TRAF6-mediated activation of xenophagy facilitates intracellular Salmonella proliferation

TRAF6-mediated activation of xenophagy facilitates intracellular Salmonella proliferation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TRAF6-mediated activation of xenophagy facilitates intracellular Salmonella proliferation Pengcheng Zhang, Mengling Huang, Chunchen Zhang, Haihua Ruan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8962384/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract TRAF6 is the unique member of the TRAF family with E3 ubiquitin ligase activity, playing a critical role in initiating autophagy and regulating its biological functions. Xenophagy is widely recognized as a specialized autophagy system. While emerging evidence has linked TRAF6 to bacterial-induced xenophagy, the underlying regulatory mechanism remains largely elusive. Following Salmonella infection, the intracellular Salmonella load in Traf6 −/− MEF cells, was lower than that in Traf6 +/+ MEF cells. After the stable Atg5 gene knockdown cell line was constructed and 3-Methyladenine(3-MA) was used to inhibit xenophagy, the results were consistent with those observed in Traf6 −/− MEF cells. The accumulation of LC3-II protein in Traf6 −/− MEF cells was significantly increased, compared with Traf6 +/+ MEF cells counterparts, suggesting xenophagy may be impaired in Traf6 −/− MEF cells. Furthermore, we identified conserved xenophagy related genes by transcriptome sequencing, which provided a molecular basis for the conclusion that xenophagy was blocked in Traf6 −/− MEF cells. Simultaneously, Western blot analysis indicated that NDP52 is the adaptor protein of cellular xenophagy. Our current findings demonstrate that TRAF6 promotes Salmonella proliferation within host cells, and notably, the expression of the receptor protein NDP52 is closely associated with this process. Depletion of TRAF6 results in reduced protein levels of DEPTOR, FOXO3, and LAMP1, whereas the protein level of LC3-II is upregulated, thereby significantly impairing the xenophagy process. TRAF6 Xenophagy Salmonella NDP52 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key member of the TNF receptor-associated factor (TRAF) protein family, is essential for regulating Toll-like receptor (TLR)-mediated signaling pathways [1] . TLRs play a pivotal role in initiating innate immunity and inducing adaptive immune responses by recognizing distinct pathogen-associated molecular patterns (PAMPs) from various pathogens [2] . As an E3 ubiquitin ligase, TRAF6 can catalyze the formation of polyubiquitin chains on target proteins, a process mediated by the ubiquitin-conjugating enzyme complex Ubc13/Uev1A [3] . This ubiquitination event is critical for activating downstream signaling pathways, including nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK), which further regulate inflammatory responses and immune activation. Salmonella , a major global foodborne pathogen, endangers public health, and host cells rely on xenophagy to selectively clear intracellular Salmonella via lysosomal degradation. Our previous observations of TRAF6-mediated ubiquitination of SopB highlight the importance of TRAF6 during Salmonella infection, where SopB suppresses host cell apoptosis by binding to TRAF6 and preventing mitochondrial reactive oxygen species (ROS) generation [4] . TRAF6 can simultaneously regulate the signal transduction induced by SPI-I effector proteins and the phosphorylation activation of Signal Transducer and Activator of Transcription 3 (STAT3); this dual regulation is involved in intracellular signal transduction cascades. Such a process facilitates the intracellular growth of Salmonella [5] .In the context of Salmonella infection, TRAF6 controls TLR4-induced autophagy through the ubiquitination of BECN1 (beclin 1). Specifically, TLR4 signaling triggers K63-linked ubiquitination of BECN1 via a mechanism dependent on TRAF6 [1] . Notably, TRAF6 and A20 function cooperatively to regulate TLR4-induced autophagy by modulating the K63 ubiquitination status of Beclin1 [6] . Yoon Min et al. [1] demonstrated that PRDX1 inhibits the ubiquitination of Beclin1—an event required for autophagy activation—by suppressing the ubiquitin ligase activity of TRAF6. This finding directly indicates that TRAF6 plays a key role in driving autophagy activation. Taken together, these results strongly demonstrate that the ubiquitin-ligase activity of TRAF6 is essential for regulating cellular autophagy activation during Salmonell a infection. Moreover, the ability of TRAF6 to coordinate both apoptotic suppression and autophagic modulation underscores its multifaceted role in facilitating Salmonella survival within host cells. Xenophagy is primarily recognized as a specialized autophagic system that clears invading pathogens, functioning as a defensive response within host cells [7] . It targets pathogens such as bacteria and viruses, which are eventually degraded by lysosomes. Similar to other forms of selective autophagy, xenophagy relies on autophagy receptors and core autophagy proteins for its execution. Key autophagy receptors—including p62 (sequestosome 1, SQSTM1), NBR1 (neighbor of BRCA1), NDP52 (nuclear dot protein 52 kDa), and optineurin—bridge ubiquitinated pathogens and microtubule-associated protein 1 light chain 3 (LC3) [8] . This interaction promotes the sequestration of invading bacteria into autophagic vesicles, thereby recruiting the autophagic machinery to eliminate intracellular bacteria [9] . Numerous studies have confirmed that autophagy, including xenophagy, mediates the degradation of intracellular bacteria. For instance, after invading HeLa cells, Staphylococcus aureus was transported to autophagosomes; microscopy observations revealed that S. aureus was encapsulated in double-membraned structures and colocalizes with LC3 [10] . Other bacterial pathogens, such as Salmonella Typhimurium , Shigella flexneri , and Listeria monocytogenes , are also targets of xenophagy [11] . Notably, Jing Zhuang et al. [12] suggested that A77 1726, the active metabolite of leflunomide, can restrict intracellular Salmonella growth in vitro by enhancing xenophagy. This finding highlights the potential of xenophagy as a modifiable target for controlling bacterial infections.However, ongoing research has revealed that some pathogens, such as Shigella and Listeria, have evolved strategies to evade xenophagic clearance. Furthermore, Salmonella has been demonstrated to hijack autophagic processes for its own replication in certain contexts [13] . For example, after infecting intestinal epithelial cells, intracellular Salmonella relies on chaperone-mediated autophagy (CMA)—specifically the host proteins LAMP-2A and Hsc73—to acquire nutrients. These proteins transport cytosolic components to lysosomes for degradation, and the resulting peptides support Salmonella ’s intracellular growth [14] . This mechanism illustrates how Salmonella can exploit autophagy to secure nutrients for proliferation. However, the specific role of xenophagy in regulating Salmonella proliferation remains poorly understood. Therefore, the objective of this study was to determine the functional role of TRAF6 in xenophagy. Our results showed that host cells activate the expression of the adaptor protein NDP52 via TRAF6, which in turn positively regulates the expression of xenophagy-related proteins DEPTOR, FOXO3, LAMP1, and LC3, and activates host cell xenophagy. We also demonstrated that TRAF6-mediated xenophagy facilitates intracellular Salmonella proliferation. MATERIALS AND METHODS Bacterial strains, cells, growth conditions The wild-type strain of Salmonella enterica serovar typhimurium LT2 (S. typhimurium LT2) used in this study was obtained from the China General Microbiological Culture Collection Center (CGMCC 7020). The human intestinal epithelial cell line (Henle-407) was purchased from the American Type Culture Collection (ATCC). Wild-type mouse embryonic fibroblasts (MEFs) and Traf6 −/− MEF cells were generously donated by Dr. Jun-ichiro Inoue and Dr. Jin Gohda from the University of Tokyo. All cells were cultured in antibiotic-free Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) at 37℃ in an atmosphere of 5% CO 2 . Bacterial infections, transfection, Western blotting and other reagents Cells at a confluency of 80% were starved in DMEM media deficient in FBS for 2 hours. Starved cells were infected with Salmonella at a multiplicity of infection (MOI) of 20(bacteria: cells = 20:1) for 1 hour. The infected cells were subsequently incubated with DMEM and 100 µg/mL gentamycin for 30 min. Finally, the infected cells were washed with prewarmed PBS and incubated with DMEM and 10µg/mL gentamycin until the indicated time. Transfection and Western blots were performed as described previously [ 15 ] . Quantification of protein bands in images of Western blots were measured with ImageJ software. Rabbit-anti-LC3, Mouse-anti-ATG5 and Rabbit-anti-FOXO3 were all purchased from Servicebio. Rabbit-anti-REDD1 and Rabbit-anti-DEPTOR were both purchased from Proteintech Group. Rabbit-anti-NDP52, Rabbit-anti-p62, Rabbit-anti-OPTN and anti-ubiquitin antibodies were all purchased from Cell Signaling Technology. Rabbit-anti-LAMP1, Rabbit-anti-NBR1, Rabbit-anti- Salmonella , Rabbit-anti- Salmonella (biotin) and Rabbit-anti-ATG9 were all purchased from abcam. Construction of a stable Atg5 gene knockdown cell Recombinant expression vector( pLV shRNA- EGFP (2A)- Puro - Atg5 -shRNA) with green fluorescence signal, Puro resistance gene, and knockdown Atg5 gene was constructed and transfected into 293T cells to obtain lentivirus plasmid system. The green fluorescence signal of the acquired lentivirus infected Henle-407 cells was observed under an inverted fluorescence microscope. Purinomycin resistance screening were used to obtain high-purity Henle-407 infected cells. The Henle-407 cell line stably knockdown the Atg5 gene was identified by Western blot assay. the landscape of transcriptome Total RNA was extracted in accordance with the TRIzol reagent (Invitrogen, Shanghai, China) instructions. Traf6 +/+ MEF and Traf6 −/− MEF was selected as cell model for the following studies. The transcriptome sequencing was performed on the RNA of the Salmonella infected Traf6 +/+ MEF and Traf6 −/− MEF at 0 h, 2 h and 8 h. A total of six groups of samples, each group of samples prepared three biological parallel experiments, was then performed on the Illumina HiSeq 4000 platform. The clean reads were obtained after discarding adaptor sequences, low-quality reads (Q-value < 20) and the reads containing more than 10% ambiguous ‘N’ bases (Table S1 ). The GC content was approximately 48% in the eighteen samples. The biological replicates produced comparable data (Table S1 ). Hisat2 software was used to compare the filtered 18 sequenced clean data with the mouse genome reference sequence version number GRCm38.p5 sequence, Total mapped value (statistics of the number of sequenced sequences that can be located on the genome): between 90.38 ~ 95.04%. The high similarity (Fig. 3 A, r > 0.84) among the three biological replicates from 18 samples demonstrated that the RNA-Seq results were consistent. Building Library Sequencing RNA was quantified using the Agilent 2100 Bioanalyzer (Agilent), NanoDrop (Thermo Fisher Scientific) and 1% agarose gel. Samples with RNA quality numbers ≥ 6 were selected for RNA library preparation in an ISO/IEC17025-accredited protocol (TruSeq RNA library preparation kit v2, Illumina, San Diego, CA). Bioinformatic Analysis Sequence information and quality were evaluated through the establishment of a library and the original sequencing obtained by high-throughput sequencing. Low quality spliced raw reads were filtered to obtain high quality clean reads for subsequent sequencing analysis. The most commonly used measure of gene expression level was FPKM(Fragments Per Kilo bases per Million reads). FPKM could detect the effects of gene length on reading and depth. Volcanic maps were used to estimate the overall distribution of differential genes. During the GO enrichment analysis, we considered p < 0.05 as significantly enriched. KEGG is a database that can systematically analyze gene function and genomic information. We used KOBAS software to conduct a holistic study on gene and expression information in the KEGG pathway. RT-qPCR of Related Genes The transcript abundance of 10 randomly selected DEGs were analyzed, including 5 up-regulated genes and 5 down regulated genes, in Salmonella infected TRAF6 deficient cells versus wild-type cells at 2 h and 8 h. Primer 5.0 software (PREMIER Biosoft, Palo Alto, CA, USA) was used to design the specific primers, as shown in Table S2 . In addition, in order to explore the RNA level of xenophagy-related factors, we used RT-qPCR to detect the expression of xenophagy-related genes in TRAF6-deficient cells and wild cells after Salmonella infection, at 0 h, 2 h, 4 h, 8 h and 16 h. Primer 5.0 software was used to design the specific primers, as shown in Table S2 , and the primers were synthesized by GENEWIZ. Statistical Analyses SPSS 16.0 statistical software was used for statistical analysis of the experimental data results, and the experimental results were expressed as 𝑥±s. Statistical significance was calculated by a two-tail distributed paired Student’s t -tests. Values of p < 0.05 were considered significantly different. RESULT TRAF6 may promotes xenophagy and causes the proliferated of intracellular Salmonella Recent reports have suggested that TRAF6 has several different functions in multiple signaling pathways [16, 17] . It is an E3 ubiquitin ligase and is widely involved in autophagy induction due to its autoubiquitination and heteroubiquitination