Salmonella-induced SIRT1 and SIRT3 are crucial for maintaining the metabolic switch in bacteria and host for successful pathogenesis
- Department of Microbiology and Cell Biology, Indian Institute of Science, India
- Centre of Infectious Disease Research, Indian Institute of Science, India
- Pharmacology Division, CSIR-Central Drug Research Institute, India
- Academy of Scientific and Innovative Research (AcSIR), India
- Adjunct Faculty, School of Biology, Indian Institute of Science Education and Research, India
Figures
Figure 1 with 1 supplement
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 …
Figure 1—figure supplement 1
Salmonella modulates the expression of SIRT1 and SIRT3 along its course of infection.
(A) Expression studies of SIRT1 and SIRT3 through immunoblotting in RAW 264.7 macrophages. (B) Representative immunoblot showing SIRT1 and SIRT3 expression at 16 hr post-infection with wildtype S. …
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Figure 1—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig1-figsupp1-data2-v1.zip
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Figure 1—figure supplement 1—source data 3
File containing original uncropped western blots for Figure 1—figure supplement 1A, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig1-figsupp1-data3-v1.zip
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Figure 1—figure supplement 1—source data 4
File containing original uncropped western blots for Figure 1—figure supplement 1B, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig1-figsupp1-data4-v1.zip
Figure 2 with 1 supplement
Effect of SIRT1 and SIRT3 knockdown in intracellular bacterial proliferation within RAW 264.7 and primary murine macrophages.
(A) Fold proliferation of S. Typhimurium within RAW 264.7 macrophages in transfected and untransfected conditions. Data is representative of N = 3, n = 3. Unpaired two-tailed Student’s t-test was …
Figure 2—figure supplement 1
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 …
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Figure 2—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 1C and D.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
File containing original uncropped western blots for Figure 2—figure supplement 1C and D, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig2-figsupp1-data2-v1.zip
Figure 3 with 4 supplements
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 (STM)-infected RAW 264.7 macrophages versus uninfected control data sets at 2 hr, 6 hr, and 16 hr time points of infection. Data is …
Figure 3—figure supplement 1
SIRT1 and SIRT3 modulate 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. …
Figure 3—figure supplement 2
Salmonella Typhimurium (STM) 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 STM-infected samples in comparison to uninfected (UI) and paraformaldehyde fixed (PFA) bacteria at the indicated …
Figure 3—figure supplement 3
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 untransfected RAW 264.7 macrophages at 16 hr post S. Typhimurium infection. Data is …
Figure 3—figure supplement 4
Effect of Sirt1 and Sirt3 knockdown or inhibition in intracellular bacterial proliferation within RAW 264.7 macrophages or peritoneal macrophages in the presence of ROS scavenger N-acetyl cysteine (NAC).
(A) Fold proliferation of S. Typhimurium within RAW 264.7 macrophages in transfected and untransfected conditions in the presence or absence of NAC (1 mM). Data are representative of N = 3, n = 3. …
Figure 4
SIRT1 mediates modulation of immune functions via deacetylation of p65 subunit of NF-κB in S. Typhimurium (STM)-infected macrophages.
