bradford (Bio-Rad) method of protein estimation. Protein samples were subjected to 12 % SDS polyacrylamide gel electrophoresis and then were transferred onto 0.45µm PVDF membrane (18V, 2 hrs). The membrane was blocked using 5% skim milk in TBST (Tris Buffered Saline containing 0.1% Tween-20) for 1hr at RT and subsequently probed with appropriate primary antibody (Table-4) for overnight at 4°C. Following wash in TBST, blot was probed with specific HRP conjugated secondary antibody for 1hr at RT. The membrane was developed using ECL (Advansta) and images were captured using ChemiDoc GE healthcare. All densitometric analysis was performed using ImageJ software.

Table 4:

Immunoprecipitation

For co-immunoprecipitation, cells were washed with PBS and were lysed in native lysis buffer containing 1% Nonidet P-40, 20 mM Tris (pH 8), 2 mM EDTA, 150 mM NaCl and protease inhibitors mixture (Roche Diagnostics) for 30 min at 4°C. Cell debris was removed by centrifugation at 10,000 rpm for 10 min and the supernatant was treated with the specific antibody against the protein to be precipitated. Antibody-lysate complexes were immunoprecipitated using Protein A/G-linked magnetic beads (MagGenome) according to the manufacturer’s protocol. Beads were extensively washed with washing buffer and denatured at 95 °C for 10 min. Denatured precipitates were subjected to SDS-PAGE (12% gel) followed by transfer to 0.45 μ PVDF membrane. The membrane was blocked using 5% skimmed milk in TBST (Tris Buffered Saline containing 0.1% Tween-20) for 1h at room temperature and eventually probed for the target primary antibodies or Anti-acetylated Lysine (Ac-K) primary antibody overnight at 4°C. The blot was probed with a specific HRP-conjugated secondary antibody for 1hr at RT after rigorous washing in TTBS. ECL (BioRad) was used for detection and images were captured using ChemiDoc GE healthcare.

ELISA

Estimation of cytokines in cell-free supernatant or in mice serum was performed according to the manufacturer’s instructions. Briefly, 96-well ELISA plates (BD Bioscience) were coated overnight with capture antibody at 4°C. Following day, plates were washed with 0.1% Tween-20 containing PBS and blocked with 10% FBS for 1 h. Following blocking, wells were washed and incubated with 100 μL of test samples for 2 h at room temperature. Subsequently, plates were washed and incubated with detection antibody and enzyme reagent for 1 h at room temperature (BD Bioscience). TMB (Sigma) was used as a substrate and reactions were stopped with 2 N H₂SO₄. For the estimation of IL-1β, pre-coated ELISA (SARD Biosciences) plates were used, and ELISA was performed as per manufacturer’s protocol. Absorbance was measured at 450 nm wavelength in Tecan Plate reader and the concentration of cytokines were interpolated from a standard curve.

Flow cytometry

After specific time points post infection, cells were washed and harvested in PBS. Following centrifugation, cell pellet was resuspended in FACS buffer comprised of 1% BSA in PBS. Blocking was performed with Fc blocker (purified Anti-mouse CD16/CD32, eBioscience) dissolved in FACS blocking buffer for 30 min on ice. Following a washing step with PBS, antibody staining was performed with PE-conjugated CD86 antibody or APC-conjugated CD206 (Thermo Scientific) for 45 min on ice. After washing in PBS, the cell pellet was resuspended in 1% PFA in PBS. Subsequently, PFA was removed, and cells were resuspended in FACS buffer and reading was taken in BD FACSVerse instrument. For flow cytometry studies in mice tissues, the harvested liver or spleen was homogenized into single-cell suspension post RBC lysis (RBC lysis Buffer, Sigma). The homogenized cell suspension was washed and resuspended in FACS buffer containing the PE-conjugated Rat anti-mouse F4/80 antibody (BD Horizon, Cat-565411) or eFluor450-conjugated anti-mouse CD11c antibody (eBioscience), or FITC-conjugated anti-mouse Ly6C antibody (BD Pharmingen) or Alexa647-conjugated anti-mouse CD45 antibody (BioLegend). Following staining protocol, the cells were washed in PBS and the cells were resuspended in FACS buffer and the FACS protocol was performed either in BD FACSVerse or CytoFLEX flow cytometer (Beckman).

