Skip to main content
NCBI home page
Gut Microbes logoLink to Gut Microbes
. 2022 Dec 1;14(1):2146979. doi: 10.1080/19490976.2022.2146979

Host acid signal controls Salmonella flagella biogenesis through CadC-YdiV axis

Weiwei Wang a,b, Yingying Yue a,b, Min Zhang b, Nannan Song a,b, Haihong Jia a,b, Yuanji Dai b, Fengyu Zhang c, Cuiling Li a,b, Bingqing Li a,b,d,e,✉
PMCID: PMC9728131  PMID: 36456534

ABSTRACT

Upon entering host cells, Salmonella quickly turns off flagella biogenesis to avoid recognition by the host immune system. However, it is not clear which host signal(s) Salmonella senses to initiate flagellum control. Here, we demonstrate that the acid signal can suppress flagella synthesis and motility of Salmonella, and this occurs after the transcription of master flagellar gene flhDC and depends on the anti-FlhDC factor YdiV. YdiV expression is activated after acid treatment. A global screen with ydiV promoter DNA and total protein from acid-treated Salmonella revealed a novel regulator of YdiV, the acid-related transcription factor CadC. Further studies showed that CadCC, the DNA binding domain of CadC, directly binds to a 33 nt region of the ydiV promoter with a 0.2 μM KD affinity. Furthermore, CadC could separate H-NS-ydiV promoter DNA complex to form CadC-DNA complex at a low concentration. Structural simulation and mutagenesis assays revealed that H43 and W106 of CadC are essential for ydiV promoter binding. No acid-induced flagellum control phenotype was observed in cadC mutant or ydiV mutant strains, suggesting that flagellum control during acid adaption is dependent on CadC and YdiV. The intracellular survival ability of cadC mutant strain decreased significantly compared with WT strain while the flagellin expression could not be effectively controlled in the cadC mutant strain when surviving within host cells. Together, our results demonstrated that acid stress acts as an important host signal to trigger Salmonella flagellum control through the CadC-YdiV-FlhDC axis, allowing Salmonella to sense a hostile environment and regulate flagellar synthesis during infection.

KEYWORDS: Salmonella typhimurium, acid signal, YdiV, CadC, FlhDC, flagellar biosynthesis, immune escape

Introduction

Salmonella is an important facultative pathogen of many animals and humans, and Salmonella infection triggers acute gastroenteritis and typhoid fever in hosts.1–3 These diseases are responsible for more than 300,000 deaths annually, presenting a significant threat to global public health, especially in developing countries.4–6

Once ingested, flagella allows Salmonella to cross the intestinal barrier and adhere to the intestinal epithelial cells.7–9 The bacterial flagellum is a complex macromolecular machine whose construction requires proteins encoded by three classes of genes.10,11 Class I comprises two genes in a single operon, flhDC, the“master switch” of all other flagellar genes. The FlhD4C2 complex acts as the transcription factor for class II genes.12,13 Products of class II flagellar genes form the flagellar basal structure and the hook-basal body complex, and class III flagellar protein are related to filament formation, flagellar rotation, and chemotaxis.11,14–16 After Salmonella enters host cells, the flagellum becomes a prime target antigen for the host immune system. Previous studies showed that the expression of flagellar antigen FliC decreased to 1/10 the original level upon Salmonella entry into host cells.17,18 Previously, three regulators were identified to be responsible for this process, YdiV, STM1697, and YdiU. YdiV, a post-transcriptional regulator, disrupts FlhDC complex structure by binding to FlhD, thereby preventing it binding to class II promoters and targeting it for proteolysis.19–21 STM1697, another post-transcriptional anti-FlhDC factor, represses flagellar synthesis by preventing FlhDC from recruiting RNA polymerase to the promoter.22 The function of FlhDC is also regulated by post-translational modification by YdiU, a modifying enzyme that catalyzes the UMPylation of the FlhC subunit to prevent FlhDC binding to flagellar genes, thus switching off flagellar biogenesis.23 Although these regulators control flagella biogenesis, it is not clear which host signals Salmonella perceives to turn on this flagella control process.

