129S6/SvEcTac female mice were housed in a pathogen-free facility. At 8 weeks old, mice were orally infected with Salmonella strains at a dose of 10⁸. At 10 days post-infection, colons and livers were collected from mice as previously described (23, 24). Briefly, the distal part of the colon was prepared as Swiss rolls, and the central part of the liver with the gallbladder was collected for histologic analysis. Tissues were embedded in cryomolds containing Tissu-Tek OCT (Sakura Finetek, Tokyo, Japan), immediately placed on dry ice, and stored at -80°C until sectioning. The remaining parts of the organs were homogenised in phosphate buffer saline (PBS) and plated on LB plates supplemented with chloramphenicol (50 μg/ml) to assess bacterial recovery. The Regional Animal Studies Ethical Committee Northern Norrland, Sweden, reference numbers A17-17 and A23-21, approved this study protocol.

At 12 weeks old, mice were exposed to a single sublethal dose of 9 Gy (total body irradiation) using a Gammacell 40 irradiator (MDS Nordion, Ottawa, ON, Canada, now provided by Best Theratronics Ottawa, ON, Canada) with Cesium-137 as the source. Radiation was given as a dose of 1Gy per min for 9 minutes. The mice were euthanised 12 hours post-irradiation, and colons and livers were collected as described above. The Regional Animal Studies Ethical Committee Northern Norrland, Sweden, reference number A25-19, approved this study protocol.

The human colonic epithelial cell lines 1CT were previously described (25). 1CT cells were cultured in complete medium, composed as follows: high‐glucose Dulbecco’s Modified Eagle (DMEM) medium/medium 199 (4530, Sigma‐Aldrich Merck, Darmstadt, Germany, at ratio 4:1), supplemented with 2% fetal bovine serum (FBS; 10270-106, Gibco ThermoFisher Scientific, Waltham, MA, USA), epidermal growth factor (EGF; 20 ng/ml, E9644, Sigma‐Aldrich Merck), hydrocortisone (1 mg/ml, H0396, Sigma‐Aldrich Merck), insulin (10 mg/ml, I9278, Sigma‐Aldrich), transferrin (2 mg/ml, T0665, Sigma‐Aldrich Merck), sodium selenite (5 nM, S5261, Sigma‐Aldrich Merck), and geneticin (G418 sulfate) sulfate (50 μg/ml, G1397, Sigma‐Aldrich Merck).

Human colonic fibroblasts (CRL-1459, ATCC, Manassas, VA, USA) were maintained in Eagle’s Minimum Essential Medium (EMEM; 30-2003, ATCC, Manassas, VA, USA) supplemented with 10% of FBS (10270-106, Gibco, ThermoFisher Scientific) and 10 μg/ml of ciprofloxacin (17850, Sigma‐Aldrich Merck).

Human PBMCs were obtained from healthy donors (Blood Bank, Umeå University Hospital) using the standard procedure of Lymphoprep centrifugation (07801, STEMCELL Technologies, Vancouver, British Columbia, Canada). The recovered cells were counted by Trypan Blue (15250-061, Gibco ThermoFisher Scientific) exclusion, revealing a routine viability of more than 99%. CD14-positive cells were isolated from PBMCs using the EasySep™ Human CD14 Positive Selection Kit II (17858, STEMCELL Technologies) following the manufacturer’s instructions. The Swedish Ethical Review authority approved the use of the donors’ blood, Ethical permit 2021-00913.

Colonic tissue models were established as previously described (26). On day 5, 2x10⁶ PBMCs or 4x10⁵ monocytes were resuspended in 150μl of RPMI 1640 (R8758, Sigma‐Aldrich Merck) supplemented with 10% FBS (10270-106, Gibco, ThermoFisher Scientific) and added to the collagen matrix. Cells were incubated for two days at 37°C in 5% CO₂ to allow sedimentation into the matrix. On day 7, 6x10⁵ 1CT cells were resuspended in 150μl DMEM complete medium (D6429, Sigma‐Aldrich Merck, Darmstadt, Germany) without antibiotics, overlaid on the fibroblast/collagen matrix and incubated for 4 hours at 37°C in 5% CO₂, after which 2 ml complete DMEM medium without antibiotics was gently added to the insert. The models were cultivated for 24 hours at 37°C in 5% CO₂ before infection.