(A) Immunoblot depicting p65 NF-κB interaction with SIRT1 post immunoprecipitation of SIRT1 in uninfected (UI) or STM-infected RAW 264.7 macrophages at 16 hr post-infection. Data is representative …
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Figure 4—source data 1
Original files for western blot analysis displayed in Figure 4A.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4B.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig4-data2-v1.zip
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Figure 4—source data 3
Original files for western blot analysis displayed in Figure 4D.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig4-data3-v1.zip
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Figure 4—source data 4
File containing original uncropped western blots for Figure 4A, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig4-data4-v1.zip
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Figure 4—source data 5
File containing original uncropped western blots for Figure 4B, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig4-data5-v1.zip
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Figure 4—source data 6
File containing original uncropped western blots for Figure 4D, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig4-data6-v1.zip
Figure 5 with 1 supplement
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 2 hr, 6 hr, and 16 hr time points of infection through nanoString. Data is representative of N = 2, n = 1. (B) P…
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Figure 5—source data 1
Original files for western blot analysis displayed in Figure 5F.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig5-data1-v1.zip
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Figure 5—source data 2
File containing original uncropped western blots for Figure 5F, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig5-data2-v1.zip
Figure 5—figure supplement 1
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) qRT PCR-mediated gene expression analysis of several host metabolic genes in Sirt1 or Sirt3 knockdown RAW 264.7 macrophages at 6 hr post-infection. Data is representative of N = 3,n = 3. …
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Figure 5—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 5—figure supplement 1D.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
File containing original uncropped western blots for Figure 5—figure supplement 1D, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig5-figsupp1-data2-v1.zip
Figure 6
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 2 hr, 6 hr, and 16 hr time points of infection through nanoString. SI, S. Typhimurium-infected; PI, …
Figure 7
SIRT1 inhibition triggers hyperacetylation of glycolytic master regulator HIF-1α within S. Typhimurium (STM)-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 STM-infected RAW 264.7 macrophages at 16 hr post-infection (derived from the same …
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Figure 7—source data 1
Original files for western blot analysis displayed in Figure 7A.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data1-v1.zip
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Figure 7—source data 2
Original files for western blot analysis displayed in Figure 7B.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data2-v1.zip
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Figure 7—source data 3
Original files for western blot analysis displayed in Figure 7D.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data3-v1.zip
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Figure 7—source data 4
Original files for western blot analysis displayed in Figure 7H.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data4-v1.zip
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Figure 7—source data 5
Original files for western blot analysis displayed in Figure 7J.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data5-v1.zip
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Figure 7—source data 6
File containing original uncropped western blots for Figure 7A, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data6-v1.zip
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Figure 7—source data 7
File containing original uncropped western blots for Figure 7B, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data7-v1.zip
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Figure 7—source data 8
File containing original uncropped western blots for Figure 7D, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data8-v1.zip
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Figure 7—source data 9
File containing original uncropped western blots for Figure 7H, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data9-v1.zip
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Figure 7—source data 10
File containing original uncropped western blots for Figure 7J, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig7-data10-v1.zip
Figure 8 with 1 supplement
Effect of SIRT1 or SIRT3 inhibition on S. Typhimurium (STM)-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 STM upon SIRT1 or SIRT3 …
Figure 8—figure supplement 1
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- to 8-week-old C57BL/6 mice (subjected to different chemical treatment – vehicle …
Figure 9 with 3 supplements
Effect of SIRT1 and SIRT3 inhibition on bacterial loads in diverse populations of S. Typhimurium-infected splenocytes.
(A) Enumeration of GFP-positive bacterial cells through flow cytometry in splenic tissues homogenate harvested from adult male 6- to 8-week-old C57BL/6 mice (subjected to different chemical …
Figure 9—figure supplement 1
Flow cytometric analysis of different splenic populations within mCherry-labelled S. Typhimurium-infected mice cohorts upon SIRT1 or SIRT3 inhibitor treatment.
(A) Enumeration of mCherry-positive bacterial cells through flow cytometry in splenic tissues homogenate harvested from adult male 6- to 8-week-old C57BL/6 mice (subjected to different chemical …
Figure 9—figure supplement 2
Flow cytometric analysis of different splenic populations within mCherry- labelled S. Typhimurium- infected mice cohorts upon SIRT1 or SIRT3 inhibitor treatment.
(A) Flow cytometric analysis of Alexa 647-conjugated CD45+ population within single cell splenic suspension isolated from adult male 6–8-week-old C57BL/6 mice (subjected to different chemical …
Figure 9—figure supplement 3
Flow cytometric analysis of different splenic populations within mCherry- labelled S. Typhimurium- infected mice cohorts upon SIRT1 or SIRT3 inhibitor treatment.
(A-B) Flow cytometric analysis of Alexa 647-conjugated CD45+ P5 (monocyte) (A), P4 (lymphocyte) (B) population (shown in Figure 9—figure supplement 2) within single cell splenic suspension isolated …
Figure 10 with 2 supplements
In vivo role of SIRT1 and SIRT3 inhibition and knockdown during S. Typhimurium infection.
(A) Representative image of haematoxylin-eosin-stained liver sections to assess the liver tissue architecture upon Salmonella infection on fifth days post-infection in different mice cohorts (UI, …
Figure 10—figure supplement 1
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 …
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Figure 10—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 10—figure supplement 1A.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig10-figsupp1-data1-v1.zip
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Figure 10—figure supplement 1—source data 2
File containing original uncropped western blots for Figure 10—figure supplement 1A, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/93125/elife-93125-fig10-figsupp1-data2-v1.zip
Figure 10—figure supplement 2
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 …
Author response image 1
Uninfected.