During infection, most Salmonella invades into host macrophages. These macrophages act as a “Trojan horse” and carry Salmonella into the systemic tissue, provoking a systemic infection.24–26 Within macrophages, Salmonella survives and replicates within the Salmonella-containing vacuole (SCV), in which Salmonella faces a hostile environment, with acidic pH of 4.5 to 5.5, low concentrations of magnesium and calcium, and the presence of antimicrobial peptides, oxygen and nitrogen-free radicals.27–30 Salmonella responds to different environmental signals and modulates related genes via a variety of mechanisms, such as two-component regulatory systems (TCS). PhoP-PhoQ is one of the best-characterized TCS. In Salmonella, PhoQ can sense environmental signals such as free radicals, low Mg2+ ions, and low pH, and activate PhoP to promote the transcription of some gene clusters with functions related to invasion, intra-macrophage survival, magnesium transport, and others.31–33 Another TCS, EnvZ-OmpR, regulates multiple cellular function including virulence, biofilm formation, and acid tolerance in response to both acid and osmotic stress.34–36 Salmonella can regulate transcription with DNA binding proteins that act as transcription repressors or activators for target genes. H-NS is a nucleoid-associated protein and acts as a master global repressor, affecting the expression of at least 250 genes in Salmonella by binding to the promoters of target genes.37–39 The inhibitory effect of H-NS can be countered by the effects of other DNA-binding proteins that can either disrupt H-NS-DNA complexes or replace H-NS on the DNA due to higher binding affinity.40,41

The Cad system is an acid-inducible degradative amino acid decarboxylase system, which helps bacteria resist environmental acid stress. The Cad system consists of cadC and cadBA operons.42–44 CadC is a membrane-spanning transcriptional activator of the cadBA operon and is normally in an inactive state. Under external acidification and exogenous lysine, CadC is rapidly activated by proteolytic cleavage. The N-terminus of CadC binds to the cadBA promoter and activates the transcription of the cadBA operon.43,45 As a result, lysine is decarboxylated to cadaverine by CadA and cadaverine is transported out of the cell by CadB, facilitating bacterial acid tolerance.46,47 CadC can also regulate other proteins involved in glycolysis, energy production, and stress tolerance in Salmonella.45

In this study, we discovered that acid stress can negatively regulate the flagellar synthesis of Salmonella. Acid-mediated flagellar control occurs after the transcription and expression of FlhDC. The expression levels of secondary and tertiary flagellar genes were significantly decreased during acid treatment while the transcription of flhDC was increased during acid treatment. Acid signal increased the expression of YdiV but not STM1697 and YdiU. YdiV mutant Salmonella loses the phenotype of acid-mediated flagellar control. Using an in vitro DNA pull-down assay of ydiV promoter, we identified the acid-initiated transcription factor CadC as a transcription regulator of YdiV. In vitro data showed that the DNA binding domain of CadC directly bound to the ydiV promoter with a 0.2 μM KD affinity. Further structural simulation and mutagenesis assays identified His43 and W106 as key amino acids for ydiV promoter binding. The cadC mutant Salmonella loses the acid-induced YdiV activation and flagellum control phenotypes. In summary, our results revealed that a novel pathway, CadC-YdiV-FlhDC, turns off flagella biogenesis response to host acid signal by which Salmonella quickly shuts down flagella synthesis to achieve immune escape within host cells.

Results

Salmonella switches off flagellum biogenesis during acid adaptation.

Previous studies suggested that Salmonella rapidly turns off flagellum synthesis after entering host cells.22,23 When entering macrophages, Salmonella-containing phagosomes acidify soon after formation. As a result, Salmonella survives in an acidic environment within host cells. We speculate that acid signal may allow Salmonella to perceive the host environment for flagellum control. To investigate this, we began to study motility and flagellum synthesis under different pH environmental conditions.

The motility behavior of Salmonella typhimurium ATCC 14028s was first assayed using semisolid agar (0.25%) plates with different pH. The results showed that Salmonella exhibits normal motility at pH 7.0, however, at pH lower than 7.0, moving ability decreases, with total loss of moving ability at pH 5.0 (Figures 1a and 1b). To determine the basis of motility loss at pH 5.0, we determined the growth of flagellar filaments of Salmonella cultured in pH 5.0 or pH 7.0 using negative-staining electron microscopy (Figures 1c and 1d). Fewer flagella were observed in Salmonella cultured in pH 5.0 than those cultures in neutral growth conditions. Overall, our data demonstrate that flagellum synthesis of Salmonella could be significantly inhibited by acid signal.

Figure 1.

Acid stress inhibits the flagellar synthesis pathway of Salmonella.