Human organotypic colonic models were infected at a MOI of 25:1 as previously described (26). After infection, 2 ml of RPMI 1640 (R8758, Sigma‐Aldrich Merck) supplemented with 10% FBS (10270-106, Gibco, ThermoFisher Scientific) and gentamicin (100μg/ml, Sigma‐Aldrich Merck) was added into each well to kill extracellular bacteria and incubated for 1 hour. The “killing medium” was then changed to “maintaining medium,” RPMI supplemented with 10% FBS and 10μg/ml gentamicin, for three additional days to avoid overgrowth of bacteria. On day 3 post-infection, 2 ml of 2M sucrose solution was added to the inner and outer chambers, and samples were incubated for 1 hour at room temperature (RT). Subsequently, the models were embedded in Tissue-Tek OCT (Sakura Finetek, Tokyo, Japan), snap-frozen, and stored at -80°C. Ten micrometer sections were cut at -20°C using the cryostat Leica CM3050S, and slides were stored at -80°C until immunofluorescence analysis.

Hematoxylin and eosin staining was employed to assess the morphology of the organotypic 3D tissue models. Briefly, slides were fixed for 20 minutes in 4% paraformaldehyde (PFA; 1004965000, Sigma‐Aldrich Merck) at RT. After fixation, the samples were washed three times for five minutes in PBS under gentle shaking. The slides were then dipped twice into hematoxylin solution (GHS216, Sigma‐Aldrich Merck) for 3 seconds and rinsed in three different baths of tap water to remove unbound dye. The samples were then submerged in an eosin Y solution (HT110116, Sigma‐Aldrich Merck) and afterward dipped once into 95% and into 99% ethanol solutions to allow the dehydration of the tissues. Finally, the slides were mounted in 40% glycerol, covered with coverslips, and stored at +4°C until imaging.

Flow cytometry analysis was performed by direct immunofluorescence analysis using the anti-human MultiMix™ Dual-Colour labeled with fluorescein isothiocyanate (FITC) (FR700, Dako, Glostrup, Denmark) and R-phycoerythrin (RPE) (FR866, Dako, Glostrup, Denmark) directed against CD45 (leukocyte marker), CD3 (T lymphocytes marker) or CD19 (B lymphocytes marker), and CD14 (monocytes marker). Unstained cells and cells labeled with the respective isotype controls (X0932, Dako, Glostrup, Denmark) were used as negative controls. A minimum of 1x10⁴ cells were analyzed using BD Accuri C6 Plus System (Becton and Dickinson, Franklin Lakes, NJ, USA) equipped with 488- and 640-nm lasers, and Accuri C6 Plus software was used to analyse the data.

For the in-situ detection at mRNA levels of Aim2, Il10, Ifnb1, Ifng, Nlrp3, and Salmonella enterica fljB, we used an RNAscope® Multiplex Fluorescent Reagent Kit (323100, Bio-techne, Oxford, UK) and performed the assay according to the manufacturer’s instructions. A positive ready-to-use probe targeting the housekeeping genes Polymerase II Subunit A (Polr2a), Peptidylpropyl isomerase B (PPIB) and Ubiquitin C (UBC) (320881, Bio-techne) and a negative control probe targeting the DapB mRNA from the Bacillus subtilis strain SMY (320871, Bio-techne) were used to assess the mRNA quality of the samples.

Following fixation and dehydration, tissue sections were pre-treated with Protease IV (Bio-techne) for 30 minutes at RT and incubated with the corresponding probe mix for 2 hours at 40°C. After subsequent steps for signal amplification, tissue sections underwent two washing steps of 2 minutes with washing buffer provided by Bio-techne at RT under gentle agitation. The signal from target and control probes was detected after incubation with the fluorescent dyes Opal 520, Opal 570, and Opal 690 (1:750 for target probes and 1:1500 for control probes, NEL810001KT, PerkinElmer, Waltham, MA, USA). All incubation steps were done at 40°C in the HybEZ™ II Oven (321719, Bio-techne). Nuclei were counterstained and mounted with DAPI/VECTASHIELD® (H-1200-100, Vector Laboratories). Images were acquired with a confocal scanning microscope (Leica TCS SP8, Leica Microsystems). The expression levels of each target probe were quantitatively evaluated using the H-score, as recommended by the manufacturer.