activities [6, 18] . Autophagy is critically associated with bacterial elimination in infectious diseases [19] . TRAF6 is functionally implicated in bactericidal activity by regulating mitochondrial reactive oxygen species (mROS) [20] and in autophagy activation through ubiquitination of BECN1 [21] . The autophagy of foreign entities, such as bacteria, viruses, and other pathogens, is called xenophagy. However, little is known about the relationship between TRAF6 and xenophagy. Therefore, we investigated the effect of TRAF6 on Salmonella replication during xenophagy. Traf6 +/+ MEF cells and Traf6 −/− MEF cells were infected with S. typhimurium LT2 at a multiplicity of infection (MOI) of 20 (bacteria:cells = 20:1). The growth curve of intracellular Salmonella was shown in Fig. 1. Compared with Traf6 +/+ MEF cells, there was no significant difference in the total number or reproduction rate of total intracellular Salmonella at the early stage(0 h-2 h) of infection. However, with the prolongation of infection time, the total number of intracellular Salmonella in Traf6 −/− MEF cells at 8 h-16 h post-infection, was significantly lower than that in Traf6 +/+ MEF cells. This result suggests that TRAF6 deficiency may inhibit xenophagy, thereby reduceing the reproduction rate of intracellular Salmonella . To further verify the effect of xenophagy on replication of Salmonella , Salmonella enumeration experiments was performed under the treatment of 3-methyladenine(3-MA), a xenophagy inhibitor. Whether Henle-407 cells were treated with 3-MA, the number of Salmonella increased slowly in a time-dependent manner(Fig. 1B). Despite this, the total number of intracellular Salmonella in Henle-407 cells treated with 3-MA, was lower than that in Henle-407 cells without 3-MA. As expected, the treatment of the xenophagy inhibitor 3-MA induced the marked inhibition of reproductive rate of Salmonella in cells (Fig. 1B). (A) Growth curve of the Salmonella in Traf6 +/+ MEF cells and Traf6 −/− MEF cells. (B) Dynamic growth curves of Salmonella in Henle-407 cells. (C) Fluorescent image of Atg5 gene knockdown cell line in Henle-407 cells. (D) Dynamic growth curves of Salmonella in siAtg5 Henle-407 cells. Additionally, knockdown of Atg5 gene(which plays a vital role in autophagy regulation) in cells can inhibit xenophagy [22] . To better understand the effect of xenophagy on replication of Salmonella , a Henle-407 cell line with stablely knockdown of Atg5 was successfully constructed(Fig. 1C, Figure S1A, Figure S1B), and the knockdown of Atg5 gene had no inhibitory effect on the reproduction ability of Henle-407 cells(Figure S1C). Compared with Henle-407 cells, there was no significant difference in the total number of total intracellular Salmonella in siAtg5 Henle-407 cells at the early stage(0 h-2 h) of infection (Fig. 1D). However, with prolonged infection time, the total number of intracellular Salmonella in siAtg5 Henle-407 cells at 8 h-16 h post-infection was significantly lower than that in wild Henle-407 cells. These results suggest that inhibition of xenophagy reduces the replication rate of total intracellular Salmonella . This means that the absence of xenophagy results in the reduction of reproductive rate of Salmonella. It is speculated that the deletion of TRAF6 causes a decrease in xenophagy, thereby leading to a reduction in the reproductive rate of Salmonella . TRAF6 activates the xenophagy of mouse embryo fibroblast We next sought to further verify the relationship between TRAF6 and xenophagy. Traf6 +/+ MEF cells and Traf6 −/− MEF cells were infected with Salmonella , and the whole cell lysate was collected at 0 h, 2 h, 4 h, 8 h, and 16 h post-infection. The detection of LC3-I/II conversion is a gold indicator of autophagy flow detection [23] . Western blot analysis indicated that, the amount of LC3-II protein gradually increased and accumulated as the infection time prolonged(Fig. 2A). Interestingly, compared with the Traf6 +/+ MEF cells, the accumulation of LC3-Ⅰ and LC3-II protein in Traf6 −/− MEF cells was significantly increased(Fig. 2A). Based on mechanistic analysis, the accumulation of LC3 in Traf6 −/− cells maybe due to an increase in synthetic sources or a decrease in degradation pathways. To address this conjecture, RT-qPCR was performed to detect the expression of LC3 mRNA in Traf6 +/+ MEF cells and Traf6 −/− MEF cells at 0 h, 2 h, 4 h, 8 h and 16 h after infection of Salmonella . Compared with Traf6 +/+ MEF cells, the LC3 mRNA level in Traf6 −/− MEF cells decreased between 0 and 16 hours post-infection. Therefore, in Traf6 −/− MEF cells, LC3-II protein accumulates in large quantities, while the transcription level of LC3 is significantly reduced. Thus, the synthesis source was reduced, and the degradation pathways are impaired, indicating that the deletion of TRAF6 leads to the inhibition of cellular xenophagy(induced by Salmonella infection). To further verify this result, Western blot was performed to detect the protein levels of LC3 in cells stimulated with the late stage inhibitor bafilomycin A1(Baf A1) of autophagy. Intracellular autophagy lysosome degradation was inhibited, and at the same time, the observed changes in protein level of LC3-II represented changes in the number of autophagosomes [23] . As shown in Fig. 2C and Fig. 2D, we found that the protein level of LC3-II in group Traf6 +/+ MEF cells treated with Baf A1 was significantly higher than those in group Traf6 −/− MEF cells treated with Baf A1, indicating that TRAF6 can promote the formation of xenophagosome. These findings indicate that TRAF6 enhances xenophagic flux. (A) Effect of TRAF6 on the expression of LC3 during Salmonella infection. GAPDH was detected as a loading control. (B) RT-qPCR determination of the relative expression of LC3 gene after Salmonella infection from 0 hours to 16 hours. * indicates P < 0.05, ** indicates P < 0.01(Student’s t -tests). (C) and (D) Detection of xenophagic flux under late autophagy inhibitor. Quantification of the fold LC3-II (relative to the levels in cells without Baf A1). Values are means (± SD) for three independent experiments. * indicates P < 0.05, ** indicates P < 0.01(Student’s t -tests). (E) Functional replenishment experiment for TRAF6 in Traf6 −/− MEF cells. Next, we transfected the plasmid pEGFP-N1-TRAF6 into Traf6 +/+ MEF cells and Traf6 −/− MEF cells. Positive transformants were infected with Salmonella , and the expression of LC3 protein was detected in infected cells. Western blot analysis showed that LC3-II levels in the Traf6 −/− MEF cells+ pEGFP-N1 group was significantly higher than those in Traf6 −/− MEF cells+ pEGFP-N1-TRAF6 group (Fig. 2E). This indicates that the TRAF6 functional complementation was successful. The result was consistent with the comparison between the Traf6 −/− MEF cells+ pEGFP-N1 group and Traf6 +/+ MEF cells+ pEGFP-N1 . It was speculated that TRAF6 enhabced the degradation of xenophagosomes. In a summary, TRAF6 promoted xenophagic flux. Next, we transfected the plasmid pEGFP-N1-TRAF6 into Traf6 +/+ MEF cells and Traf6 −/− MEF cells. Positive transfectants were infected with Salmonella , and LC3 protein expression was detected in the infected cells. Western blot analysis showed that LC3-II levels in the Traf6 −/− MEF cells+pEGFP-N1 group were significantly higher than those in the Traf6 −/− MEF cells+pEGFP-N1-TRAF6 group (Fig. 2E). This indicates that TRAF6 functional complementation was successful. The result was consistent with the comparison between the Traf6 −/− MEF cells+pEGFP-N1 group and the Traf6 +/+ MEF cells+pEGFP-N1 group. It was speculated that TRAF6 enhanced the degradation of xenophagosomes. In summary, TRAF6 promoted xenophagic flux. TRAF6 plays an important role in bacterial carcinogenesis Our results demonstrated that TRAF6 promoted xenophagic flux during Salmonella infection. However, the molecular mechanisms by which TRAF6 participates in xenophagic activity remain poorly understood. To further investigate the molecular mechanisms underlying the TRAF6-regulated xenophagy pathway, six RNA libraries were constructed (at 0 h, 2 h and 8 h post-infection of Traf6 +/+ MEF cells and Traf6 −/− MEF cells with Samonella ). High inter-sample reproducibility (Pearson’s r > 0.99 among biological replicates) confirmed the robustness of the transcriptional datasets (Fig. 3A). Pearson correlation analysis revealed substantially lower correlation coefficients between Traf6 +/+ MEF cells and Traf6 −/− MEF cells (r = 0.72–0.89), indicating that TRAF6 deficiency induces a systematic rewiring of the host transcriptome across infection time points. Principal component analysis (PCA) resolved these differences further: genotype constituted the dominant source of transcriptional variance (PC1, 25.2%), with Traf6 +/+ MEF cells and Traf6 −/− MEF cells populations segregating into distinct clusters (Figure S2). While Traf6 +/+ MEF cells exhibited a coordinated temporal trajectory within the PC1-PC2 coordinate system, Traf6 −/− MEF cells deviated markedly along PC2 (21.8% variance) at 8 hours post-infection, occupying a unique transcriptional space indicative of dysregulated late-phase immune signaling. These data establish TRAF6 as a critical node governing the temporal dynamics of infection-induced transcriptional responses, whose absence precipitates an aberrant, genotype-specific transcriptomic state at late infection stages. We identified differentially expressed genes (DEGs) by analyzing samples collected at different time points post-infection, comparing Traf6 −/− MEF cells and Traf6 +/+ MEF cells infected with Salmonella . Compared with Traf6 +/+ MEF cells infected with Salmonella , Traf6 −/− MEF cells exhibited 2861 and 2617 DEGs at 2 hours and 8 hours post-infection, respectively. Among these DEGs, there were 1,759 and 1,455 upregulated genes, and 1,102 and 1,162 downregulated genes at the two time points (Fig. 3B, Fig. 3C, Fig. 3D). The number of DEGs differed significantly between the 2 h and 8 h groups. (A) Pairwise correlation matrix of transcriptomic profiles across Traf6 genotypes and infection time points. Pearson correlation heatmap of RNA-seq data from Traf6 +/+ MEF cells and Traf6 −/− MEF cells collected at 2 and 8 hours post-infection. Rows and columns represent individual samples grouped by genotype, time point, and biological replicate (A, B, C; n = 3 per group). (B) The number of DEGs in the comparison of the groups, the orange bars indicate the up-regulated genes, and the blue bars indicate the down-regulated genes. (C) Volcano plot map of Traf6 +/+ -2h vs Traf6 −/− -2h. (D) Volcano plot map of Traf6 +/+ -8 h vs Traf6 −/− -8 h. (E) and (F) Venny plot analysis screens 2 h DEGs and 8 h DEGs. To validate the accuracy and reproducibility of RNA-Seq data, we assessed the transcript levels of 10 randomly selected differentially expressed genes (DEGs) via RT-qPCR, including 5 upregulated and 5 downregulated genes from the RNA-Seq dataset (Additional Files 1 and 2). All 5 upregulated genes exhibited significant upregulation in Salmonella -infected Traf6 −/− MEF cells at 2 h and 8 h post-infection(Figure S3A and Figure S3B). Moreover, all 5 downregulated genes showed significant downregulation in Salmonella -infected Traf6 −/− MEF cells at 2 h and 8 h post-infection(Figure S3A and Figure S3B). The results were consistent with those from the RNA-Seq data. confirming the reliability of transcriptomic changes in gene expression. In order to quantify the DEGs in Traf6 +/+ MEF cells and Traf6 −/− MEF cells at 2 h and 8 h post- Salmonella infection, Venny plot analysis was performed(Fig. 3E, 3F). Specifically, the left blue portion represents DEGs at 2 h post-infection after excluding common DEGs in the uninfected group, totaling 1,229 DEGs. Similarly, the right orange portion corresponds to 1,035 DEGs identified at 8 h post- Salmonella infection (Fig. 3F). KEGG enrichment analysis was subsequently conducted to screen key genes on the important pathways. The pathways with significant changes ( p -value ≤ 0.05) in Traf6 −/− MEF cells were identified using the KEGG database. The top 20 most significantly enriched KEGG pathways at 2 h and 8 h post- Salmonella infection are shown in Figure S4 (full datasets are provided in Additional Files 3 and 4). The ‘MicroRNAs in cancer’, ‘Human papillomavirus infection’, ‘Focal adhesion’, ‘Pathways in cancer’, and ‘AGE-RAGE signaling pathway in diabetic complications’ pathways were the most prominently represented. It shows that TRAF6 plays an important role in bacterial carcinogenesis. Two xenophagy-related pathways were screened out from the results of KEGG enrichment, specifically Autophagy-animal(ko04140) and Mitophagy-animal(ko04137) signaling pathway(Table 1). Meanwhile, gene expression was referenced based on gene expression abundance as measured by FPKM values, with a threshold of “1”used to determine gene detectability. Using this criterion, we screened 6 DEGs, namely LC3 , c-Jun , Ddit4 , Deptor , Lamp1 and FoxO3 (Table 2). Table 1 Statistics of DEGs in KEGG pathway related to xenophagy Salmonella infection 2 h Salmonella infection 8 h Gene log 2 Fold Change Description Gene log 2 Fold Change Description Autophagy-animal(ko04140) Deptor 1.65196 DEP domain containing MTOR-interacting protein Deptor 1.64494 DEP domain containing MTOR-interacting protein Lamp1 -1.05129 lysosomal-associated membrane protein 1 Lamp1 -1.01769 lysosomal-associated membrane protein 1 LC3 -1.19786 gamma-aminobutyric acid (GABA) A receptor-associated protein-like 2 Prkaa2 -1.07725 protein kinase, AMP-activated, α-2 catalytic subunit Pik3cd 1.30285 phosphatidylinositol 3-kinase catalytic delta polypeptide —— —— —— Ddit4 1.19923 DNA-damage-inducible transcript 4 —— —— —— Mitophagy–animal(ko04137) Jun -1.23794 jun proto-oncogene FoxO3 -1.03999 Forkhead box O3 LC3 -1.19786 gamma-aminobutyric acid (GABA) A receptor-associated protein-like 2 —— —— —— Note: —— means no signal Table 2 FPKM values of Xenophage-related DEGs Gene Protein Salmonella infection 2 h Salmonella infection 8 h Traf6 +/+ Traf6 −/− TrafF6 +/+ Traf6 −/− LC3 LC3 61.52 27.05 —— Jun c-JUN 100.10 43.51 —— Ddit4 REDD1 11.55 27.72 —— Pik3cd PI3K 0.22 0.61 —— Deptor DEPTOR 1.83 6.04 1.40 4.39 Lamp1 LAMP1 659.01 327.11 594.76 291.15 Prkaa2 AMPK —— 0.66 0.30 FoxO3 FOXO3 —— 12.20 5.88 Note: —— means no signal TRAF6 can activate the c-Jun pathway by promoting the phosphorylation and activation of JNK, or directly interact with c-Jun to enhance its stability by inhibiting degradation. The ROS-induced JNK/c-Jun pathway activates SIRT1 and triggers autophagy as an adaptive response to protect cells from fluoride-induced damage [24] . REDD1 regulates cell growth, proliferation and survival by inhibiting the activity of mammalian target of rapamycin complex 1 (mTORC1) [25, 26] . mTORC1 negatively regulates autophagy. DEPTOR is a negative regulator of mTORC1 and mTORC2 signaling pathways, which inhibits the kinase activity of both complexes [27] . FOXO3, a transcriptional activator, can recognize and bind to the DNA sequence 5'-[AG]TAAA[TC]A-3' and regulate different processes, such as apoptosis and autophagy [28] . LAMP1, a lysosomal-associated membrane glycoprotein 1, localized to the xenolysosomal membrane, is positively correlated with the level of xenophagy [29] . Xenophagy was blocked in Traf6 −/− MEF cells We next investigated the effect of TRAF6 on the xenophagy signaling pathway. Traf6 +/+ MEF cells and Traf6 −/− MEF cells were infected with Salmonella , and cell lysates were harvested at 0 h, 2 h, 4 h, 8 h, and 16 h post-infection for Western blot analysis. (A) The expression of REDD1 was detected at protein level. GAPDH was detected as a loading control. (B) Protein level of DEPTOR, FOXO3 and LAMP1. GAPDH was detected as a loading control. There was no difference in the protein levels of REDD1 between Traf6 −/− MEF cells and Traf6 +/+ MEF cells, indicating that REDD1 is not involved in the xenophagy regulated by TRAF6(Fig. 4A). Furthermore, the results also indicate that TRAF6 does not affect cell proliferation. Transcriptome analysis revealed that, following Salmonella infection, the transcription level of Deptor in Traf6 −/− MEF cells was significantly higher than that in Traf6 +/+ MEF cells (Table 1). But the protein level of DEPTOR in Traf6 −/− cells decreased, which resulted in mTOR activation and hindered xenophagy(Fig. 4B). Compared with Traf6 +/+ MEF cells, the transcriptional level of FoxO3 in Traf6 −/− MEF cells was lower at 8 hours(Table 1). Accordingly, FoxO3 protein levels exhibited a brief increase at 2 h followed by a sustained decrease, indicating that the xenophagy was initially induced but subsequently blocked in Traf6 −/− MEF cells (Fig. 4B). Compared with Salmonella -infected Traf6 +/+ MEF cells, both of the transcriptional levels of LAMP1 and protein levels of LAMP1 in Traf6 −/− MEF cells, showed a brief increase followed by a sustained decrease (Fig. 4B, Table 1), further supporting that xenophagy was initially induced and was blocked in Traf6 −/− MEF cells. Collectively, the above results indicate that the knockout of TRAF6 negatively impacts the protein levels of DEPTOR, FOXO3 and LAMP1, leading to the inability to further form xenophagy in Traf6 −/− MEF cells. Given the blockade of xenophagy in TRAF6-deficient cells and the associated dysregulation of DEPTOR, FOXO3, and LAMP1, what molecular events drive the failure of xenophagic flux under these conditions, and how are they linked to the loss of TRAF6 function? NDP52 was an adaptor in xenophagy regulated by TRAF6 Over the past decade, it has been increasingly recognized that autophagy/xenophagy exerts a pivotal role in antimicrobial immunity. Diverse molecular mechanisms underlying xenophagic target recognition have been identified. Adaptor proteins such as p62, NDP52, and NBR1 selectively recognize PAMPs and danger signals, which are essential for the effective clearance of pathogens [30] . To further explore reason of that xenophagy was blocked in Traf6 −/− MEF cells, Western blot was performed to examine detect the four adaptor proteins, including p62, OPTN, NBR1 and NDP52 [31] . The results showed that there was no significant difference in the expression of p62, OPTN, and NBR1 in Traf6 +/+ MEF cells and Traf6 −/− MEF cells. Notably, the protein level of NDP52 in Traf6 ⁻/⁻ MEF cells exhibited a transient increase within 2 hours post-Salmonella infection, followed by a sustained decline(Fig. 5A), a pattern consistent with the dynamic changes in DEPTOR, FOXO3, and LAMP1 protein levels(Fig. 4B). Western blot analysis indicates that NDP52, as an adaptor protein, is involved in TRAF6-mediated xenophagy induced by Salmonella infection. Regarding transcriptional levels, NDP52 expression was initially lower in Traf6 −/− cells compared to Traf6 +/+ MEF cells(0–6 h), followed by higher expression at 8 h and 16 h(Fig. 5C). NDP52 may act as a key receptor in the xenophagy process influenced by TRAF6. Ubiquitination of NDP52 plays an important role in autophagy/ xenophagy [32, 33] . The immunoprecipitation technique was performed to detect the ubiquitination of NDP52. Polyubiquitinated NDP52 was readily detectable 2 h post- Salmonella infection (Fig. 5B) The amount of ubiquitinated NDP52 gradually increased and peaked at 8 h post-infection, in Traf6 +/+ MEF cells. In comparison, minimal NDP52 ubiquitination was detected in Salmonella -infected Traf6 −/− MEF cells(Fig. 5B), indicating a requirement for TRAF6 in the ubiquitination of NDP52 during Salmonella infection. These findings suggest that TRAF6 promotes xenophagosome formation during Salmonella infection, in which NDP52 plays a crucial role as a receptor for xenophage formation. (A) Detection of adaptor proteins in xenophagy (B) NDP52 undergoes ubiquitination during xenophagy, caused by TRAF6. (C) The transcription level of NDP52 in Traf6 −/− MEF cells relative to that in Traf6 +/+ MEF cells. * indicates P < 0.05, ** indicates P < 0.01(Student’s t -tests) Discussion Xenophagy, a critical form of selective autophagy, exerts a crucial role in the host's innate immune defense, with TRAF6 functioning as a key regulator of xenophagic activity. In the present study, we report that the loss of TRAF6 results in the inhibition of cell xenophagy induced by Salmonella infection [ 4 , 5 , 34 ] . Upon infecting host cells, Salmonella proliferated within the cells and triggered the xenophagy pathway of the cells. If xenophagy was inhibited, the proliferation of Salmonella would be hindered. In this study, Henle-407 cells treated with 3-MA and subsequently infected with Salmonella exhibited a significant reduction in intracellular Salmonella burden compared to untreated counterparts (Fig. 1 ). Furthermore, at 16 h post- Salmonell a infection, Henle-407 cells transfected with siAtg5 showed a markedly lower intracellular Salmonella load than wild-type Henle-407 cells (Fig. 1 ). In Traf6 −/− cells, where xenophagy is blocked, LC3 protein levels were significantly increased, accompanied by a notable decrease in intracellular Salmonella (Fig. 1 , Fig. 2 ). These results show that TRAF6 can promote xenophagy flux. However, the specific mechanism of which TRAF6 affects xenophagy remain to be further explored. Through high-throughput transcriptome sequencing analysis of mRNA expression profiles in Traf6 ⁺/⁺ and Traf6 ⁻/⁻ MEF cells infected with Salmonella at 0, 2, and 8 hours, we identified six candidate genes: LC3, c-JUN, Ddit4, Deptor, LAMP1, and FoxO3 (Table 2 ), which are involved in the Autophagy-animal (ko04140) and Mitophagy-animal (ko04137) signaling pathways. As depicted in Fig. 4 , in TRAF6-deficient MEF cells, the protein levels of xenophagy-related genes, including Deptor, FoxO3, and LAMP1, were significantly reduced. These findings are consistent with previous reports [ 27 , 28 ] . Furthermore, we identified NDP52 as an adaptor protein associated with TRAF6-mediated regulation of Salmonella -induced xenophagy. It is well established that the research on xenophagy has been based on the mixed count of two types of intracellular bacteria, which makes it difficult to elaborate and accurately describe the function of xenophagy during Salmonella infection. Because the reproduction modes of the two types of bacteria are completely different, and the biological significance of xenophagy to these two types of bacteria is also different. The proportion of the two types of Salmonella at different infection time points has changed greatly, especially in cytosolic Salmonella . The proportion of cytosolic Salmonella has been at a lower proportion throughout the infection process. In contrast, vacuolar Salmonella has been the predominant form of intracellular Salmonella . This may be because the protein in the cytoplasm is transported to the lysosome for degradation by Chaperone-mediated Autophagy (CMA), and the degraded polypeptides provide nutrients for the reproduction of intracellular Salmonella [ 14 ] . 3-MA is a commonly autophagy inhibitor that acts by mediating Phosphatidylinositol 3-kinase/protein kinase the mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway, which affects cell growth, proliferation, survival and xenophagy [ 35 ] . Salmonella blocks or delays apoptosis of infected cells by activating Akt. 3-MA inhibits the phosphorylation of Akt by inhibiting PI3K, thereby blocking the Akt/mTOR signaling pathway, inhibiting xenophagy and promoting apoptosis [ 36 ] . After xenophagy was inhibited by 3-MA, both cytosolic and vacuolar Salmonella exhibited a lower burden compared to that in normal cells(Fig. 6 ). Although the total number of intracellular Salmonella was increasing, the proportion of the two types of Salmonella varied significantly at different infection time points. Especially, the cytosolic Salmonella population continued to increase throughout the entire infection process. In contrast, vacuolar Salmonella was the predominant form of intracellular Salmonella in the early stages of infection. However, the proportion of the vacuolar Salmonella decreased at the later stage of infection. These results show that xenophagy may facilitate the replication of cytosolic Salmonella . (A) The number of intracellular Salmonella in Henle-407 treated with 3-MA. (B) The number of intracellular Salmonella in siAtg5 -Henle-407. (C and D) The proportion of the two types of Salmonella in different cells. Atg5 mediates the conjugation of LC3 to autophagosomes, thereby preventing lysosome clearance of intracellular Salmonella . Atg5 and Tecpr1 target Shigella to autophagosomes by binding to the Shigella effector protein IcsA [ 37 ] . In this study, the Henle-407 cell line, stably knocked down the Atg5 gene, was constructed via a lentiviral system. Our results indicate that xenophagy exerts distinct biological effects on the intracellular replication of these two Salmonella populations (Fig. 6 ). In siAtg5- transfected Henle-407 cells, the counts of both cytosolic and vacuolar Salmonella were lower than those in Henle-407 cells. This observation may be attributed to the loss of xenophagy, which leads to the disruption of Salmonella -containing vacuoles (SCVs) and the subsequent loss of the proton gradient across the SCV membrane. These changes significantly reduces the expression of Salmonella T3SS SPI-2 effector protein and the reproduction of bacteria in cells [ 38 ] . We believe that future studies should focus on three key directions to deepen understanding of xenophagy in Salmonella infection and boost its translational value. First, it is critical to further dissect the specific molecular mechanisms by which TRAF6 regulates NDP52 ubiquitination—including identifying pivotal E3 ubiquitin ligases, deubiquitinases, and interacting proteins—to fully delineate the TRAF6-NDP52-mediated xenophagy regulatory axis. Second, systematic studies are needed to clarify the molecular basis for xenophagy’s differential effects on cytosolic versus vacuolar Salmonella , with particular emphasis on how reciprocal regulation between Salmonella -containing vacuole (SCV) integrity and xenophagy shapes bacterial intracellular survival. Third, building on the key molecules identified here (e.g., NDP52, TRAF6, SPI-2 effectors), targeted validation studies should assess their feasibility as therapeutic targets for salmonellosis. Collectively, these efforts will not only deepen mechanistic insights into host-xenophagy- Salmonella crosstalk but also lay a solid experimental foundation for developing novel antibacterial strategies against salmonellosis. Declarations Author Contributions Pengcheng Zhang: Conceptualization, Methodology, Investigation, Visualization, Writing - original draft, Writing - review & editing. Mengling Huang: Investigation. Methodology, Writing - original draft. Chunchen Zhang : Investigation, Methodology. Haihua Ruan: Conceptualization, Funding acquisition, Methodology, Resources, Writing - review & editing, Supervision. Funding This work was funded mainly by the National Natural Science Foundation of China (Nos. 31870122) Data Availability Statement The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors. 