Figure 1.

(A and B) The motility of the wild-type strain was measured using 0.25% semisolid agar plates at pH 5, 6, and 7, respectively. (C and D) The numbers of flagella of the wild-type strain were observed using negative-staining electron microscopy (EM). Data are shown as the means and SEM, n=3. ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., P > 0.05.

Open in a new tab

Acid-mediated flagellum control occurs after flhDC transcription.

To further probe the effect of acidity on the expression of flagellar genes in Salmonella, RNA-sequencing analysis was performed in Salmonella cultured with or without acid treatment (Figure 2 and Fig.S1). The results showed that the mRNA level was significantly increased for class I flagellar genes flhD and flhC after acid treatment (Figures 2a and 2b). However, there was severely reduced mRNA level of 18 class II and class III flagellar genes after acid treatment (Figures 2a and 2b). To verify the RNA-sequencing results, the transcription level of three flagellar genes (flhD, fliA, and fliC) of Salmonella before and after acid treatment was further detected by RT-PCR. The gapdh gene was used as an internal control and its expression was not affected by acid stress (Fig. S2). Consistent with the RNA-sequencing results, the transcription level of flhD was upregulated after acid treatment (Figure 2c), and the transcription level of fliA and fliC was markedly decreased under acid stress compared to under non-stress conditions (Figures 2d and 2e). Finally, the expression of FliC, the major flagellum antigen, was determined by immunoblot analysis of the treated and untreated samples. Consistent with the RT-PCR results, the protein level of FliC was significantly reduced after acid treatment (figures 2f and 2g). The above data collectively demonstrate that acid-related flagellum control occurs after class I flagellar gene flhDC transcription. Previous studies showed that three proteins, YdiV, YdiU, and STM1697, negatively regulate flagellum biogenesis by inhibiting the ability of FlhDC complex to regulate transcription.20,22 Data analysis of the above RNA-sequencing results demonstrated that the expression of ydiV is largely up-regulated, while expression of ydiU and stm1697 was almost unchanged or even down-regulated (Figure 2h). Therefore, YdiV was chosen as the focus of further study.

Figure 2.

Acid stress inhibits the expression of flagellum after flhDC transcription.

Figure 2.

(A, B and H) Differentially expressed genes of wild-type strain under normal or acid stress conditions. (A) Volcano plots were generated to visualize the transcriptomic results. Red and green points indicate upregulated and downregulated genes, respectively. Genes related to flagellar synthesis are labeled. (B) Heatmap showing relative transcript abundances of flagellar synthesis-related genes in the wild-type strain. (C to E) The expression levels of flhD (C), fliA (D), and fliC (E) in the wild-type strain under normal and acid stress conditions were measured by qRT-PCR. (F) The protein levels of FliC and DnaK after acid treatment were determined by Western blotting. DnaK was used as a loading control. (G) The relative FliC levels compared with DnaK were quantified by grayscale for three independent experiments. The gray values were obtained using ImageJ. (H) The relative transcription levels of flagellar regulators genes (ydiV, ydiU and stm1697) and cadC gene. Data are shown as the means and SEM, n=3. ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., P > 0.05.

Open in a new tab

Flagellum control during acid adaptation is dependent on YdiV.

The above data clearly demonstrated that the acid signal can inhibit flagellar expression in Salmonella and this regulation occurs after flhDC transcription. YdiV may be involved in the regulation of acid-flagellar inhibition in Salmonella. The increased expression of ydiV was dramatic, with increases of 21.6- and 31.0-fold 1 and 2 h after wild-type Salmonella entry into host cells, respectively (Figure 3a).

Figure 3.

YdiV is required for flagellar control of Salmonella under acid stress.

Figure 3.

(A) Salmonella upregulates YdiV expression during invasion. The intracellular mRNA levels of ydiV before and after Salmonella invasion were detected in the wild-type strain by qRT-PCR. (B to D) The transcription and protein levels of YdiV in Salmonella cultivated under acid condition were detected by qRT-PCR and western blot. (B) The protein levels of YdiV in wild-type strain after normal or acid (acetic acid; 2-mercaptoethanesulfonic acid, MES; Hydrochloric acid, HCl) treatment were determined by Western blotting. DnaK was used as a loading control. (C) The relative YdiV levels compared with the levels of DnaK were quantified by grayscale in three independent experiments. The gray values were obtained using ImageJ. (D) The transcription levels of YdiV in the wild-type strain under normal and acid stress conditions were detected by qRT-PCR. (E and F) The motility of the wild-type and ∆ydiV strains were measured using 0.25% semisolid agar plates at pH 5.5 and 7. Scale bars shows 1 cm. (G to I) The expression levels of flhD (G), fliA (H), and fliC (I) in the ∆ydiV strain under normal and acid stress conditions were measured by qRT-PCR. Data are shown as the means and SEM, n=3. ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., P > 0.05.