For the co-detection of target mRNA probes (Salmonella enterica fljB) and protein (γH2AX), we used the RNA-Protein Co-Detection Ancillary Kit (323180, Bio-techne), following manufacturers’ instructions. Briefly, tissue sections were fixed in pre-chill 4% PFA (Sigma‐Aldrich Merck) for 15 minutes at +4°C and dehydrated in sequential immersions of 50%, 70%, and 100% ethanol. Tissue sections were further incubated with the corresponding primary antibody overnight at +4°C, followed by a washing step in 0.1% Triton X-100 in PBS and post-primary fixation in 4% PFA (Sigma‐Aldrich Merck) for 30 minutes at RT. Sections were then pre-treated with Protease IV for 30 minutes at RT and incubated with the corresponding probe mix for 2h at 40°C. Subsequent hybridization steps and probe signal detection with fluorescent dyes were done as described in the previous section. This was followed by incubation with the appropriate secondary antibody to detect the protein of interest for 30 minutes at RT in a humidified chamber. Sections were counterstained with DAPI/VECTASHIELD® (H-1200-100, Vector Laboratories). Images were acquired with a confocal scanning microscope (Leica TCS SP8, Leica Microsystems).

All the statistical analyses were performed using Prism 7 and Graph Pad Software. The significance of the difference between the three experimental groups was determined by ANOVA with Tukey’s post-test. p-values <0.05 were considered significant; * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001. If not indicated, differences were not statistically significant.

Principal component analysis (PCA) was performed to create an overview of all assessed parameters, investigate data integrity, identify potential outliers, and explore possible trends and groupings of the samples (29). All multivariate data analysis and model plots were performed in SIMCA 16.0 (Sartorius Stedim Data Analytics AB, Umeå, Sweden).

In the liver, increased levels of leukocytes (CD45 positive cells) were detected in all the infected animals compared to the uninfected group, with higher cell infiltration levels in mice infected with the genotoxigenic strain ( Figure 1 ). Conversely, levels of leukocytes in the colon of mice infected with the MC1 TT strain were significantly lower than those observed in mice infected with the control strain MC1 ΔcdtB ( Figure 1 ).

In line with pronounced hepatic leukocyte infiltration, co-immunostaining with macrophage marker F4/80 and mannose receptor CD206, which identifies anti-inflammatory M2-like macrophages, showed a significant decrease in the number of M2-like macrophages recruited to the liver upon infection with genotoxigenic Salmonella ( Figure 2A ).

RNAscope™ was used to assess the mRNA levels of the pro-inflammatory cytokine Ifng and the anti-inflammatory cytokine Il10 in the liver. Specific positive and negative probes were used to evaluate the quality and sensitivity of the assays ( Supplementary Figure S1 ). Levels of Il10 were lower than those observed for Ifng ( Supplementary Figure S2A ), and higher values of both mRNAs were present in mice infected with the genotoxigenic strain ( Supplementary Figure S2A ). However, no difference in the Ifng/Il10 ratio was observed independent of the presence of the typhoid toxin ( Figure 2B ).

To evaluate tissue differences in the host response to the genotoxigenic strain, we assessed the mRNA levels of inflammasome sensors, which are key effectors in driving inflammation (30). We focused on Absent in Melanoma 2 (Aim2) and the nucleotide-binding oligomerization domain-like receptor (NLR) Nlrp3. These sensors were selected due to their activation by different stimuli: AIM2 is activated by cytosolic double-strand DNA (22) and NLRP3 responds to intracellular damage (e.g., sensing of K⁺ efflux) (31). Upon infection, a toxin-dependent induction of Aim2 mRNA was observed in the colon ( Figure 3A ) despite similar levels of bacterial recovery in the two infected groups (24). In the liver of infected mice, the enhanced Aim2 expression was toxin-independent ( Figure 3A ). The infection did not significantly change Nlrp3 expression in colonic tissue, while higher Nlrp3 levels were detected in the hepatic tissue after infection with the MC1 TT strain ( Figure 3A ).

Aim2 is an interferon-stimulated gene (ISG) (30). Therefore, we evaluated the co-expression of Aim2 and Ifnb1 mRNAs in the colon and liver. Significant co-expression levels of Ifnb1 and Aim2 mRNAs were observed exclusively in the colon of mice infected with the genotoxigenic strain. In contrast, Ifnb1 and Nlrp3 co-expression was not observed in any analyzed tissues or conditions ( Figure 3B ). A tendency for increased Ifnb1 mRNA levels was present in the colonic tissue of infected animals ( Supplementary Figure S2B ). However, this trend was not observed in the liver samples ( Supplementary Figure S2B ).