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Supplementary Files Additionalfile3.xlsx Additionalfile4.xlsx Additionalfile2.xlsx Additionalfile1.xlsx FigureS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 06 May, 2026 Reviewers invited by journal 05 May, 2026 Editor assigned by journal 26 Feb, 2026 Submission checks completed at journal 26 Feb, 2026 First submitted to journal 24 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8962384","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635804921,"identity":"6b7feab7-e69a-4786-9dcf-3e813c39e891","order_by":0,"name":"Pengcheng Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACCTiL+QCUk0C0FrbEBlK18Bg2MBCjRX5287GHX9sO25uzn/n+wKLmMAM/e44Bw88duLUY3DmWbizbdpjZsid3Y4PEscMMkj1vDBh7z+DRIpFjJi257TCbwQ3ejQ2SDYcZDG7kGDAztuFx2Iz8byAtPAY3eB6CtdgT0sJwI4dN8uO2wxJALYwQWyQIaDG4kWYmzfgv3cCyJ81whsSxdB6JM88KDvbidVjyM8kfZ6yBIXb4wWeJGms5/vbkjQ9+4nMYEDDzgKwDMYCRBGIzHMCvgYGB8QdUC+MHQkpHwSgYBaNgRAIA+aVPrW38WIYAAAAASUVORK5CYII=","orcid":"","institution":"Tianjin University of Commerce","correspondingAuthor":true,"prefix":"","firstName":"Pengcheng","middleName":"","lastName":"Zhang","suffix":""},{"id":635804922,"identity":"8107149f-1a7e-4320-94df-96363f028db4","order_by":1,"name":"Mengling Huang","email":"","orcid":"","institution":"Tianjin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Mengling","middleName":"","lastName":"Huang","suffix":""},{"id":635804925,"identity":"aad7fb52-3245-4101-937c-12178f62305e","order_by":2,"name":"Chunchen Zhang","email":"","orcid":"","institution":"Tianjin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Chunchen","middleName":"","lastName":"Zhang","suffix":""},{"id":635804926,"identity":"22f8a675-51a0-4a48-8bb4-f44bb0f8bc98","order_by":3,"name":"Haihua Ruan","email":"","orcid":"","institution":"Tianjin University of Commerce","correspondingAuthor":false,"prefix":"","firstName":"Haihua","middleName":"","lastName":"Ruan","suffix":""}],"badges":[],"createdAt":"2026-02-25 02:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8962384/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8962384/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109222273,"identity":"79b13c64-78b7-4c94-b3d9-93724c12535f","added_by":"auto","created_at":"2026-05-13 21:06:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":244869,"visible":true,"origin":"","legend":"\u003cp\u003eXenophagy promotes the proliferation of intracellular \u003cem\u003eSalmonella.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Growth curve of the \u003cem\u003eSalmonella\u003c/em\u003e in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e MEF cells. (B) Dynamic growth curves of \u003cem\u003eSalmonella\u003c/em\u003e in Henle-407 cells. (C) Fluorescent image of \u003cem\u003eAtg5\u003c/em\u003e gene knockdown cell line in Henle-407 cells. (D) Dynamic growth curves of \u003cem\u003eSalmonella\u003c/em\u003e in \u003cem\u003esiAtg5\u003c/em\u003e Henle-407 cells.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/9e321e45369890e04d86351e.jpg"},{"id":109222327,"identity":"1d475d18-69a0-438a-82ed-22be4c3cfca9","added_by":"auto","created_at":"2026-05-13 21:07:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":280665,"visible":true,"origin":"","legend":"\u003cp\u003eTRAF6 enhances the xenophagy of mouse embryo fibroblast.\u003c/p\u003e\n\u003cp\u003e(A) Effect of TRAF6 on the expression of LC3 during \u003cem\u003eSalmonella\u003c/em\u003e infection. GAPDH was detected as a loading control. (B) RT-qPCR determination of the relative expression of \u003cem\u003eLC3\u003c/em\u003e gene after\u003cem\u003e Salmonella\u003c/em\u003e infection from 0 hours to 16 hours. * indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01(Student’s \u003cem\u003et\u003c/em\u003e-tests). (C) and (D) Detection of xenophagic flux under late autophagy inhibitor. Quantification of the fold LC3-II (relative to the levels in cells without Baf A1). Values are means (± SD) for three independent experiments. * indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01(Student’s \u003cem\u003et\u003c/em\u003e-tests). (E) Functional replenishment experiment for TRAF6 in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e MEF cells.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/35e6eedf848f6d9082454cca.jpg"},{"id":109222330,"identity":"49c8290f-1ae0-49c4-90e1-c463fd968725","added_by":"auto","created_at":"2026-05-13 21:07:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":413145,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome sequencing data statistics.\u003c/p\u003e\n\u003cp\u003e(A) Pairwise correlation matrix of transcriptomic profiles across Traf6 genotypes and infection time points. Pearson correlation heatmap of RNA-seq data from \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e MEF cells collected at 2 and 8 hours post-infection. Rows and columns represent individual samples grouped by genotype, time point, and biological replicate (A, B, C; n = 3 per group). (B) The number of DEGs in the comparison of the groups, the orange bars indicate the up-regulated genes, and the blue bars indicate the down-regulated genes. (C) Volcano plot map of Traf6\u003csup\u003e+/+\u003c/sup\u003e-2h vs Traf6\u003csup\u003e-/-\u003c/sup\u003e-2h. (D) Volcano plot map of Traf6\u003csup\u003e+/+\u003c/sup\u003e-8 h vs Traf6\u003csup\u003e-/-\u003c/sup\u003e-8 h. (E) and (F) Venny plot analysis screens 2 h DEGs and 8 h DEGs.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/bd9b423a5a0227b9b0e8361e.jpg"},{"id":109222328,"identity":"9f370f71-129e-474d-a461-3b54be80fe7c","added_by":"auto","created_at":"2026-05-13 21:07:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48493,"visible":true,"origin":"","legend":"\u003cp\u003eExpression verification of xenophagy-related DEGs.\u003c/p\u003e\n\u003cp\u003e(A) The expression of REDD1 was detected at protein level. GAPDH was detected as a loading control. (B) Protein level of DEPTOR, FOXO3 and LAMP1. GAPDH was detected as a loading control.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/f66d289326854d23207a40aa.jpg"},{"id":109222313,"identity":"8947467d-639b-4a59-a9ef-ab6507d5be1c","added_by":"auto","created_at":"2026-05-13 21:07:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":231302,"visible":true,"origin":"","legend":"\u003cp\u003eNDP52 is an adaptor protein related to TRAF6 function in the xenophagy induced by \u003cem\u003eSalmonella\u003c/em\u003e infection.\u003c/p\u003e\n\u003cp\u003e(A) Detection of adaptor proteins in xenophagy (B) NDP52 undergoes ubiquitination during xenophagy, caused by TRAF6. (C) The transcription level of \u003cem\u003eNDP52\u003c/em\u003e in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e MEF cells relative to that in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+ \u003c/em\u003e\u003c/sup\u003eMEF cells. * indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01(Student’s \u003cem\u003et\u003c/em\u003e-tests)\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/8b6fc5d30309c33020a00d08.jpg"},{"id":109222315,"identity":"4537de3e-4b48-4cf6-889c-fd7a8f45af88","added_by":"auto","created_at":"2026-05-13 21:07:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":318253,"visible":true,"origin":"","legend":"\u003cp\u003eXenophagy facilitates cytosolic \u003cem\u003eSalmonella\u003c/em\u003e to reproduction.\u003c/p\u003e\n\u003cp\u003e(A) The number of intracellular \u003cem\u003eSalmonella\u003c/em\u003ein Henle-407 treated with 3-MA. (B) The number of intracellular \u003cem\u003eSalmonella\u003c/em\u003ein \u003cem\u003esiAtg5\u003c/em\u003e-Henle-407. (C and D) The proportion of the two types of \u003cem\u003eSalmonella\u003c/em\u003ein different cells.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/8abdeff7e3996e369e8afaf3.jpg"},{"id":109249916,"identity":"04e231d3-4a08-46ed-9e4d-25bd941cb12a","added_by":"auto","created_at":"2026-05-14 09:05:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1959668,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/064be777-ac22-4643-b666-2c7efdb6f83f.pdf"},{"id":109222309,"identity":"6a811a61-6f2f-4a8e-9a52-96c005b28662","added_by":"auto","created_at":"2026-05-13 21:07:14","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15317,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/3a6fec4e70a9c096b9a90f26.xlsx"},{"id":109222321,"identity":"8f814ac9-233e-4e17-84e3-248c27266ad3","added_by":"auto","created_at":"2026-05-13 21:07:20","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16456,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/b532c1577876d14af4df900c.xlsx"},{"id":109222335,"identity":"d0799886-1df3-4ac2-9f38-01730ede0dfc","added_by":"auto","created_at":"2026-05-13 21:07:31","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9071146,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/69632686d3c238f4988ce08c.xlsx"},{"id":109222329,"identity":"8cd9d8d2-660f-4363-b240-8c6d217930cf","added_by":"auto","created_at":"2026-05-13 21:07:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9088769,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/8bbe66dc2c251d90bf58097e.xlsx"},{"id":109222326,"identity":"76a8dc76-26d3-4216-a895-b4925a5b2db5","added_by":"auto","created_at":"2026-05-13 21:07:21","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1471076,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8962384/v1/7f3aa698a4f9ca071e069b6b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"TRAF6-mediated activation of xenophagy facilitates intracellular Salmonella proliferation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTumor necrosis factor receptor-associated factor 6 (TRAF6), a key member of the TNF receptor-associated factor (TRAF) protein family, is essential for regulating Toll-like receptor (TLR)-mediated signaling pathways\u003csup\u003e[1]\u003c/sup\u003e. TLRs play a pivotal role in initiating innate immunity and inducing adaptive immune responses by recognizing distinct pathogen-associated molecular patterns (PAMPs) from various pathogens\u003csup\u003e[2]\u003c/sup\u003e. As an E3 ubiquitin ligase, TRAF6 can catalyze the formation of polyubiquitin chains on target proteins, a process mediated by the ubiquitin-conjugating enzyme complex Ubc13/Uev1A\u003csup\u003e[3]\u003c/sup\u003e. This ubiquitination event is critical for activating downstream signaling pathways, including nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK), which further regulate inflammatory responses and immune activation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e, a major global foodborne pathogen, endangers public health, and host cells rely on xenophagy to selectively clear intracellular \u003cem\u003eSalmonella\u003c/em\u003e via lysosomal degradation. Our previous observations of TRAF6-mediated ubiquitination of SopB highlight the importance of TRAF6 during \u003cem\u003eSalmonella\u003c/em\u003e infection, where SopB suppresses host cell apoptosis by binding to TRAF6 and preventing mitochondrial reactive oxygen species (ROS) generation\u003csup\u003e[4]\u003c/sup\u003e. TRAF6 can simultaneously regulate the signal transduction induced by SPI-I effector proteins and the phosphorylation activation of Signal Transducer and Activator of Transcription 3 (STAT3); this dual regulation is involved in intracellular signal transduction cascades. Such a process facilitates the intracellular growth of \u003cem\u003eSalmonella\u003c/em\u003e\u003csup\u003e[5]\u003c/sup\u003e.In the context of \u003cem\u003eSalmonella\u003c/em\u003e infection, TRAF6 controls TLR4-induced autophagy through the ubiquitination of BECN1 (beclin 1). Specifically, TLR4 signaling triggers K63-linked ubiquitination of BECN1 via a mechanism dependent on TRAF6\u003csup\u003e[1]\u003c/sup\u003e. Notably, TRAF6 and A20 function cooperatively to regulate TLR4-induced autophagy by modulating the K63 ubiquitination status of Beclin1\u003csup\u003e[6]\u003c/sup\u003e. Yoon Min et al.\u003csup\u003e[1]\u003c/sup\u003e demonstrated that PRDX1 inhibits the ubiquitination of Beclin1—an event required for autophagy activation—by suppressing the ubiquitin ligase activity of TRAF6. This finding directly indicates that TRAF6 plays a key role in driving autophagy activation. Taken together, these results strongly demonstrate that the ubiquitin-ligase activity of TRAF6 is essential for regulating cellular autophagy activation during \u003cem\u003eSalmonell\u003c/em\u003ea infection. Moreover, the ability of TRAF6 to coordinate both apoptotic suppression and autophagic modulation underscores its multifaceted role in facilitating \u003cem\u003eSalmonella\u003c/em\u003e survival within host cells.\u003c/p\u003e\n\u003cp\u003eXenophagy is primarily recognized as a specialized autophagic system that clears invading pathogens, functioning as a defensive response within host cells\u003csup\u003e[7]\u003c/sup\u003e. It targets pathogens such as bacteria and viruses, which are eventually degraded by lysosomes. Similar to other forms of selective autophagy, xenophagy relies on autophagy receptors and core autophagy proteins for its execution. Key autophagy receptors—including p62 (sequestosome 1, SQSTM1), NBR1 (neighbor of BRCA1), NDP52 (nuclear dot protein 52 kDa), and optineurin—bridge ubiquitinated pathogens and microtubule-associated protein 1 light chain 3 (LC3)\u003csup\u003e[8]\u003c/sup\u003e. This interaction promotes the sequestration of invading bacteria into autophagic vesicles, thereby recruiting the autophagic machinery to eliminate intracellular bacteria\u003csup\u003e[9]\u003c/sup\u003e. Numerous studies have confirmed that autophagy, including xenophagy, mediates the degradation of intracellular bacteria. For instance, after invading HeLa cells, Staphylococcus aureus was transported to autophagosomes; microscopy observations revealed that S. aureus was encapsulated in double-membraned structures and colocalizes with LC3\u003csup\u003e[10]\u003c/sup\u003e. Other bacterial pathogens, such as \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e, \u003cem\u003eShigella flexneri\u003c/em\u003e, and \u003cem\u003eListeria monocytogenes\u003c/em\u003e, are also targets of xenophagy\u003csup\u003e[11]\u003c/sup\u003e. Notably, Jing Zhuang et al.