Open in a new tab

To further determine whether acid can induce the expression of ydiV, the protein level of YdiV was detected after acid treatment (Figures 3b and 3c). Considering that the acid stress faced by Salmonella in the process of invading the host includes organic and inorganic acids, we used these two different types of acids in the experiment. As expected, YdiV was barely expressed when Salmonella was cultured in LB medium or under inorganic acid stress (HCl), whereas expression increased to a high level under organic acid stress (acetic acid and MES). The special dissociation constant and cell membrane permeability of organic acids are better than inorganic acids. Therefore, acetic acid was selected as the acid pressure in subsequent experiment. The mRNA level of ydiV was detected by RT-PCR at different time points upon acid treatment, and expression increased 5.1-, 6.9-, 3.0-, and 2.8- fold, respectively, when detected 0.5, 1, 1.5, and 2 h upon acid treatment (Figure 3d). To determine whether YdiV is related to flagellum control under acidic conditions, we next assessed the expression of flagellar genes (flhD, fliA, fliC) in ∆ydiV strain during acid adaptation. The expression levels of flhD, fliA and fliC were all remarkably up-regulated after acid treatment (Figure 3g-i), indicating that ∆ydiV lost the acid-mediated flagellum control phenotype. Furthermore, we monitored the motility behavior of WT and ∆ydiV strains at different pH. There was no obvious difference in motility behavior of WT and ∆ydiV strains at pH7.0. However, the ∆ydiV did not exhibit the severe motility defect that was observed in WT strain under acidic pH condition (Figures 3e and 3f). Together, these results suggest that YdiV exerts an important role in flagellum control upon encountering acid stress.

The acid-related transcription regulator CadC binds to ydiV promoter.

Previous studies have shown that Salmonella does not express YdiV in nutrient-rich medium, but nutrition deficiency or osmotic pressure signal will initiate the expression of YdiV.20,21 Our data revealed that acid signal up-regulated the expression of YdiV by an unknown mechanism. By analysis of genome location, we found a long non-coding region (313 bp) upstream of the ydiV gene (Fig. S3A), so we speculated that some transcription factors might play a regulatory role in acid-related YdiV expression. To identify the putative transcription regulator, we performed a DNA pull-down assay with the 313 bp promoter DNA region of ydiV and total cellular protein of Salmonella cultured under acidic condition (Fig. S3B). Mass spectrometry identified several transcription factors, including the acid-related regulator CadC and the global transcription repressor H-NS (Figure 4a and Fig. S4A). Previously, H-NS was found to be a positive regulator of Salmonella motility by silencing ydiV expression. However, CadC is an acid-activated transcription factor and was not previously found to be related to ydiV. We purified the DNA-binding domains of CadC (CadCc, CadC carboxy terminus, 1–160 amino acid) and H-NS, and confirmed their interaction with the ydiV promoter via electrophoretic mobility shift assay (EMSA) (Fig. S5). The EMSA results showed that both CadCc and H-NS bound to the DNA fragment, with DNA shift detected at molar ratio of CadCc: DNA above 2 and a molar ration of H-NS above 4 (Figure 4b and Fig. S4B), indicating CadCc and H-NS directly bind to the ydiV promoter. To more precisely determine the specific locations on the ydiV promoter that interact with CadC or H-NS, in vitro DNase I footprinting assays were performed in the presence and absence of CadCc or H-NS. The results showed that a 33 nt region of the ydiV promoter was protected by CadCc-binding and two regions (51 nt and 34 nt) were protected by H-NS (Figure 4c and Fig. S4C). There was a 19 nt overlap (TCATCATATATAATGATAT) region protected in the presence of both CadCc and H-NS. Furthermore, the interaction dynamics were determined with the 19 nt overlap DNA and the two proteins using the ForteBio Octet detection system. The results revealed that both CadCc and H-NS bound to this DNA with similar KD values (0.203 ± 0.0084 μmoL for CadCc, 0.533 ± 0.0127 μmoL for H-NS) (Figure 4d and Fig. S4D). However, dissociation equilibrium of H-NS to target DNA was achieved much more quickly than for CadCc, with Kdis of 0.0145/s for H-NS and 0.00247/s for CadCc (Figure 4d and Fig. S4D), indicating the binding of CadC to target DNA is more stable than binding of H-NS. The result of EMSA competition assay further proves that CadC can dissociate the H-NS-ydiV promoter DNA comlex (Figure 4e). When the concentration of CadCc is 18.75 µM, it begins to bind to ydiV promoter competitively with H-NS, and when the concentration of CadCc is above 150 µM, it competes completely with H-NS to form CadCc-DNA complex. This data further supports our conclusion that H-NS inhibits ydiV transcription, while CadC removes this repression through competitive binding with ydiV promoter.