For DCFDA staining, one hour before the indicated time point of infection, cells were incubated with 10µM DCFDA containing DMEM media at 37°C humidified incubator with 5% CO₂for 45 min. Post incubation, cells were washed and harvested in PBS. Readings were measured in BD FACSVerse instrument.

All analysis was done using BD FACSuite software.

Phenol Red-Hydrogen Peroxidase Assay

Post 48hr of transfection, cells were infected with STM culture at an MOI of 10. Cells were incubated with Phenol Red solution containing hydrogen peroxidase enzyme (8.5U/ml). At, the designated time points post-infection, the supernatant was collected, and the absorbance was taken at 610 nm in Tecan Plate reader. The exogenously produced H2O2 was quantified using a standard curve of known concentration of H₂O₂.

Gene expression studies by nanoString nCounter technology

Total RNA was isolated at specific time points post infection by using TRIzol (Takara) as per manufacturer’s protocol. Quantification of RNA was performed in Nano Drop (Thermo-Fischer scientific) and Qubit Bioanalyzer (Agilent 2100 Bioanalyzer). Quality of isolated RNA was detected by performing 2% agarose gel electrophoresis. Post quality check, samples were subjected to nanoString nCounter technology (theraCUES). This technology allows multi-plex, spatially resolved RNA expression quantification with appropriate probes designed against the target gene.

Lactate Estimation Assay

Cell-supernatant was harvested at the specific time-points post-infection and the lactate content of the sample was estimated using the Lactate Assay Kit (Sigma, Catalog Number-MAK064) as per manufacturer’s protocol. Briefly, 50µl of the sample was added to each 96 well-plate and each of the well 50µl of the master reaction mix containing 46µl of lactate assay buffer, 2µl of lactate enzyme mix, and 2µl of lactate probe was added. After the addition of the master reaction mix to the sample, they were mixed by horizontal shaker or via pipetting. The plate was incubated for 30 minutes in dark at room temperature. Post incubation, absorbance was measured at 570nm. The lactate content of the sample was estimated from the lactate standard curve ranging from 0-10 nmole/µl.

Fatty Acid Oxidation Assay

At 16h post-infection, cell pellets were harvested and stored at-80°C. The fatty acid oxidation assay protocol was followed as per manufacturer’s instructions (AssayGenie Fatty Acid Oxidation (FAO) Assay Kit, Catalogue Code-BR00001). Briefly, cell pellets were lysed using 1X Cell lysis buffer (provided in the kit) and the cell supernatant were obtained post centrifugation of the cell lysate in a cold microfuge at 14,000rpm for 5min. The protein content of the cell supernatants was estimated using Bradford (Bio-Rad) method. 20µl of the protein sample was added to each 96-well plate in duplicate on ice. Each sample was treated with 50µl of control solution and 50µl of reaction solution by swiftly adding one 50µl of control solution to one set of wells and 50µl of reaction solution to the other set of wells. The contents were gently mixed for 10s. The plate is covered and incubated in a 37°C incubator for 30-60 min (without CO₂). After incubation cherry red colour appears in the wells. The O.D. is measured at 492nm using a plate reader at 30 min,60 min or 120 min. The control well reading was subtracted from the reaction well reading for each sample for each time point. The subtracted reading is used for enzyme activity calculation by considering the incubation time.

Animal Experiment

For all experiments, 6–8 weeks old C57BL/6 or gp91phox-/− mice were used. For organ burden analysis, 6 weeks old C57BL/6 or gp91phox-/− mice were infected with 10⁷ CFU bacteria via oral gavaging. For bacterial enumeration via flow cytometry, mice were infected with 10⁷ GFP-expressing bacteria orally. Infected mice were intraperitoneally injected on every other day with either 1 mg/kg body weight of SIRT1 inhibitor EX-527 (Sigma-Aldrich) [32], or SIRT3 inhibitor 3TYP (Selleck Chemical) [33] or SIRT1 activator SRT1720 (Calbiochem, Sigma-Aldrich) [34] or treated with vehicle alone. 5 days post infection, mice were sacrificed, and bacterial organ load was estimated by plating the tissue homogenates on SS agar plates. For calculating percentage survival, 6 weeks old C57BL/6 mice were infected with 10⁸ bacteria orally and monitored till fatality. For flow cytometry studies, the harvested liver or spleen were homogenized into single cell suspension and were subjected to flow cytometry. The animal experiments were carried out in accordance with the approved guidelines of the institutional animal ethics committee at the Indian Institute of Science, Bangalore, India (Registration No: 48/1999/CPCSEA). All procedures involving the use of animals were performed according to the Institutional Animal Ethics Committee (IAEC)-approved protocol.