Infection promoted a significant increase in cells positive for the DDR marker γH2AX (a phosphorylated form of histone H2AX at serine 139) in both tissues in a toxin-independent manner ( Figure 4A ). Since both oxidative-induced and TT-induced DNA damage promotes H2AX phosphorylation (7, 32, 33), we performed additional experiments to assess the type of DNA damage induced in the colonic and hepatic tissues upon infection.

Oxidative-induced DNA damage was measured by the detection of 8-dihydro-8-oxoguanine (8-oxoG) using immunofluorescence analysis. Low numbers of positive cells were present in uninfected tissues in both organs ( Figure 4B ). The fluorescence intensity of each positive cell was quantified, and enhanced fluorescence intensity was observed in the colon of mice infected with the control strain compared to mice infected with the genotoxigenic strain ( Figure 4C ), despite a similar percentage of 8-oxoG positive cells ( Figure 4B ). Conversely, in the liver, the cell fluorescence intensity was higher in the MC1 TT-infected mice compared to mice infected with the MC1 ΔcdtB Salmonella ( Figure 4C ). Interestingly, we observed that the few 8-oxoG-positive cells in the liver of uninfected mice had a higher fluorescence intensity than the levels observed in the colon ( Figure 4C ).

As a next step, we investigated whether there was a correlation between the presence of genotoxigenic Salmonella and DNA damage response by assessing the proximity of the bacterium to cells activating the DDR. We established a combined protein/mRNA protocol to detect the levels of γH2AX by immunofluorescence in combination with the mRNA probe specific for the flagellin subunit fljB of S. enterica. Significantly higher levels of fljB mRNA were detected in infected samples compared to uninfected tissues, independent of the Salmonella strain used, supporting the specificity of the probe for Salmonella and lack of cross-reactivity with the colonic microbiota ( Supplementary Figure S2C ). We found a higher percentage of γH2AX-positive cells in proximity to the flagellin subunit fljB upon infection with the MC1 TT bacterium in the colon. In contrast, this proximity was not observed in the colon of mice infected with the MC1 ΔcdtB strain or in infected liver samples ( Figure 4D ).

To complete the analysis of the DDR response, the levels of expression of the tumour suppressor protein p53 were investigated by immunofluorescence analysis. Higher percentage of p53-positive cells was detected in the colon of infected mice compared to the uninfected controls or hepatic samples ( Figures 5A, B ). However, p53 exhibited a nuclear localisation in the colonic tissue of mice infected with the genotoxigenic strain, while mice infected with the control strain showed a more dominant cytosolic p53 localization ( Figure 5C ). When detected in the liver, p53 exhibited an exclusively nuclear localization ( Figure 5B ).

To evaluate whether the intestinal immune response induced by genotoxigenic Salmonella was comparable to that caused by other agents that induce DNA breaks, we assessed the response produced by ionizing radiation (IR). We analyzed the infiltration of leukocytes and macrophages and the induction of γH2AX in the colon and liver of mice exposed to a single sublethal radiation dose of 9 Gy 12 hours post-treatment. We did not observe any signs of inflammatory response regarding leukocyte infiltration or changes in the proportion of M2-like CD206-positive macrophages in the colon or liver. However, differences in macrophage populations were observed, with a significant decrease in F4/80 positive cells in hepatic tissues upon irradiation ( Supplementary Figure S3 ). As expected, significantly higher levels of γH2AX were present in both tissues upon irradiation. These data show that the presence of DNA breaks, neither in the colon nor in the liver, does not promote a strong inflammatory response within the analysed time.

An unbiased Principal Component Analysis was performed by combining all the parameters analysed in the study, including immune markers (CD45 for leukocytes, F4/80 for macrophages, CD206 for M2-like macrophages, Ifng and Il10 mRNAs), markers for DNA damage (γH2AX, 8-oxo-G), and markers for DNA damage innate immune response (mRNA for type I interferon and the DNA-damage sensor AIM2). The PCA analysis shows that mice infected with the MC1 TT strain are well-separated from those infected with the isogenic MC1 ΔcdtB strain and clustered close to the non-infected mice in the colon ( Figure 6 ). However, the pattern observed in the liver samples shows that both infected groups are clustered very closely and are well-separated from the uninfected group ( Figure 6 ).