Figure 4.

CadC binds to the promoter region of ydiV.

Figure 4.

(A) Domain profile and sequence of CadC. Red sequence indicates the fragment identified by MS. (B) EMSA result for CadCc and ydiV promoter DNA; 5 µM ydiV promoter DNA was pre-incubated with different ratios of CadCc for 10 min. Mixtures were analyzed by native 5% polyacrylamide gel at 4 °C and then stained with GelRed. The ratio of protein to DNA ranged from 0 to 18. A sample containing only CadCc was used as a control. (C) DNase I footprinting assays of the ydiV promoter with CadCc. The promoter fragment of ydiV was PCR amplified and labeled with FAM, incubated with increasing amounts of purified CadCc, and then subjected to DNase I footprinting assay. DNase I digestion reactions were analyzed by ABI 3500XL DNA analyzer. Protected regions are boxed and marked with positions. The upper picture shows the control reactions (no added protein). Protected regions are shown in dotted squares. (D) Biolayer interferometry (BLI) analysis of the binding capacity of CadCc to overlapping DNA. Assessment of BLI response signal (nm) for CadCc protein binding to DNA. (E) CadC and H-NS compete for binding to the promoter region of ydiV. Competitive EMSA using the 313-bp ydiV promoter and the proteins H-NS (constant concentration) and CadCc (variable concentration). The sample containing CadCc+DNA and H-NS+DNA were added as control.

Open in a new tab

H43 and W106 of CadC are essential for ydiV promoter-binding.

To further characterize the interaction between CadC and target DNA, a molecular docking analysis between CadCc and target DNA was performed. To do this, a structural model of Salmonella CadC was built using SWISS Model based on the structure of Escherichia coli CadC (PDB code 5JU7), which has 76.19% amino acid sequence identity to Salmonella CadC. Then, the structural model of Salmonella CadCc-DNA complex was generated using the rigid-body docking program Zdock. The complex model of CadCc and DNA showed a good match in both shape and energy, and the highest score model contains four CadCc molecules on one target DNA molecule (Figure 5a). Further interaction surface analysis demonstrated that residues R7, R32, H42, H43, R60 and W106 of CadC could potentially interact with target DNA (Figure 5b). To determine the role of these residues in target DNA binding, six CadCc mutants (R7A, R32A, H42A, H43A, R60A, and W106A) were engineered into an expression vector and purified from E. coli BL21(DE3). Then, the DNA binding ability of the purified mutant proteins was investigated using EMSA (Figures 5c and 5d). Compared with native CadCc, the R7A and R32A mutants exhibited largely reduced DNA-binding ability to the ydiV promoter, while the H43A and W106A mutants lost almost all their DNA-binding ability to the ydiV promoter, indicating that H43 and W106 are the key amino acids for the CadC-ydiV promoter interaction. Sequence alignment of CadC homologues showed that H43 and W106 are highly conserved, indicating that the mechanism of CadC activation of ydiV expression under acidic conditions may be widespread in bacteria (Fig. S6). Interestingly, the ydiV promoter sequences of Salmonella and E. coli are completely different, and the ydiV promoter of E. coli does not have the same DNA sequence that interacts with CadC in the Salmonella ydiV promoter (Fig. S7). Consistent with the results of sequence alignment, EMSA results showed that both CadCC and H-NS could not bind to E. coli ydiV promoter (Fig. S8).

Figure 5.

Interaction models between CadCc and target DNA.

Figure 5.