Author response image 2
Vehicle control infected.
Author response image 3
EX-527 infected.
Author response image 4
3TYP infected.
Author response image 5
SRT 1720 infected.
Author response image 6
Alternate blot images for Fig. 7B-C.
Author response image 7
Alternate blot images for Fig. 7I, J.
Author response image 8
Author response image 9
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background | Salmonella enterica serovar Typhimurium ATCC strain 14028S (STM) | ATCC strain 14028S – gfted by Prof. M. Hensel | ATCC strain 14028S | |
| Recombinant DNA reagent | pFPV:GFP-AmpR | Laboratory stock | ||
| Recombinant DNA reagent | pFPV:mCherry-AmpR | Laboratory stock | ||
| Recombinant DNA reagent | pLKO.2: shRNA-AmpR | Laboratory stock – gifted by Prof. Subba Rao | ||
| Recombinant DNA reagent | STM ∆ invC-KanR | Laboratory stock | ||
| Recombinant DNA reagent | STM ∆ ssaV-ChlR | Laboratory stock | ||
| Recombinant DNA reagent | STM ∆ steE-KanR | Laboratory stock | ||
| Cell line | RAW 264.7; macrophage; mouse (Mus musculus) | ATCC | ATCC-TIB-71 | |
| Antibody | SIRT1 polyclonal | Thermo Fisher Scientific | Cat# PA5-85921 | 1:1000 dilution (western), 1:100 (immunoprecipitation) |
| Antibody | SIRT3 monoclonal | Thermo Fisher Scientific | Cat# PA5-13222 | 1:1000 dilution (western), 1:100 (immunoprecipitation) |
| Antibody | CD86 monoclonal | Thermo Fisher Scientific | Cat# 12-086282PE | 1:100 dilution |
| Antibody | CD206 monoclonal | Thermo Fisher Scientific | Cat# 17-2069-42 | 1:100 dilution |
| Antibody | F4/80 monoclonal antibody | BD Horizon | Cat# 565411 | 1:100 dilution |
| Antibody | HIF-1α monoclonal | Santa Cruz | sc-13515 SCBT | 1:1000 dilution (western), 1:100 (immunoprecipitation) |
| Antibody | NF-κβ p65 monoclonal | CST (D14E12) | #8242 | 1:1000 dilution (western), 1:100 (immunoprecipitation) |
| Antibody | Acetylated-lysine antibody polyclonal | CST | Cat# 9441 | 1:1000 dilution |
| Antibody | PDHA1 monoclonal | ABclonal | Cat# A13687 | 1:1000 dilution (western), 1:100 (immunoprecipitation) |
| Antibody | HADHA monoclonal | ABclonal | Cat# A5346 | 1:1000 dilution |
| Antibody | ACOX1 monoclonal | ABclonal | Cat# A8091 | 1:1000 dilution |
| Antibody | PGK monoclonal | ABclonal | Cat# A12686 | 1:1000 dilution |
| Antibody | PFK monoclonal | ABclonal | Cat# A5477 | 1:1000 dilution |
| Antibody | β actin monoclonal | Sigma | A3854 | 1:50,000 dilution |
| Commercial assay or kit | Lactate Assay Kit | Sigma | Cat# MAK064 | |
| Commercial assay or kit | Fatty acid oxidation (FAO) Assay Kit | AssayGenie | Catalogue code BR00001 | |
| Commercial assay or kit | IL-6 | BD Bioscience | Cat# 555240 | |
| Commercial assay or kit | IL-10 | BD Bioscience | Cat# 555252 | |
| Commercial assay or kit | IL-1β | SARD Biosciences | Cat# SB-EKM1085 | |
| Antibody | CD16/32 monoclonal | eBioscience | Cat# 14-0161-82 | 1:100 dilution |
| Antibody | eFluor450-conjugated anti-mouse CD11c monoclonal | eBioscience | Cat# 48-0116-42 | 1:100 dilution |
| Antibody | FITC-conjugated anti-mouse Ly6C antibody monoclonal | BD Pharmingen | Cat# 553104 | 1:100 dilution |
| Antibody | Alexa 647-conjugated anti-mouse CD45 antibody mooclonal | BioLegend | Cat# 103124 | 1:100 dilution |
| Chemical compound, drug | SIRT1 inhibitor EX-527 | Sigma-Aldrich | E7034-5MG | |
| Chemical compound, drug | SIRT3 inhibitor 3TYP | Selleck Chemical | Cat# S8628 | |
| Chemical compound, drug | SIRT1 activator SRT1720 | Sigma-Aldrich | 567860-10MG | |
| Chemical compound, drug | N-Acetyl cysteine | Sigma-Aldrich | A9165 | |
| Chemical compound, drug | PEI Max 4000 | Polysciences | Cat# 24765 | |
| Chemical compound | Protease Inhibitor Cocktail | Roche | 04693132001 | |
| Chemical compound, drug | Saponin | Sigma | SAE0073 | |
| Chemical compound, drug | Chetomin | Sigma | C9623 | |
| Chemical compound, drug | SS agar | Himedia | M-108 | |
| Chemical compound, drug | Skimmed milk | Himedia | GRM-1254 |
Table 1
List of shRNA used for knockdown.
