Fig 1.
Identification of factors regulating the expression of IpaJ using Tn-Seq.
(A) Relative growth of the C79-13-PipaJ-Cm vs. WT strains grown in LB medium with serially diluted concentrations of Cm. (B) Schematic representation of the Tn-Seq strategy used to identify regulators controlling ipaJ expression and secretion. A transposon library was created in the S. Pullorum C79-13 strain by inserting a Cm resistance gene downstream of the ipaJ start codon. The library was grown in LB medium in the presence or absence of Cm. The sites and abundance of transposon insertions under these two conditions were compared. (C) Volcano plot showing the output/input FC in reads of genes, as revealed by Tn-Seq analysis. The genes of interest were highlighted with cut-offs of |log2(FC)| > 1 and P-value < 0.05. (D) Thirteen regulatory factors potentially promoted the expression of IpaJ.
Fig 2.
SPI-1 regulates the secretion of IpaJ.
(A) Immunoblotting analysis of secreted IpaJ proteins isolated from cultured LB medium supernatant. The mutants SPI-1 (ΔSPI-1), SPI-2 (ΔSPI-2), SPI-19(ΔSPI-19) or SPI-1 and SPI-2 (ΔSPI-1ΔSPI-2) were used to detect whether the secretion of IpaJ is dependent on SPI-1, SPI-2 or SPI-19. The ipaJ-deleted strain (ΔpSPI12) and the WT/Complementary strains (C79-13 and ΔpSPI12-pipaJ) were used as the negative and positive controls, respectively. The S. Enteritidis P125109 strain transformed with pBR322-ipaJ (P125109-pipaJ) was also used as another positive control for P125109. (B-C) Western blot analysis of samples from bacterial cultures grown in LB medium at 37°C with an OD600 value of 0.5. Bacterial cell lysates (B) and secreted proteins from the supernatants (C) were subjected to Western blot analysis. (D) Immunofluorescence microscopic analysis of T3SS-1-dependent translocation of IpaJ. HD-11 cells were infected with the WT strain or ΔSPI-1 or ΔSPI-2 at an MOI of 200. Red fluorescence represents bacteria carrying pRFP plasmid, green fluorescence represents the expression of IpaJ.
Fig 3.
The regulator ItrA promotes ipaJ expression.
(A) Schematic representation of the DNA pull-down assay to screen out the direct regulator binding to the promoter of ipaJ (PipaJ). Biotin-PipaJ (sample) and biotin-ipaJorf (negative control) fragments were used as the bait DNA. (B). The MS results of identified potential regulators binding to PipaJ. Protein-encoding genes without assigned names were displayed using SPN with a genetic code number. (C) Western blot analysis of IpaJ expression in WT and mutant strains cultured in LB medium at 37°C. DnaK was used as the control. Results are representative of three independent experiments. (*) Indicates the significant differences in IpaJ expression levels in different strains vs. the C79-13 group. ****: P < 0.0001. (D) Western blot analysis of IpaJ expression and secretion in WT, C79-13-ΔitrA, and C79-13-ΔitrA-pitrA strains cultured in LB medium at 37°C. (E) qRT-PCR analysis of ipaJ in WT, C79-13-ΔitrA, and C79-13-ΔitrA-pitrA strains cultured in LB medium at 37°C. ***: P < 0.001.
Fig 4.
ItrA directly binds to the ipaJ promoter region.
(A) FAM-labeled probes from PipaJ or ipaJorf were used for EMSA with 0, 0.1, 1, 2, 4 or 8 μM of purified rHis-ItrA. (B) FAM-labeled probes from PipaJ were incubated with 2μM purified rHis-ItrA in the presence of 25- and 50- fold excess of unlabeled PipaJ competitors. (C) Distribution of the four gradient fragments in the PipaJ region. The length of each of the four fragments is 100 bp. (D) Four FAM-labelled fragments were subjected to an EMSA assay using 2 μM rHis-ItrA. (E) FAM-labeled probes (P-240~-91 and P-90~-41) were used for EMSA assay with 0, 0.1, 1, 2, 4, or 8 μM purified rHis-ItrA.
Fig 5.
Phosphoproteomic profiles of proteins involved in dysregulated pathways.
(A) Quantitative volcano map of differentially phosphosites between ΔipaJ and WT infected groups. (B) Motif enrichment heat maps of amino acids upstream and downstream of all the identified phosphosites. (C) The predominant phosphorylation motif. (D) The subcellular localization of the differentially phosphorylated proteins between the two groups (ΔipaJ/WT). (E) The distribution of the differentially phosphorylated proteins in GO secondary annotation between the two groups. (F) The enriched KEGG pathways related to the differentially phosphorylated proteins between the two groups. (G) The domain enrichment of differentially phosphorylated proteins between the two groups.
Fig 6.
IpaJ inhibits the activation of the MAPK pathway
(A) Graphical illustration of the differentially modified phosphoproteins using STRING (confidence score > 0.9). (B) Cluster analysis of K-means-based cytoskeleton-associated proteins. Regulated proteins were annotated, clustered, interacted and MAPK-associated cluster isolated. (C) Western blot analysis of signaling molecules (MEK and ERK) in the MAPK pathway in HeLa cells infected with WT or mutant strains at 6 h post-infection. (D) The HeLa cells infected with different S. Pullorum strains at 6 h post-infection were collected and subjected to IP using anti-Ras antibody. Ras expression levels and ubiquitination status were analyzed via Western blot using anti-Ras and antiubiquitin antibodies, respectively. (E) Interaction between IpaJ and Ras using Co-IP assay. The anti-Ras antibody was used to capture proteins from HeLa cells transfected with pCMV-HA-ipaJ or pCMV-HA plasmids. The captured proteins were subjected to Western blot analysis using the anti-IpaJ antibody. **: P < 0.01; ***: P < 0.001.
Fig 7.
Schematic illustration of IpaJ as a T3SS1 effector protein that can suppress cellular immune responses during Salmonella infection.
The DeoR family regulator ItrA directly binds to the promoter of ipaJ and initiates its expression. The expressed IpaJ is then secreted into host cells or bacterial culture supernatants depending on SPI-1/T3SS1, which is regulated by HilA and HilD. As an effector protein of SPI-1, IpaJ can prevent the ubiquitination and degradation of IκBα in the NF-κB signaling pathway and inhibit the phosphorylation of MEK and ERK in the MAPK signaling pathway through deubiquination of Ras, thereby downregulating proinflammatory responses, cellular growth and differentiation, cell survival, and apoptosis.