(A) The model of the CadCc(DNA)4 complex. The four monomers of CadCc protein are colored in green, light blue, purple, and blue. The DNA molecule bound to the four CadCc monomers is constructed by bases A, T, G, and C, which are colored red, dark blue, emerald green, and yellow, respectively. (B) Detailed interactions between CadCc and DNA of the CadCc-DNA complex. The residues of the DNA binding environment (R7, R32, H42, H43, R60 and W106) are shown as sticks. (C) Binding ability of CadCc mutants to the promoter DNA, as detected by EMSA. The concentration for all CadCc proteins was 25 µM, with DNA at a concentration of 5 μM. Control: reaction system without CadCc in any form. (D) The corresponding band densitometry quantified from the results of EMSA.

Open in a new tab

CadC is crucial for acid-mediated YdiV activation and flagellum control.

Since our data clearly demonstrated CadCc directly binds to the ydiV promoter in vitro, we speculated that CadC may participate in acid-mediated regulation of YdiV expression. To test this, we constructed a cadC knockout strain of Salmonella (∆cadC) and then assessed the expression of ydiV using RT-PCR in WT and ∆cadC strains before and after acid treatment. The results showed that the expression of ydiV was markedly increased 2.49 times in the WT strain after acid treatment compared to the level before. However, in the ∆cadC strain after acid treatment, the expression of ydiV was slightly declined to 0.47 times the level before acid treatment (Figure 6). Overall, there was a 5.3-fold difference in expression of ydiV in the WT strain compared to the ∆cadC strain under the same acidic condition. This indicates that CadC plays an important role in ydiV-expression regulation under acidic condition since flagellum control during acid adaptation is dependent on YdiV and the expression of YdiV is induced by acid in a CadC-dependent manner. Next, flagellum growth was observed in WT, ∆cadC, and ∆ydiV strains under acidic conditions using negative-staining electron microscopy (Figure 7). Fewer flagella were observed in WT Salmonella cultured in pH 5.0; however, multiple flagellar filaments were observed in ∆cadC and ∆ydiV under pH 5.0 acidic condition (~6 flagella for ∆cadC and ~11 flagella ∆ydiV). The loss of the acid-related flagellum control phenotype in the ∆cadC and ∆ydiV strains indicated CadC and YdiV are key factors in the regulation of this process. Collectively, our results demonstrate that the CadC-YdiV axis is essential for acid-related flagellum control.

Figure 6.

CadC exerts a positive effect on YdiV expression under acid stress.

Figure 6.

The transcription levels of YdiV in wild-type and ∆ydiV strains under normal or acid stress conditions were detected by qRT-PCR. Data are shown as means and SEM, n=3. ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., P > 0.05.

Open in a new tab

Figure 7.

The flagellar biogenesis phenotypes of ∆cadC and ∆ydiV strains under acid stress condition.

Figure 7.

(A to C) The numbers of flagella of the wild-type (A), ∆cadC (B) and ∆ydiV (C) strains were observed using negative-staining electron microscopy (EM). (D) Data are shown as the means and SEM, n=3. ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., P > 0.05.

Open in a new tab

CadC activates YdiV expression and flagellar control upon Salmonella entry into host cells.

Considering that CadC is a key regulator of Salmonella in response to acid stress, we speculate that it may affect Salmonella survival within host cells. Therefore, we used WT or ∆cadC to infect RAW264.7 cells with the same multiplicity of infection (MOI). At 2 h, 4 h and 6 h post-infection, RAW264.7 cells were lysed and then the number of bacteria in the cells was determined by counting CFU. The results showed that the survival rate of ΔcadC strain in RAW264.7 cells from 2 h to 6 h after infection were significantly lower than that of WT, suggesting CadC works in the whole infection period (Figure 8a). To further explore the mechanism of the effect of CadC on the intracellular survival of Salmonella, we used qRT-PCR to detect the transcription levels of ydiV and fliC in WT and ΔcadC strains at different post-infection time. Compared with that before invasion, the expression of ydiV in WT and ΔcadC strains after 4 h infection was significantly increased 433-fold and 139-fold, respectively. More importantly, the expression of ydiV in ΔcadC strain is only 1/3 of that in WT strain (Figure 8b). Then, the transcription level of fliC was detected 2 h or 4 h post infection. The results showed that the transcription level of fliC remained almost unchanged between 2 h and 4 h after WT Salmonella entering host cells, while the transcription of fliC increased significantly in ΔcadC strain at 4 h post infection than 2 h post infection (Figure 8c-d). To sum up, in host cells, CadC induces the expression of YdiV by sensing acid signals and then inhibits flagella synthesis, thus realizing Salmonella immune escape.