\u003csup\u003e[12]\u003c/sup\u003e suggested that A77 1726, the active metabolite of leflunomide, can restrict intracellular \u003cem\u003eSalmonella\u003c/em\u003e growth in vitro by enhancing xenophagy. This finding highlights the potential of xenophagy as a modifiable target for controlling bacterial infections.However, ongoing research has revealed that some pathogens, such as Shigella and Listeria, have evolved strategies to evade xenophagic clearance. Furthermore, \u003cem\u003eSalmonella\u003c/em\u003e has been demonstrated to hijack autophagic processes for its own replication in certain contexts\u003csup\u003e[13]\u003c/sup\u003e. For example, after infecting intestinal epithelial cells, intracellular \u003cem\u003eSalmonella\u003c/em\u003e relies on chaperone-mediated autophagy (CMA)—specifically the host proteins LAMP-2A and Hsc73—to acquire nutrients. These proteins transport cytosolic components to lysosomes for degradation, and the resulting peptides support \u003cem\u003eSalmonella\u003c/em\u003e’s intracellular growth\u003csup\u003e[14]\u003c/sup\u003e. This mechanism illustrates how \u003cem\u003eSalmonella\u003c/em\u003e can exploit autophagy to secure nutrients for proliferation. However, the specific role of xenophagy in regulating \u003cem\u003eSalmonella\u003c/em\u003e proliferation remains poorly understood.\u003c/p\u003e\n\u003cp\u003eTherefore, the objective of this study was to determine the functional role of TRAF6 in xenophagy. Our results showed that host cells activate the expression of the adaptor protein NDP52 via TRAF6, which in turn positively regulates the expression of xenophagy-related proteins DEPTOR, FOXO3, LAMP1, and LC3, and activates host cell xenophagy. We also demonstrated that TRAF6-mediated xenophagy facilitates intracellular \u003cem\u003eSalmonella\u003c/em\u003e proliferation.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains, cells, growth conditions\u003c/h2\u003e \u003cp\u003eThe wild-type strain of \u003cem\u003eSalmonella\u003c/em\u003e enterica serovar typhimurium LT2 (S. typhimurium LT2) used in this study was obtained from the China General Microbiological Culture Collection Center (CGMCC 7020). The human intestinal epithelial cell line (Henle-407) was purchased from the American Type Culture Collection (ATCC). Wild-type mouse embryonic fibroblasts (MEFs) and Traf6\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MEF cells were generously donated by Dr. Jun-ichiro Inoue and Dr. Jin Gohda from the University of Tokyo. All cells were cultured in antibiotic-free Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) at 37℃ in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial infections, transfection, Western blotting and other reagents\u003c/h2\u003e \u003cp\u003eCells at a confluency of 80% were starved in DMEM media deficient in FBS for 2 hours. Starved cells were infected with \u003cem\u003eSalmonella\u003c/em\u003e at a multiplicity of infection (MOI) of 20(bacteria: cells\u0026thinsp;=\u0026thinsp;20:1) for 1 hour. The infected cells were subsequently incubated with DMEM and 100 \u0026micro;g/mL gentamycin for 30 min. Finally, the infected cells were washed with prewarmed PBS and incubated with DMEM and 10\u0026micro;g/mL gentamycin until the indicated time. Transfection and Western blots were performed as described previously\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Quantification of protein bands in images of Western blots were measured with ImageJ software. Rabbit-anti-LC3, Mouse-anti-ATG5 and Rabbit-anti-FOXO3 were all purchased from Servicebio. Rabbit-anti-REDD1 and Rabbit-anti-DEPTOR were both purchased from Proteintech Group. Rabbit-anti-NDP52, Rabbit-anti-p62, Rabbit-anti-OPTN and anti-ubiquitin antibodies were all purchased from Cell Signaling Technology. Rabbit-anti-LAMP1, Rabbit-anti-NBR1, Rabbit-anti-\u003cem\u003eSalmonella\u003c/em\u003e, Rabbit-anti-\u003cem\u003eSalmonella\u003c/em\u003e(biotin) and Rabbit-anti-ATG9 were all purchased from abcam.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of a stable\u003c/b\u003e \u003cb\u003eAtg5\u003c/b\u003e \u003cb\u003egene knockdown cell\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRecombinant expression vector(\u003cem\u003epLV\u003c/em\u003eshRNA-\u003cem\u003eEGFP\u003c/em\u003e(2A)-\u003cem\u003ePuro\u003c/em\u003e-\u003cem\u003eAtg5\u003c/em\u003e-shRNA) with green fluorescence signal, Puro resistance gene, and knockdown \u003cem\u003eAtg5\u003c/em\u003e gene was constructed and transfected into 293T cells to obtain lentivirus plasmid system. The green fluorescence signal of the acquired lentivirus infected Henle-407 cells was observed under an inverted fluorescence microscope. Purinomycin resistance screening were used to obtain high-purity Henle-407 infected cells. The Henle-407 cell line stably knockdown the \u003cem\u003eAtg5\u003c/em\u003e gene was identified by Western blot assay.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ethe landscape of transcriptome\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted in accordance with the TRIzol reagent (Invitrogen, Shanghai, China) instructions. \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF was selected as cell model for the following studies. The transcriptome sequencing was performed on the RNA of the \u003cem\u003eSalmonella\u003c/em\u003e infected \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF at 0 h, 2 h and 8 h. A total of six groups of samples, each group of samples prepared three biological parallel experiments, was then performed on the Illumina HiSeq 4000 platform. The clean reads were obtained after discarding adaptor sequences, low-quality reads (Q-value\u0026thinsp;\u0026lt;\u0026thinsp;20) and the reads containing more than 10% ambiguous \u0026lsquo;N\u0026rsquo; bases (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The GC content was approximately 48% in the eighteen samples. The biological replicates produced comparable data (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Hisat2 software was used to compare the filtered 18 sequenced clean data with the mouse genome reference sequence version number GRCm38.p5 sequence, Total mapped value (statistics of the number of sequenced sequences that can be located on the genome): between 90.38\u0026thinsp;~\u0026thinsp;95.04%. The high similarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, r\u0026thinsp;\u0026gt;\u0026thinsp;0.84) among the three biological replicates from 18 samples demonstrated that the RNA-Seq results were consistent.\u003c/p\u003e\n\u003ch3\u003eBuilding Library Sequencing\u003c/h3\u003e\n\u003cp\u003eRNA was quantified using the Agilent 2100 Bioanalyzer (Agilent), NanoDrop (Thermo Fisher Scientific) and 1% agarose gel. Samples with RNA quality numbers\u0026thinsp;\u0026ge;\u0026thinsp;6 were selected for RNA library preparation in an ISO/IEC17025-accredited protocol (TruSeq RNA library preparation kit v2, Illumina, San Diego, CA).\u003c/p\u003e\n\u003ch3\u003eBioinformatic Analysis\u003c/h3\u003e\n\u003cp\u003eSequence information and quality were evaluated through the establishment of a library and the original sequencing obtained by high-throughput sequencing. Low quality spliced raw reads were filtered to obtain high quality clean reads for subsequent sequencing analysis. The most commonly used measure of gene expression level was FPKM(Fragments Per Kilo bases per Million reads). FPKM could detect the effects of gene length on reading and depth. Volcanic maps were used to estimate the overall distribution of differential genes. During the GO enrichment analysis, we considered \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as significantly enriched. KEGG is a database that can systematically analyze gene function and genomic information. We used KOBAS software to conduct a holistic study on gene and expression information in the KEGG pathway.\u003c/p\u003e\n\u003ch3\u003eRT-qPCR of Related Genes\u003c/h3\u003e\n\u003cp\u003eThe transcript abundance of 10 randomly selected DEGs were analyzed, including 5 up-regulated genes and 5 down regulated genes, in \u003cem\u003eSalmonella\u003c/em\u003e infected TRAF6 deficient cells versus wild-type cells at 2 h and 8 h. Primer 5.0 software (PREMIER Biosoft, Palo Alto, CA, USA) was used to design the specific primers, as shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. In addition, in order to explore the RNA level of xenophagy-related factors, we used RT-qPCR to detect the expression of xenophagy-related genes in TRAF6-deficient cells and wild cells after \u003cem\u003eSalmonella\u003c/em\u003e infection, at 0 h, 2 h, 4 h, 8 h and 16 h. Primer 5.0 software was used to design the specific primers, as shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, and the primers were synthesized by GENEWIZ.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eSPSS 16.0 statistical software was used for statistical analysis of the experimental data results, and the experimental results were expressed as \u0026#119909;\u0026plusmn;s. Statistical significance was calculated by a two-tail distributed paired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests. Values of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significantly different.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULT","content":"\u003cp\u003e\u003cstrong\u003eTRAF6 may promotes xenophagy and causes the proliferated of intracellular\u003c/strong\u003e \u003cstrong\u003eSalmonella\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecent reports have suggested that TRAF6 has several different functions in multiple signaling pathways\u003csup\u003e[16, 17]\u003c/sup\u003e. It is an E3 ubiquitin ligase and is widely involved in autophagy induction due to its autoubiquitination and heteroubiquitination activities\u003csup\u003e[6, 18]\u003c/sup\u003e. Autophagy is critically associated with bacterial elimination in infectious diseases\u003csup\u003e[19]\u003c/sup\u003e. TRAF6 is functionally implicated in bactericidal activity by regulating mitochondrial reactive oxygen species (mROS)\u003csup\u003e[20]\u003c/sup\u003e and in autophagy activation through ubiquitination of BECN1\u003csup\u003e[21]\u003c/sup\u003e. The autophagy of foreign entities, such as bacteria, viruses, and other pathogens, is called xenophagy. However, little is known about the relationship between TRAF6 and xenophagy.\u003c/p\u003e\n\u003cp\u003eTherefore, we investigated the effect of TRAF6 on \u003cem\u003eSalmonella\u003c/em\u003e replication during xenophagy. \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells were infected with \u003cem\u003eS. typhimurium\u003c/em\u003e LT2 at a multiplicity of infection (MOI) of 20 (bacteria:cells\u0026thinsp;=\u0026thinsp;20:1). The growth curve of intracellular \u003cem\u003eSalmonella\u003c/em\u003e was shown in Fig.\u0026nbsp;1. Compared with \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells, there was no significant difference in the total number or reproduction rate of total intracellular \u003cem\u003eSalmonella\u003c/em\u003e at the early stage(0 h-2 h) of infection. However, with the prolongation of infection time, the total number of intracellular \u003cem\u003eSalmonella\u003c/em\u003e in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells at 8 h-16 h post-infection, was significantly lower than that in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells. This result suggests that TRAF6 deficiency may inhibit xenophagy, thereby reduceing the reproduction rate of intracellular \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo further verify the effect of xenophagy on replication of \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e enumeration experiments was performed under the treatment of 3-methyladenine(3-MA), a xenophagy inhibitor. Whether Henle-407 cells were treated with 3-MA, the number of \u003cem\u003eSalmonella\u003c/em\u003e increased slowly in a time-dependent manner(Fig.\u0026nbsp;1B). Despite this, the total number of intracellular \u003cem\u003eSalmonella\u003c/em\u003e in Henle-407 cells treated with 3-MA, was lower than that in Henle-407 cells without 3-MA. As expected, the treatment of the xenophagy inhibitor 3-MA induced the marked inhibition of reproductive rate of \u003cem\u003eSalmonella\u003c/em\u003e in cells (Fig.\u0026nbsp;1B).\u003c/p\u003e\n\u003cp\u003e(A) Growth curve of the \u003cem\u003eSalmonella\u003c/em\u003e in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells. (B) Dynamic growth curves of \u003cem\u003eSalmonella\u003c/em\u003e in Henle-407 cells. (C) Fluorescent image of \u003cem\u003eAtg5\u003c/em\u003e gene knockdown cell line in Henle-407 cells. (D) Dynamic growth curves of \u003cem\u003eSalmonella\u003c/em\u003e in \u003cem\u003esiAtg5\u003c/em\u003e Henle-407 cells.\u003c/p\u003e\n\u003cp\u003eAdditionally, knockdown of \u003cem\u003eAtg5\u003c/em\u003e gene(which plays a vital role in autophagy regulation) in cells can inhibit xenophagy\u003csup\u003e[22]\u003c/sup\u003e. To better understand the effect of xenophagy on replication of \u003cem\u003eSalmonella\u003c/em\u003e, a Henle-407 cell line with stablely knockdown of \u003cem\u003eAtg5\u003c/em\u003e was successfully constructed(Fig.\u0026nbsp;1C, Figure S1A, Figure S1B), and the knockdown of \u003cem\u003eAtg5\u003c/em\u003e gene had no inhibitory effect on the reproduction ability of Henle-407 cells(Figure S1C). Compared with Henle-407 cells, there was no significant difference in the total number of total intracellular \u003cem\u003eSalmonella\u003c/em\u003e in \u003cem\u003esiAtg5\u003c/em\u003e Henle-407 cells at the early stage(0 h-2 h) of infection (Fig.