| Sirtuins | Construct ID | TRC ID | Sequence (5’–3’) |
|---|---|---|---|
| Sirt1 | C4 | TRCN0000218734 | GTACCGGCATGAAGTGCCTCAGATATTACTCGAGTAATATCTGAGGCACTTCATGTTTTTTG |
| Sirt1 | C12 | TRCN0000018979 | CCGGGCAAAGCCTTTCTGAATCTATCTCGAGATAGATTCAGAAAGGCTTTGCTTTTT |
| Sirt3 | E8 | TRCN0000038889 | CCGGCCCAACGTCACTCACTACTTTCTCGAGAAAGTAGTGAGTGACGTTGGGTTTTTG |
| Sirt3 | E12 | TRCN0000038893 | CCGGCCACCTGCACAGTCTGCCAAACTCGAGTTTGGCAGACTGTGCAGGTGGTTTTTG |
Table 2
List of primers.
| Primer name | Sequence (5’–3’) |
|---|---|
| Sirt1 FP | ACAAAGTTGACTGTGAAGCTGTAC |
| Sirt1 RP | GTTCATCAGCTGGGCACCTA |
| Sirt3 FP | GCTGCTTCTGCGGCTCTATAC |
| Sirt3 RP | GAAGGACCTTCGACAGACCGT |
| Ppard FP | CACAACGCTATCCGCTTTGG |
| Ppard RP | ATGCTCCGGGCCTTCTTTTT |
| Actb FP | CAGCAAGCAGGAGTACGATG |
| Actb RP | GCAGCTCAGTAACAGTCCG |
| Acadl FP | CTTGGGAAGAGCAAGCGTACT |
| Acadl RP | CTGTTCTTTTGTGCCGTAATTCG |
| Hadha FP | AGCAACACGAATATCACAGGAAG |
| Hadha RP | AGGCACACCCACCATTTTGG |
| Acox1 FP | TCGAAGCCAGCGTTACGAG |
| Acox1 RP | ATCTCCGTCTGGGCGTAGG |
| Pdha1 FP | TGTCGGTTCCCAGTCCATCA |
| Pdha1 RP | CGTTTCCTTTTCACAGCACATGA |
| Pfkl FP | GAACTACGCACACTTGACCAT |
| Pfkl RP | CTCCAAAACAAAGGTCCTCTGG |
| ptsG FP | TATCTGGGCTTCTTTGCGGG |
| ptsG RP | ACCAGGCAACGCTCGATAAA |
| fadA FP | TCTGGGATTGATGGAGCAGA |
| fadA RP | AGACCAATACACATCGTCGC |
| fadB FP | GTCCCCGAAGAGCAGTTAAG |
| fadB RP | CCAGTTTAGTTTCCGGCAGA |
| aceA FP | CGATCTGGTATGGTGCGAAA |
| aceA RP | TTCTGCCAGTTGAAGGATGG |
| aceB FP | AGCGTTTCAATCAACAGGGT |
| aceB RP | CCCGTATTTTTACCTGCCGA |
| Salmonella 16S rRNA FP | GTGAGGTAACGGCTCACCAA |
| Salmonella 16S rRNA RP | TAACCGCAACACCTTCCTCC |