To assess whether the presence of immune cells influences the activation of DDR upon infection with the genotoxigenic strain in a human setting, we modified the organotypic 3D models previously developed in our laboratory (26) by embedding freshly isolated peripheral blood mononuclear cells (PBMCs) (2x10⁶ cells/model) or freshly purified monocytes (4x10⁵ cells/model) in the collagen matrix. Phenotypic characterization of the PBMC population, performed by flow cytometry analysis, showed that more than 90% of the cells were positive for the leukocyte marker CD45, with T lymphocytes (CD3 positive) representing the predominant population (62%), followed by monocytes (CD14 positive, 19%) and B lymphocytes (CD19 positive, 3.5%) ( Supplementary Figure S4A ). Upon isolation, 80% of the monocyte population was CD14 positive, with a small percentage (7.3%) of T lymphocytes (CD3 positive) ( Supplementary Figure S4A ).

The numbers of cells embedded into the matrix were selected based on previous studies (34). The models were left uninfected or infected with the MC1 ΔcdtB or the MC1 TT for 72h to mimic an acute infection. Figure 7A shows that the addition of PBMCs did not significantly alter the model’s general architecture as assessed by Haematoxylin and Eosin (H&E) staining in uninfected or infected samples. However, in the presence of freshly isolated monocytes, the epithelial layer became more fragile upon infection and occasionally presented a multi-layered structure.

Activation of the DNA damage response was assessed by immunofluorescence analysis using markers for the phosphorylation of two early effectors of the DNA damage sensing machinery, KAP1 (pKAP1) and γH2AX (35, 36), and formation of 53BP1 foci at the site of the damaged DNA, which enables recruitment of repair effectors (37). As expected, we detected a significant increase of all the markers upon infection with the MC1 TT strain in the absence of immune cells ( Figure 7B ), confirming our previous results (26). In the presence of PBMCs, we still observed a toxin-induced increase of the DDR signature, but this was accompanied by increased levels of all the markers tested in uninfected and MC1 ΔcdtB infected cells ( Figure 7B and Supplementary Figure S4B ). This suggests that the addition of PBMCs promoted non-toxin-induced DNA damage, possibly due to induction of immune cell-induced oxidative damage. In contrast, the loss of a specific TT-induced DNA break-dependent response was associated with the presence of monocytes ( Figure 7B and Supplementary Figure S4B ).

These findings indicate that toxin-mediated DNA damage activates a default response to DNA-damaging agents in the colonic mucosa’s tolerogenic environment, suppressing S. Thyphimurium-induced inflammation. However, this effect is lost in the liver, where bacterium-induced inflammation is the predominant response.

The anti-inflammatory effect exerted by the typhoid toxin may be dependent on the tolerogenic environment of the intestinal mucosa, characterised by the almost exclusive presence of M2-like macrophages under homeostatic conditions (uninfected mice) (24), compared to approximately 40% in the liver ( Figure 2 ). The tolerogenic environment may cause an initial inhibitory effect, later supported by a sustained chronic production of type I IFNs, stimulated by the presence of cytoplasmic DNA fragments via the cGAS/STING/IFN-I axis (30, 38). Chronic production of type I IFNs promotes immunosuppression via diverse effects on immune cells (39), including inhibition of dendritic cells (DCs), with expansion and polarization of DCs subsets with an IL10-secreting suppressor phenotype; reduced IFNγ release from NK cells, an essential cytokine against intracellular bacteria such as Salmonella (40, 41); reduced recruitment of neutrophils, via inhibition of the chemokines CXCL1 and CXCL2; activation of myeloid-derived suppressor cells (MDSCs). This pattern is compatible with several observations we have made previously (24). We did not detect neutrophil influx, but a higher frequency of MDSC-induced cell types, such as M2-like macrophages and T regulatory cells (42). In addition, prolonged induction of type I IFNs due to toxin-dependent DNA damage may contribute to reduced levels of Ifng detected in the colon of mice infected with the genotoxigenic strain (23).