\u0026nbsp;1D). However, with prolonged infection time, the total number of intracellular \u003cem\u003eSalmonella\u003c/em\u003e in \u003cem\u003esiAtg5\u003c/em\u003e Henle-407 cells at 8 h-16 h post-infection was significantly lower than that in wild Henle-407 cells. These results suggest that inhibition of xenophagy reduces the replication rate of total intracellular \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThis means that the absence of xenophagy results in the reduction of reproductive rate of \u003cem\u003eSalmonella.\u003c/em\u003e It is speculated that the deletion of \u003cem\u003eTRAF6\u003c/em\u003e causes a decrease in xenophagy, thereby leading to a reduction in the reproductive rate of \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eTRAF6 activates the xenophagy of mouse embryo fibroblast\u003c/h3\u003e\n\u003cp\u003eWe next sought to further verify the relationship between TRAF6 and xenophagy. \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells were infected with \u003cem\u003eSalmonella\u003c/em\u003e, and the whole cell lysate was collected at 0 h, 2 h, 4 h, 8 h, and 16 h post-infection. The detection of LC3-I/II conversion is a gold indicator of autophagy flow detection\u003csup\u003e[23]\u003c/sup\u003e. Western blot analysis indicated that, the amount of LC3-II protein gradually increased and accumulated as the infection time prolonged(Fig.\u0026nbsp;2A). Interestingly, compared with the \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells, the accumulation of LC3-Ⅰ and LC3-II protein in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells was significantly increased(Fig.\u0026nbsp;2A). Based on mechanistic analysis, the accumulation of LC3 in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells maybe due to an increase in synthetic sources or a decrease in degradation pathways.\u003c/p\u003e\n\u003cp\u003eTo address this conjecture, RT-qPCR was performed to detect the expression of \u003cem\u003eLC3\u003c/em\u003e mRNA in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells at 0 h, 2 h, 4 h, 8 h and 16 h after infection of \u003cem\u003eSalmonella\u003c/em\u003e. Compared with \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells, the LC3 mRNA level in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells decreased between 0 and 16 hours post-infection. Therefore, in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells, LC3-II protein accumulates in large quantities, while the transcription level of \u003cem\u003eLC3\u003c/em\u003e is significantly reduced. Thus, the synthesis source was reduced, and the degradation pathways are impaired, indicating that the deletion of TRAF6 leads to the inhibition of cellular xenophagy(induced by \u003cem\u003eSalmonella\u003c/em\u003e infection).\u003c/p\u003e\n\u003cp\u003eTo further verify this result, Western blot was performed to detect the protein levels of LC3 in cells stimulated with the late stage inhibitor bafilomycin A1(Baf A1) of autophagy. Intracellular autophagy lysosome degradation was inhibited, and at the same time, the observed changes in protein level of LC3-II represented changes in the number of autophagosomes\u003csup\u003e[23]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;2C and Fig.\u0026nbsp;2D, we found that the protein level of LC3-II in group \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells treated with Baf A1 was significantly higher than those in group \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells treated with Baf A1, indicating that TRAF6 can promote the formation of xenophagosome. These findings indicate that TRAF6 enhances xenophagic flux.\u003c/p\u003e\n\u003cp\u003e(A) Effect of TRAF6 on the expression of LC3 during \u003cem\u003eSalmonella\u003c/em\u003e infection. GAPDH was detected as a loading control. (B) RT-qPCR determination of the relative expression of \u003cem\u003eLC3\u003c/em\u003e gene after \u003cem\u003eSalmonella\u003c/em\u003e infection from 0 hours to 16 hours. * indicates \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** indicates \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01(Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests). (C) and (D) Detection of xenophagic flux under late autophagy inhibitor. Quantification of the fold LC3-II (relative to the levels in cells without Baf A1). Values are means (\u0026plusmn;\u0026thinsp;SD) for three independent experiments. * indicates \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** indicates \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01(Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests). (E) Functional replenishment experiment for TRAF6 in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells.\u003c/p\u003e\n\u003cp\u003eNext, we transfected the plasmid \u003cem\u003epEGFP-N1-TRAF6\u003c/em\u003e into \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells. Positive transformants were infected with \u003cem\u003eSalmonella\u003c/em\u003e, and the expression of LC3 protein was detected in infected cells. Western blot analysis showed that LC3-II levels in the \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells+\u003cem\u003epEGFP-N1\u003c/em\u003e group was significantly higher than those in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells+\u003cem\u003epEGFP-N1-TRAF6\u003c/em\u003e group (Fig.\u0026nbsp;2E). This indicates that the TRAF6 functional complementation was successful. The result was consistent with the comparison between the \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells+\u003cem\u003epEGFP-N1\u003c/em\u003e group and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells+\u003cem\u003epEGFP-N1\u003c/em\u003e. It was speculated that TRAF6 enhabced the degradation of xenophagosomes. In a summary, TRAF6 promoted xenophagic flux.\u003c/p\u003e\n\u003cp\u003eNext, we transfected the plasmid pEGFP-N1-TRAF6 into Traf6\u003csup\u003e+/+\u003c/sup\u003e MEF cells and Traf6\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MEF cells. Positive transfectants were infected with \u003cem\u003eSalmonella\u003c/em\u003e, and LC3 protein expression was detected in the infected cells. Western blot analysis showed that LC3-II levels in the Traf6\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MEF cells+pEGFP-N1 group were significantly higher than those in the Traf6\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MEF cells+pEGFP-N1-TRAF6 group (Fig.\u0026nbsp;2E). This indicates that TRAF6 functional complementation was successful. The result was consistent with the comparison between the Traf6\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MEF cells+pEGFP-N1 group and the Traf6\u003csup\u003e+/+\u003c/sup\u003e MEF cells+pEGFP-N1 group. It was speculated that TRAF6 enhanced the degradation of xenophagosomes. In summary, TRAF6 promoted xenophagic flux.\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eTRAF6 plays an important role in bacterial carcinogenesis\u003c/h2\u003e\n \u003cp\u003eOur results demonstrated that TRAF6 promoted xenophagic flux during \u003cem\u003eSalmonella\u003c/em\u003e infection. However, the molecular mechanisms by which TRAF6 participates in xenophagic activity remain poorly understood. To further investigate the molecular mechanisms underlying the TRAF6-regulated xenophagy pathway, six RNA libraries were constructed (at 0 h, 2 h and 8 h post-infection of \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells with \u003cem\u003eSamonella\u003c/em\u003e).\u003c/p\u003e\n \u003cp\u003eHigh inter-sample reproducibility (Pearson\u0026rsquo;s r\u0026thinsp;\u0026gt;\u0026thinsp;0.99 among biological replicates) confirmed the robustness of the transcriptional datasets (Fig.\u0026nbsp;3A). Pearson correlation analysis revealed substantially lower correlation coefficients between \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells (r\u0026thinsp;=\u0026thinsp;0.72\u0026ndash;0.89), indicating that TRAF6 deficiency induces a systematic rewiring of the host transcriptome across infection time points. Principal component analysis (PCA) resolved these differences further: genotype constituted the dominant source of transcriptional variance (PC1, 25.2%), with \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells populations segregating into distinct clusters (Figure S2). While \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells exhibited a coordinated temporal trajectory within the PC1-PC2 coordinate system, \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells deviated markedly along PC2 (21.8% variance) at 8 hours post-infection, occupying a unique transcriptional space indicative of dysregulated late-phase immune signaling. These data establish TRAF6 as a critical node governing the temporal dynamics of infection-induced transcriptional responses, whose absence precipitates an aberrant, genotype-specific transcriptomic state at late infection stages.\u003c/p\u003e\n \u003cp\u003eWe identified differentially expressed genes (DEGs) by analyzing samples collected at different time points post-infection, comparing \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells infected with \u003cem\u003eSalmonella\u003c/em\u003e. Compared with \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells infected with \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells exhibited 2861 and 2617 DEGs at 2 hours and 8 hours post-infection, respectively. Among these DEGs, there were 1,759 and 1,455 upregulated genes, and 1,102 and 1,162 downregulated genes at the two time points (Fig.\u0026nbsp;3B, Fig.\u0026nbsp;3C, Fig.\u0026nbsp;3D). The number of DEGs differed significantly between the 2 h and 8 h groups.\u003c/p\u003e\n \u003cp\u003e(A) Pairwise correlation matrix of transcriptomic profiles across Traf6 genotypes and infection time points. Pearson correlation heatmap of RNA-seq data from \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells collected at 2 and 8 hours post-infection. Rows and columns represent individual samples grouped by genotype, time point, and biological replicate (A, B, C; n\u0026thinsp;=\u0026thinsp;3 per group). (B) The number of DEGs in the comparison of the groups, the orange bars indicate the up-regulated genes, and the blue bars indicate the down-regulated genes. (C) Volcano plot map of Traf6\u003csup\u003e+/+\u003c/sup\u003e-2h vs Traf6\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-2h. (D) Volcano plot map of Traf6\u003csup\u003e+/+\u003c/sup\u003e-8 h vs Traf6\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-8 h. (E) and (F) Venny plot analysis screens 2 h DEGs and 8 h DEGs.\u003c/p\u003e\n \u003cp\u003eTo validate the accuracy and reproducibility of RNA-Seq data, we assessed the transcript levels of 10 randomly selected differentially expressed genes (DEGs) via RT-qPCR, including 5 upregulated and 5 downregulated genes from the RNA-Seq dataset (Additional Files 1 and 2). All 5 upregulated genes exhibited significant upregulation in \u003cem\u003eSalmonella\u003c/em\u003e-infected \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells at 2 h and 8 h post-infection(Figure S3A and Figure S3B). Moreover, all 5 downregulated genes showed significant downregulation in \u003cem\u003eSalmonella\u003c/em\u003e-infected \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells at 2 h and 8 h post-infection(Figure S3A and Figure S3B). The results were consistent with those from the RNA-Seq data. confirming the reliability of transcriptomic changes in gene expression.\u003c/p\u003e\n \u003cp\u003eIn order to quantify the DEGs in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells at 2 h and 8 h post-\u003cem\u003eSalmonella\u003c/em\u003e infection, Venny plot analysis was performed(Fig.\u0026nbsp;3E, 3F). Specifically, the left blue portion represents DEGs at 2 h post-infection after excluding common DEGs in the uninfected group, totaling 1,229 DEGs. Similarly, the right orange portion corresponds to 1,035 DEGs identified at 8 h post-\u003cem\u003eSalmonella\u003c/em\u003e infection (Fig.\u0026nbsp;3F). KEGG enrichment analysis was subsequently conducted to screen key genes on the important pathways. The pathways with significant changes (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05) in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells were identified using the KEGG database. The top 20 most significantly enriched KEGG pathways at 2 h and 8 h post-\u003cem\u003eSalmonella\u003c/em\u003e infection are shown in Figure S4 (full datasets are provided in Additional Files 3 and 4). The \u0026lsquo;MicroRNAs in cancer\u0026rsquo;, \u0026lsquo;Human papillomavirus infection\u0026rsquo;, \u0026lsquo;Focal adhesion\u0026rsquo;, \u0026lsquo;Pathways in cancer\u0026rsquo;, and \u0026lsquo;AGE-RAGE signaling pathway in diabetic complications\u0026rsquo; pathways were the most prominently represented. It shows that TRAF6 plays an important role in bacterial carcinogenesis.\u003c/p\u003e\n \u003cp\u003eTwo xenophagy-related pathways were screened out from the results of KEGG enrichment, specifically Autophagy-animal(ko04140) and Mitophagy-animal(ko04137) signaling pathway(Table\u0026nbsp;1). Meanwhile, gene expression was referenced based on gene expression abundance as measured by FPKM values, with a threshold of \u0026ldquo;1\u0026rdquo;used to determine gene detectability. Using this criterion, we screened 6 DEGs, namely \u003cem\u003eLC3\u003c/em\u003e, \u003cem\u003ec-Jun\u003c/em\u003e, \u003cem\u003eDdit4\u003c/em\u003e, \u003cem\u003eDeptor\u003c/em\u003e, \u003cem\u003eLamp1\u003c/em\u003e and \u003cem\u003eFoxO3\u003c/em\u003e (Table\u0026nbsp;2).