In vivo knockdown

For in vivo knockdown adeno-associated virus serotype 6 (AAV6) was used. AAV6 viruses were produced in HEK293T cells. Briefly, HEK293T cells were transfected with AAV plasmid encoding shRNAs targeting SIRT1, SIRT3 and scramble control under U6 promoter together with helper plasmid using PEI Max 40000 (Polysciences USA). Next day transfection medium was removed, and fresh culture medium was added. Forty-eight hours later, medium was collected, and fresh culture medium was added and incubated for next forty-eight hours. Again, culture medium was collected and cells were harvested by trypsinization. Collected medium containing secreted AAV viral particles was incubated with 40% PEG 8000 overnight. Precipitated viral particles were then collected by centrifugation and re-suspended in PBS. Cells containing AAV viral particles were then lysed with citrate buffer and incubated with 40% PEG 8000 and processed similarly to medium for viral particle precipitation. Precipitated viral particles were then cleaned with chloroform and loaded on iodixanol gradient for further cleaning using ultracentrifugation. Cleaned viral particles were collected from the forty percent gradient and loaded on Amicon 100K cut-off columns. Purified AAV particles were then titrated by real-time PCR. For infection of AAV6, the mice were intravenously injected with a volume of 200 μl containing approximately 10¹² viral particles harbouring the respective scrambled or shRNA constructs. The mice were infected at 7^th day after injection of the virus with 10⁶ cfu units of S. Typhimurium orally. Fifth day post-infection, mice were euthanized, and dissected out for organ harvesting and blood collection. The knockdown was validated by performing western blotting and qPCR of the harvested liver tissue (Fig. S10).

Haematoxylin and Eosin Staining

6-8 weeks old C57BL/6 mice were infected with 10⁷ bacteria orally. Infected mice were intraperitoneally injected every alternate with either 1 mg/kg body weight of SIRT1 inhibitor EX-527, or SIRT3 inhibitor 3TYP or SIRT1 activator SRT1720 or treated with vehicle alone. 5 days post infection, mice were euthanized, and livers were collected and fixed using 3.5% paraformaldehyde. The fixed liver was then dehydrated using a gradually increasing concentration of ethanol and embedded in paraffin. 5μm sections were collected on coated plates. Sections were further rehydrated and then stained with hematoxylin and eosin. Images were collected in a Leica microscope. 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.

Statistical analysis

Data were analyzed and graphed using the GraphPad Prism 8 software (San Diego, CA). Statistical significance was determined by Student’s t-test or Two-way ANOVA and Bonferroni post-t-test to obtain p values. Adjusted p-values below 0.05 are considered statistically significant. The results are expressed as mean ± SD or SEM of three independent experiments.

Acknowledgements

We duly thank Prof. Subba Rao Gangi Setty and Prof. Michael Hensel for providing us with the shRNA knockdown constructs and the S. Typhimurium 14028S strain, respectively.

Funding

This work was supported by the DAE SRC fellowship (DAE00195) and DBT-IISc partnership umbrella program for advanced research in biological sciences and Bioengineering to DC. Infrastructure support from ICMR (Centre for Advanced Study in Molecular Medicine), DST (FIST), and UGC (special assistance) is highly acknowledged. DH sincerely acknowledges the CSIR-SPM fellowship for her financial support. SKG is supported by Ramalingaswami Re-entry Fellowship BT/RLF/re-entry/14/2019 from DBT, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contribution

DH and DC have conceptualized the study. DH has contributed to the experiment designing, visualization, methodology, investigation, formal analysis, literature survey, validation, writing (original draft), reviewing, and editing of the manuscript. RSR has performed the histopathological scoring of the liver sections and the retro-orbital injection of the adenoviral constructs in mice. ADC and SKG have prepared and provided AAV6, for in vivo mice knockdown experiments. DC has contributed to the funding acquisition, project administration, and overall supervision of the work. All authors approved the final version of the manuscript.

Declaration of interest

The authors are unaware of any conflicting interests and thereby declare no conflict of interest.