The role of immune microenvironment on the modulation of the toxin-induced DDR is supported by the organotypic model ( Figure 7 ), where we observed a general increase of DDR markers in samples left uninfected or infected with the control strain MC1 ΔcdtB upon addition of the PBMCs. The observed effect may result from a PBMC-mediated inflammatory response and consequent production of DNA-damaging ROS. This observation aligns with a previous report showing that adding PBMCs promoted a 2- to 6-fold increase in pro-inflammatory cytokines IL6 and IL8 in a 3D oral mucosa model (34). Interestingly, the presence of myeloid cells promoted a loss of the toxin-induced DDR pattern associated with an alteration of the epithelial structure ( Figure 7 ). These findings suggest that the host response to the bacterium is overwhelming in a non-tolerogenic environment, promoting tissue destruction, and masking the detection of the toxin-induced DDR. However, we cannot exclude that the different colonic and hepatic types of immune responses upon infection with the MC1 TT strain might be due to the 10-fold higher bacterial recovery in the liver (24), which may promote a more robust inflammatory response that covers the toxin-mediated DNA damage.

The absence of leukocyte influx in the colon and liver of mice exposed to a single dose of radiation ( Supplementary Figure S3 ) indicates that the hepatic inflammatory response seen in mice infected with the genotoxigenic strain is mainly due to the bacteria-induced host response. The response observed in irradiated mice was unexpected but in agreement with previous results reporting a fast upregulation of the inflammatory cytokines and chemokines in rats with a dose of 25 Gy, associated with a transient increase of recruitment of neutrophils into the portal area but not into the hepatic parenchyma within a period of 3 to 6h post-irradiation (43). Thus, it is likely that, depending on the irradiation dose, the primary tissue response is to activate DNA repair, promoting a very limited, short-lived, and controlled inflammatory response to avoid further tissue damage, and that bacterial genotoxins hijack this response to ensure a stealth crossing of the intestinal barrier.

We observed that the upregulation of the inflammasome sensor Aim2 was uncoupled from an increased inflammatory response in the colon of mice infected with the genotoxigenic strain ( Figure 3 ). It has been previously shown that upregulation of AIM2 can activate a RalB-dependent autophagosome formation and recruitment of K63 polyubiquitinated AIM2 inflammasomes for degradation, resulting in reduced maturation of IL-1β β (44). This activity is independent of the inflammasome adaptor ASC and pro-inflammatory cysteine caspase-1 and is uncoupled from induced proinflammatory cell death. This negative feedback loop may explain why increased levels of Aim2 do not correlate with an inflammatory response in the colon of mice infected with the genotoxigenic strain.

We observed enhanced Aim2 levels in the liver of infected mice in the absence of co-expression with its inducer Ifnb1 ( Figure 2 ). Thus, AIM2 expression may be regulated in an IFN-I-independent manner. The nuclear factor E2-related factor-2 (Nrf2), a key transcription factor that regulates the cellular antioxidant responses, can promote AIM2 transcription (45, 46), which is consistent with the increased oxidative state associated with the bacteria-induced hepatic inflammation ( Figures 1 and 4 ).

The product of the tumour suppressor gene TP53 is a transcription factor activated in response to DNA damage. Stabilization of p53 determines the fate of damaged cells by inducing either cell cycle arrest, allowing DNA repair, or apoptosis promoting cell death (47). Inflammation is also associated with higher p53 levels, likely due to the production of ROS and subsequent oxidative-induced DNA damage (48, 49). Therefore, it is unsurprising that we observed significantly higher levels of p53 stabilization in the colon of mice infected with the control MC1 ΔcdtB strain ( Figure 5 ), which aligns with the increased levels of γH2AX staining ( Figure 4 ). This was also expected in the colonic tissue of mice infected with the genotoxigenic strain, which causes DNA fragmentation (24).

Interestingly, p53 showed prevalent nuclear localization in mice infected with the MC1 TT strain and a more dominant cytosolic distribution in mice infected with the control strain ( Figure 5 ). The cytosolic fraction of p53 may regulate several processes, including autophagy and induction of ROS scavengers (50, 51). In the context of a strong inflammatory response observed in the colon of MC1 ΔcdtB infected mice, the cytosolic fraction of p53 may activate negative feedback mechanisms to control the bacterium and prevent irreparable tissue damage.

Surprisingly, we found very low levels of p53 stabilization in the liver of the infected mice in a strain-independent manner despite the strong inflammation and bacteria-induced DNA damage ( Figure 5 ). One possible explanation is that the levels of p53 in inflamed colon and liver are orchestrated in a tissue-dependent manner by the presence of different cellular subsets and/or different patterns of chemokines/cytokines in a cell non-autonomous manner.