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eStatistics of DEGs in KEGG pathway related to xenophagy\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e infection 2 h\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e\n \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e infection 8 h\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003elog\u003csub\u003e2\u003c/sub\u003eFold Change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003elog\u003csub\u003e2\u003c/sub\u003eFold Change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\n \u003cp\u003eAutophagy-animal(ko04140)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eDeptor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1.65196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eDEP domain containing MTOR-interacting protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u003cem\u003eDeptor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e1.64494\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\n \u003cp\u003eDEP domain containing MTOR-interacting protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eLamp1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e-1.05129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003elysosomal-associated membrane protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u003cem\u003eLamp1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-1.01769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\n \u003cp\u003elysosomal-associated membrane protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eLC3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e-1.19786\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003egamma-aminobutyric acid (GABA) A receptor-associated protein-like 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u003cem\u003ePrkaa2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e-1.07725\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\n \u003cp\u003eprotein kinase, AMP-activated, \u0026alpha;-2 catalytic subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003ePik3cd\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1.30285\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003ephosphatidylinositol 3-kinase catalytic delta polypeptide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eDdit4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e1.19923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eDNA-damage-inducible transcript 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\n \u003cp\u003eMitophagy\u0026ndash;animal(ko04137)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eJun\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e-1.23794\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003ejun proto-oncogene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u003cem\u003eFoxO3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e-1.03999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003eForkhead box O3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eLC3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e-1.19786\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003egamma-aminobutyric acid (GABA) A receptor-associated protein-like 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003cp\u003eNote: \u0026mdash;\u0026mdash; means no signal\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eFPKM values of Xenophage-related DEGs\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e infection 2 h\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e infection 8 h\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e\u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e\u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e\u003cem\u003eTrafF6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e\u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eLC3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eLC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e61.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e27.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eJun\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003ec-JUN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e100.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e43.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eDdit4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eREDD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e11.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e27.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003ePik3cd\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003ePI3K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eDeptor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDEPTOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e6.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e4.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eLamp1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eLAMP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e659.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e327.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e594.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e291.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003ePrkaa2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAMPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cem\u003eFoxO3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eFOXO3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\n \u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e12.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e5.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\u003c/div\u003e\n \u003cdiv\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003cp\u003eNote: \u0026mdash;\u0026mdash; means no signal\u003c/p\u003e\n \u003cp\u003eTRAF6 can activate the c-Jun pathway by promoting the phosphorylation and activation of JNK, or directly interact with c-Jun to enhance its stability by inhibiting degradation. The ROS-induced JNK/c-Jun pathway activates SIRT1 and triggers autophagy as an adaptive response to protect cells from fluoride-induced damage\u003csup\u003e[24]\u003c/sup\u003e. REDD1 regulates cell growth, proliferation and survival by inhibiting the activity of mammalian target of rapamycin complex 1 (mTORC1)\u003csup\u003e[25, 26]\u003c/sup\u003e. mTORC1 negatively regulates autophagy. DEPTOR is a negative regulator of mTORC1 and mTORC2 signaling pathways, which inhibits the kinase activity of both complexes\u003csup\u003e[27]\u003c/sup\u003e. FOXO3, a transcriptional activator, can recognize and bind to the DNA sequence 5\u0026apos;-[AG]TAAA[TC]A-3\u0026apos; and regulate different processes, such as apoptosis and autophagy\u003csup\u003e[28]\u003c/sup\u003e. LAMP1, a lysosomal-associated membrane glycoprotein 1, localized to the xenolysosomal membrane, is positively correlated with the level of xenophagy\u003csup\u003e[29]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eXenophagy was blocked in\u003c/strong\u003e \u003cstrong\u003eTraf6\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026minus;/\u0026minus;\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003eMEF cells\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWe next investigated the effect of TRAF6 on the xenophagy signaling pathway. \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells were infected with \u003cem\u003eSalmonella\u003c/em\u003e, and cell lysates were harvested at 0 h, 2 h, 4 h, 8 h, and 16 h post-infection for Western blot analysis.\u003c/p\u003e\n \u003cp\u003e(A) The expression of REDD1 was detected at protein level. GAPDH was detected as a loading control. (B) Protein level of DEPTOR, FOXO3 and LAMP1. GAPDH was detected as a loading control.\u003c/p\u003e\n \u003cp\u003eThere was no difference in the protein levels of REDD1 between \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells, indicating that REDD1 is not involved in the xenophagy regulated by TRAF6(Fig.\u0026nbsp;4A). Furthermore, the results also indicate that TRAF6 does not affect cell proliferation. Transcriptome analysis revealed that, following \u003cem\u003eSalmonella\u003c/em\u003e infection, the transcription level of \u003cem\u003eDeptor\u003c/em\u003e in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells was significantly higher than that in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells (Table\u0026nbsp;1). But the protein level of DEPTOR in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells decreased, which resulted in mTOR activation and hindered xenophagy(Fig.\u0026nbsp;4B). Compared with \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells, the transcriptional level of \u003cem\u003eFoxO3\u003c/em\u003e in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells was lower at 8 hours(Table\u0026nbsp;1). Accordingly, FoxO3 protein levels exhibited a brief increase at 2 h followed by a sustained decrease, indicating that the xenophagy was initially induced but subsequently blocked in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells (Fig.\u0026nbsp;4B). Compared with \u003cem\u003eSalmonella\u003c/em\u003e-infected \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells, both of the transcriptional levels of \u003cem\u003eLAMP1\u003c/em\u003e and protein levels of LAMP1 in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells, showed a brief increase followed by a sustained decrease (Fig.\u0026nbsp;4B, Table\u0026nbsp;1), further supporting that xenophagy was initially induced and was blocked in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells. Collectively, the above results indicate that the knockout of \u003cem\u003eTRAF6\u003c/em\u003e negatively impacts the protein levels of DEPTOR, FOXO3 and LAMP1, leading to the inability to further form xenophagy in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells.\u003c/p\u003e\n \u003cp\u003eGiven the blockade of xenophagy in TRAF6-deficient cells and the associated dysregulation of DEPTOR, FOXO3, and LAMP1, what molecular events drive the failure of xenophagic flux under these conditions, and how are they linked to the loss of TRAF6 function?\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eNDP52 was an adaptor in xenophagy regulated by TRAF6\u003c/h2\u003e\n \u003cp\u003eOver the past decade, it has been increasingly recognized that autophagy/xenophagy exerts a pivotal role in antimicrobial immunity. Diverse molecular mechanisms underlying xenophagic target recognition have been identified. Adaptor proteins such as p62, NDP52, and NBR1 selectively recognize PAMPs and danger signals, which are essential for the effective clearance of pathogens\u003csup\u003e[30]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eTo further explore reason of that xenophagy was blocked in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells, Western blot was performed to examine detect the four adaptor proteins, including p62, OPTN, NBR1 and NDP52\u003csup\u003e[31]\u003c/sup\u003e. The results showed that there was no significant difference in the expression of p62, OPTN, and NBR1 in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells and \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells.\u003c/p\u003e\n \u003cp\u003eNotably, the protein level of NDP52 in Traf6\u003csup\u003e⁻/⁻\u003c/sup\u003e MEF cells exhibited a transient increase within 2 hours post-Salmonella infection, followed by a sustained decline(Fig.\u0026nbsp;5A), a pattern consistent with the dynamic changes in DEPTOR, FOXO3, and LAMP1 protein levels(Fig.\u0026nbsp;4B). Western blot analysis indicates that NDP52, as an adaptor protein, is involved in TRAF6-mediated xenophagy induced by \u003cem\u003eSalmonella\u003c/em\u003e infection. Regarding transcriptional levels, NDP52 expression was initially lower in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells compared to \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells(0\u0026ndash;6 h), followed by higher expression at 8 h and 16 h(Fig.\u0026nbsp;5C). NDP52 may act as a key receptor in the xenophagy process influenced by TRAF6.\u003c/p\u003e\n \u003cp\u003eUbiquitination of NDP52 plays an important role in autophagy/ xenophagy\u003csup\u003e[32, 33]\u003c/sup\u003e. The immunoprecipitation technique was performed to detect the ubiquitination of NDP52. Polyubiquitinated NDP52 was readily detectable 2 h post-\u003cem\u003eSalmonella\u003c/em\u003e infection (Fig.\u0026nbsp;5B) The amount of ubiquitinated NDP52 gradually increased and peaked at 8 h post-infection, in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells. In comparison, minimal NDP52 ubiquitination was detected in \u003cem\u003eSalmonella\u003c/em\u003e-infected \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells(Fig.\u0026nbsp;5B), indicating a requirement for TRAF6 in the ubiquitination of NDP52 during \u003cem\u003eSalmonella\u003c/em\u003e infection.\u003c/p\u003e\n \u003cp\u003eThese findings suggest that TRAF6 promotes xenophagosome formation during \u003cem\u003eSalmonella\u003c/em\u003e infection, in which NDP52 plays a crucial role as a receptor for xenophage formation.