Switching off Bacterial Flagellar Biogenesis by YdiU-Mediated UMPylation of FlhDC

Abstract

Bacterial flagellin activates the host immune system and triggers pyroptosis. Salmonella reduces flagellin expression when it survives within host cells. Here, we found that the UMPylator YdiU significantly altered the Salmonella flagellar biogenesis process upon host cell entry. The expression levels of class II and class III flagellar genes, but not the class I flagellar genes flhDC, were dramatically increased in a ΔydiU strain compared to wild-type (WT) Salmonella in a host-simulating environment. A direct interaction between YdiU and FlhDC was detected by bacterial two-hybrid assay. Furthermore, YdiU efficiently catalyzed the UMPylation of FlhC but not FlhD, FliA, or FliC. UMPylation of FlhC completely eliminated its DNA-binding activity. In vivo experiments showed that YdiU was required and sufficient for Salmonella flagellar control within host cells. Mice infected with the ΔydiU strain died much earlier than WT strain-infected mice and developed much more severe inflammation and injury in organs and much higher levels of cytokines in blood, demonstrating that early host death induced by the ΔydiU strain is probably due to excessive inflammation. Our results indicate that YdiU acts as an essential factor of Salmonella to mediate host immune escape. IMPORTANCE Salmonella is an important facultative pathogen of foodborne illness and typhoid fever in humans. Flagella allow bacterial motility and are required for Salmonella to successfully invade the host cells. In parallel, flagellin triggers the host immune system. Salmonella reduces flagellar biogenesis to avoid detection within host cells by a largely unknown mechanism. Here, we report that the UMPylator YdiU inhibits flagellin expression in response to host signals in an UMPylation-dependent manner. The target of YdiU is the major flagellar transcription factor FlhDC. YdiU UMPylates the FlhC subunit on its Ser31 residue and prevents FlhDC from binding to flagellar genes, thus switching off flagellar biogenesis. Our results reveal a novel mechanism by which Salmonella adopts posttranslational modification to shut down flagellar synthesis as a strategy to achieve immune escape.

Keywords: FlhDC; Salmonella; UMPylation; YdiU; flagellar gene regulation.

FIG 1

YdiU inhibits the flagellar synthesis pathway of Salmonella. (A and B) Differentially expressed flagellar proteins of WT and ΔydiU under iron-limited conditions. (A) Heat map of the fold changes of flagellum-related proteins. (B) Total volcano plots of the proteomic results. The blue and red spots represent the downregulated and upregulated proteins, respectively. Flagellum-related proteins are labeled. (C) The mRNA levels of four flagellar genes in WT and ΔydiU strains under iron-limited conditions were separately determined by qRT-PCR. (D) The motility behavior of WT and ΔydiU strains was measured with soft-agar plates using iron-limited medium. The above-described experiments were performed as three replicates or five replicates (D), and means and standard errors of the means (SEM; error bars) are presented. ***, P < 0.001; **, P < 0.01; n.s., not significant (P > 0.05).

FIG 2

YdiU inhibits the expression of FliC in an UMPylation activity-dependent manner. (A) The motility behavior of four Salmonella strains was measured with soft-agar plates using iron-limited medium. (B) The numbers of flagella of the strains were observed using negative-staining electron microscopy (EM). The above-described experiments were performed as five replicates, and the means and SEM are presented. ***, P < 0.001; n.s., P > 0.05. (C) The protein levels of FlhD, FlhC, FliC, and YdiU were determined by Western blotting. GapA was used as a loading control. (D) The FliC levels relative to that of GapA were quantified by grayscale in three independent experiments. The gray values were obtained using ImageJ.

FIG 3

YdiU UMPylates the FlhC subunit of FlhDC. (A) In vitro UMPylation assays of FlhDC, FliA, and FliC by YdiU were separately performed with biotin-16-UTP. The UMPylated proteins were detected by streptavidin-HRP blotting, and total proteins were visualized by Ponceau S staining. (B) DNA binding does not inhibit the in vitro UMPylation of FlhC by YdiU. The DNA-bound FlhDC (DB-FlhDC) was prepared by incubation with excess target DNA for 10 min and used for in vitro UMPylation assays. The UMPylated proteins were detected by streptavidin-HRP blotting, and total proteins were visualized by Ponceau S staining.

FIG 4

The UMPylated sites of FlhC are located at the DNA-binding region. (A) In vitro UMPylation assays of the four FlhDC proteins were separately performed with biotin-16-UTP. The UMPylated proteins were detected by streptavidin HRP blotting, and total proteins were visualized by Ponceau S staining. (B) UMPylation sites of FlhC were identified by mass spectrometry in the four FlhDC proteins. (C) Structural presentation showing that S31 and S50 of FlhC are located in the DNA binding region.

FIG 5

UMPylated FlhDC loses the ability to bind target DNA. EMSAs were performed for native FlhDC and UMPylated FlhDC with target DNA pflhB (promoter of flhB). In these assays, 10 nM FAM-labeled pflhB DNA was mixed with the indicated concentration of native FlhDC or UMPylated FlhDC for 10 min and then analyzed by EMSA. The same samples were analyzed by SDS-PAGE stained with Coomassie brilliant blue. The experiment was repeated three times, and a representative image is shown.

FIG 6

YdiU is required for flagellar control upon the entry of Salmonella into host cells. (A) The mRNA levels of ydiU before and 6 h after Salmonella invasion into HT-29 cells were detected in WT and ΔydiU strains by qRT-PCR. (B to E) The mRNA levels of flhD (B), fliA (C), fliZ (D), and fliC (E) before and 6 h after Salmonella invasion were detected in WT and ΔydiU strains by qRT-PCR. (F) Model illustrating YdiU-mediated flagellar regulation. The above-described experiments were performed as three replicates, and the means and SEM are presented. ***, P < 0.001; n.s., P > 0.05.

FIG 7

YdiU facilitates Salmonella escape from the host immune system. (A) BALB/c mice were infected with equal numbers of WT and ΔydiU strains by intraperitoneal injection. Each group included five mice, and survival was monitored. (B) Pathological analysis of wild-type- and ΔydiU strain-infected small intestines was conducted. Tissues were collected at 12 h postinfection (hpi). The small intestine of an uninfected mouse was used as a negative control. (C) Blind histopathology scoring of inflammation in infected small intestines showing average scores for individual animals (squares). (D to J) The levels of indicated immune factors were detected by Meso Scale Discovery (MSD) assays with the serum of mice at 6 hpi and 12 hpi. Data are from three mice per group, and means and SEM are presented. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (compared with the wild-type strain).

FIG 8

Model of YdiU-mediated flagellar regulation during Salmonella infection. When Salmonella organisms are living in an extracellular environment, unmodified FlhDC binds to the promoter region of flagellar genes and recruits RNA polymerase to promote flagellar biogenesis. After Salmonella enters host cells, YdiU is expressed and UMPylates the FlhC subunit of the FlhDC complex. UMPylated FlhDC loses the ability to bind DNA, resulting in repression of the expression of flagellar genes to shut down flagellar biogenesis. With this decrease of flagellar antigen, Salmonella can successfully escape the host immune system.