\u003c/p\u003e\n \u003cp\u003e(A) Detection of adaptor proteins in xenophagy (B) NDP52 undergoes ubiquitination during xenophagy, caused by TRAF6. (C) The transcription level of \u003cem\u003eNDP52\u003c/em\u003e in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells relative to that in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells. * indicates \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** indicates \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01(Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests)\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eXenophagy, a critical form of selective autophagy, exerts a crucial role in the host's innate immune defense, with TRAF6 functioning as a key regulator of xenophagic activity. In the present study, we report that the loss of TRAF6 results in the inhibition of cell xenophagy induced by \u003cem\u003eSalmonella\u003c/em\u003e infection\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Upon infecting host cells, \u003cem\u003eSalmonella\u003c/em\u003e proliferated within the cells and triggered the xenophagy pathway of the cells. If xenophagy was inhibited, the proliferation of \u003cem\u003eSalmonella\u003c/em\u003e would be hindered. In this study, Henle-407 cells treated with 3-MA and subsequently infected with \u003cem\u003eSalmonella\u003c/em\u003e exhibited a significant reduction in intracellular \u003cem\u003eSalmonella\u003c/em\u003e burden compared to untreated counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, at 16 h post-\u003cem\u003eSalmonell\u003c/em\u003ea infection, Henle-407 cells transfected with \u003cem\u003esiAtg5\u003c/em\u003e showed a markedly lower intracellular \u003cem\u003eSalmonella\u003c/em\u003e load than wild-type Henle-407 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells, where xenophagy is blocked, LC3 protein levels were significantly increased, accompanied by a notable decrease in intracellular \u003cem\u003eSalmonella\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results show that TRAF6 can promote xenophagy flux. However, the specific mechanism of which TRAF6 affects xenophagy remain to be further explored.\u003c/p\u003e \u003cp\u003eThrough high-throughput transcriptome sequencing analysis of mRNA expression profiles in Traf6\u003csup\u003e⁺/⁺\u003c/sup\u003e and Traf6\u003csup\u003e⁻/⁻\u003c/sup\u003e MEF cells infected with \u003cem\u003eSalmonella\u003c/em\u003e at 0, 2, and 8 hours, we identified six candidate genes: LC3, c-JUN, Ddit4, Deptor, LAMP1, and FoxO3 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which are involved in the Autophagy-animal (ko04140) and Mitophagy-animal (ko04137) signaling pathways. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, in TRAF6-deficient MEF cells, the protein levels of xenophagy-related genes, including Deptor, FoxO3, and LAMP1, were significantly reduced. These findings are consistent with previous reports\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Furthermore, we identified NDP52 as an adaptor protein associated with TRAF6-mediated regulation of \u003cem\u003eSalmonella\u003c/em\u003e-induced xenophagy.\u003c/p\u003e \u003cp\u003eIt is well established that the research on xenophagy has been based on the mixed count of two types of intracellular bacteria, which makes it difficult to elaborate and accurately describe the function of xenophagy during \u003cem\u003eSalmonella\u003c/em\u003e infection. Because the reproduction modes of the two types of bacteria are completely different, and the biological significance of xenophagy to these two types of bacteria is also different. The proportion of the two types of \u003cem\u003eSalmonella\u003c/em\u003e at different infection time points has changed greatly, especially in cytosolic \u003cem\u003eSalmonella\u003c/em\u003e. The proportion of cytosolic \u003cem\u003eSalmonella\u003c/em\u003e has been at a lower proportion throughout the infection process. In contrast, vacuolar \u003cem\u003eSalmonella\u003c/em\u003e has been the predominant form of intracellular \u003cem\u003eSalmonella\u003c/em\u003e. This may be because the protein in the cytoplasm is transported to the lysosome for degradation by Chaperone-mediated Autophagy (CMA), and the degraded polypeptides provide nutrients for the reproduction of intracellular \u003cem\u003eSalmonella\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e3-MA is a commonly autophagy inhibitor that acts by mediating Phosphatidylinositol 3-kinase/protein kinase the mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway, which affects cell growth, proliferation, survival and xenophagy\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eSalmonella\u003c/em\u003e blocks or delays apoptosis of infected cells by activating Akt. 3-MA inhibits the phosphorylation of Akt by inhibiting PI3K, thereby blocking the Akt/mTOR signaling pathway, inhibiting xenophagy and promoting apoptosis\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. After xenophagy was inhibited by 3-MA, both cytosolic and vacuolar \u003cem\u003eSalmonella\u003c/em\u003e exhibited a lower burden compared to that in normal cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Although the total number of intracellular \u003cem\u003eSalmonella\u003c/em\u003e was increasing, the proportion of the two types of \u003cem\u003eSalmonella\u003c/em\u003e varied significantly at different infection time points. Especially, the cytosolic \u003cem\u003eSalmonella\u003c/em\u003e population continued to increase throughout the entire infection process. In contrast, vacuolar \u003cem\u003eSalmonella\u003c/em\u003e was the predominant form of intracellular \u003cem\u003eSalmonella\u003c/em\u003e in the early stages of infection. However, the proportion of the vacuolar \u003cem\u003eSalmonella\u003c/em\u003e decreased at the later stage of infection. These results show that xenophagy may facilitate the replication of cytosolic \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) The number of intracellular \u003cem\u003eSalmonella\u003c/em\u003e in Henle-407 treated with 3-MA. (B) The number of intracellular \u003cem\u003eSalmonella\u003c/em\u003e in \u003cem\u003esiAtg5\u003c/em\u003e-Henle-407. (C and D) The proportion of the two types of \u003cem\u003eSalmonella\u003c/em\u003e in different cells.\u003c/p\u003e \u003cp\u003eAtg5 mediates the conjugation of LC3 to autophagosomes, thereby preventing lysosome clearance of intracellular \u003cem\u003eSalmonella\u003c/em\u003e. Atg5 and Tecpr1 target \u003cem\u003eShigella\u003c/em\u003e to autophagosomes by binding to the \u003cem\u003eShigella\u003c/em\u003e effector protein IcsA\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In this study, the Henle-407 cell line, stably knocked down the \u003cem\u003eAtg5\u003c/em\u003e gene, was constructed via a lentiviral system. Our results indicate that xenophagy exerts distinct biological effects on the intracellular replication of these two \u003cem\u003eSalmonella\u003c/em\u003e populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In \u003cem\u003esiAtg5-\u003c/em\u003etransfected Henle-407 cells, the counts of both cytosolic and vacuolar \u003cem\u003eSalmonella\u003c/em\u003e were lower than those in Henle-407 cells. This observation may be attributed to the loss of xenophagy, which leads to the disruption of \u003cem\u003eSalmonella\u003c/em\u003e-containing vacuoles (SCVs) and the subsequent loss of the proton gradient across the SCV membrane. These changes significantly reduces the expression of \u003cem\u003eSalmonella\u003c/em\u003e T3SS SPI-2 effector protein and the reproduction of bacteria in cells\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe believe that future studies should focus on three key directions to deepen understanding of xenophagy in \u003cem\u003eSalmonella\u003c/em\u003e infection and boost its translational value. First, it is critical to further dissect the specific molecular mechanisms by which TRAF6 regulates NDP52 ubiquitination\u0026mdash;including identifying pivotal E3 ubiquitin ligases, deubiquitinases, and interacting proteins\u0026mdash;to fully delineate the TRAF6-NDP52-mediated xenophagy regulatory axis. Second, systematic studies are needed to clarify the molecular basis for xenophagy\u0026rsquo;s differential effects on cytosolic versus vacuolar \u003cem\u003eSalmonella\u003c/em\u003e, with particular emphasis on how reciprocal regulation between \u003cem\u003eSalmonella\u003c/em\u003e-containing vacuole (SCV) integrity and xenophagy shapes bacterial intracellular survival. Third, building on the key molecules identified here (e.g., NDP52, TRAF6, SPI-2 effectors), targeted validation studies should assess their feasibility as therapeutic targets for salmonellosis. Collectively, these efforts will not only deepen mechanistic insights into host-xenophagy-\u003cem\u003eSalmonella\u003c/em\u003e crosstalk but also lay a solid experimental foundation for developing novel antibacterial strategies against salmonellosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePengcheng Zhang:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Investigation, Visualization, Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMengling Huang:\u003c/strong\u003e Investigation. Methodology, Writing - original draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChunchen Zhang\u003c/strong\u003e: Investigation, Methodology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHaihua Ruan:\u003c/strong\u003e Conceptualization,\u0026nbsp;Funding acquisition, Methodology, Resources, Writing - review \u0026amp; editing, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded mainly by the National Natural Science Foundation of China (Nos. 31870122)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenerative AI statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no Gen AI was used in the creation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMin Y, Kim MJ, Lee S et al (2018) Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation. 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Cell Host Microbe 18:527\u0026ndash;537. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chom.2015.10.015\u003c/span\u003e\u003cspan address=\"10.1016/j.chom.2015.10.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-microbiology-and-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mmim","sideBox":"Learn more about [Medical Microbiology and Immunology](http://link.springer.com/journal/430)","snPcode":"430","submissionUrl":"https://submission.nature.com/new-submission/430/3","title":"Medical Microbiology and Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TRAF6, Xenophagy, Salmonella, NDP52","lastPublishedDoi":"10.21203/rs.3.rs-8962384/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8962384/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTRAF6 is the unique member of the TRAF family with E3 ubiquitin ligase activity, playing a critical role in initiating autophagy and regulating its biological functions. Xenophagy is widely recognized as a specialized autophagy system. While emerging evidence has linked TRAF6 to bacterial-induced xenophagy, the underlying regulatory mechanism remains largely elusive. Following \u003cem\u003eSalmonella\u003c/em\u003e infection, the intracellular \u003cem\u003eSalmonella\u003c/em\u003e load in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells, was lower than that in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells. After the stable \u003cem\u003eAtg5\u003c/em\u003e gene knockdown cell line was constructed and 3-Methyladenine(3-MA) was used to inhibit xenophagy, the results were consistent with those observed in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells. The accumulation of LC3-II protein in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells was significantly increased, compared with \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e MEF cells counterparts, suggesting xenophagy may be impaired in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells. Furthermore, we identified conserved xenophagy related genes by transcriptome sequencing, which provided a molecular basis for the conclusion that xenophagy was blocked in \u003cem\u003eTraf6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e MEF cells. Simultaneously, Western blot analysis indicated that NDP52 is the adaptor protein of cellular xenophagy. Our current findings demonstrate that TRAF6 promotes \u003cem\u003eSalmonella\u003c/em\u003e proliferation within host cells, and notably, the expression of the receptor protein NDP52 is closely associated with this process. Depletion of TRAF6 results in reduced protein levels of DEPTOR, FOXO3, and LAMP1, whereas the protein level of LC3-II is upregulated, thereby significantly impairing the xenophagy process.\u003c/p\u003e","manuscriptTitle":"TRAF6-mediated activation of xenophagy facilitates intracellular Salmonella proliferation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 18:05:46","doi":"10.21203/rs.3.rs-8962384/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"177553299993097541844664033525559639250","date":"2026-05-07T00:47:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-05T08:17:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-26T07:45:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-26T07:44:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Microbiology and Immunology","date":"2026-02-25T02:36:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-microbiology-and-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mmim","sideBox":"Learn more about [Medical Microbiology and Immunology](http://link.springer.com/journal/430)","snPcode":"430","submissionUrl":"https://submission.nature.com/new-submission/430/3","title":"Medical Microbiology and Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5b3497e3-c2ac-4062-8150-eea35df6dce9","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"177553299993097541844664033525559639250","date":"2026-05-07T00:47:07+00:00","index":12,"fulltext":""},{"type":"reviewersInvited","content":"5","date":"2026-05-05T08:17:31+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T18:05:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 18:05:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8962384","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8962384","identity":"rs-8962384","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}