Figure 8.

CadC is required for flagellar control of Salmonella within host cells.

Figure 8.

(A) Intracellular survival capability of WT and ΔcadC strains within RAW264.7 cells 2, 4, 6h post-infection. (B) The transcription levels of ydiV before and after Salmonella WT or ∆cadC invasion into RAW 264.7 cells were detected by qRT-PCR. (C) The mRNA levels of fliC were detected in WT by qRT-PCR at 2h and 4 h after WT Salmonella entry into RAW264.7 cells. (D) The mRNA levels of fliC were detected by qRT-PCR at 2h and 4 h after ΔcadC entry into RAW264.7 cells. Data are shown as means and SEM, n=3. ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., P > 0.05.

Open in a new tab

Discussion

Flagellum is an important motility organ, enabling movement of bacteria. The construction of bacterial flagella involves the synthesis of dozens of flagellar proteins in a highly energy-consuming process. In the initial stage of Salmonella infection, flagellum-mediated motility facilitates Salmonella gastrointestinal colonization of preferred sites.48–51 However, once Salmonella enters host cells, flagella are key in triggering of the host’s immune response. Interestingly, Salmonella can quickly turn off flagellum synthesis after entering host cells.42,52 How Salmonella senses the host cell environment and controls flagellum synthesis has not been fully determined.

After Salmonella entry, infected host cells recognize clues from the pathogens and undergo a series of changes, including acidification of the SCV.53 Therefore, acidic condition may be the signal that Salmonella perceives to sense the host environment. However, in different bacteria, there are varying effects of acid on flagellum synthesis. Previous research reported that Escherichia coli and Vibrio parahaemolyticus enhanced flagellum synthesis under acidic conditions.54–56 However, the regulation of flagellum synthesis in Salmonella under acidic conditions remains controversial. Some reports observed inhibited flagellum synthesis under acidic conditions, while another study has reported enhanced flagellum synthesis in Salmonella under acidic conditions.57–60 In this study, we determined that Salmonella effectively turns off flagellum synthesis under acidic conditions, which is consistent with the conclusions of earlier work. We further found that acid-related flagellum control occurred after flhDC transcription, and identified an important role of the anti-FlhDC factor YdiV. The acid-inducible transcription factor CadC can specifically bind to ydiV promoter as evidenced by DNA pull down of Salmonella ydiV promoter DNA with a whole-protein lysate of Salmonella cultured in acid condition. CadC is a previously characterized acid-inducible transcription factor that can regulate the expression levels of cadA and cadB genes. This is the first report of CadC-related regulation of the ydiV gene. Interestingly, a previous study reported that CadC increases the expression of FliC protein under acidic condition, which is opposite to our findings in this study.59 In that study, only weak movement of wild-type Salmonella was detected even at pH 7.0. Comparing the method of motility assay, we found that they used the overnight culture and then spotted on semisolid medium plates at different pH (10 mM lysine). While the wild-type Salmonella we used was in the logarithmic phase and the culture plate did not contain 10 mM lysine. The above reasons may lead to inconsistent experiment results.

Our previous study showed that YdiV regulates flagellum biogenesis and iron absorption.19,61 Previous literature shows that H-NS mediates the silencing of ydiV gene under normal growth conditions.62 It has also been reported that YdiV expression is regulated by signals such as osmotic pressure and nutritional deficiency.20,21 This finding of the regulatory effect of acid on the expression of YdiV, and our results reveal CadC as an acid-sensing regulator of YdiV expression. Based on our experimental results, we propose a mechanistic model of acid-mediated flagellum control (Figure 9). Before Salmonella enters the host cell, H-NS binds to the promoter region of the ydiv gene to block transcription, so there is little expression of YdiV protein and Salmonella produce flagellum. Using their flagella, Salmonella can invade host cells. After Salmonella enters a host cell, the host cell acidifies rapidly. Under acidic conditions, CadC on the cell membrane is cut off by protease, releasing the DNA-binding domain of CadC into the cytoplasm. CadC then binds to the promoter region of ydiV with a high binding affinity, slowly replacing H-NS near the ydiV promoter, thus gradually relieving the expression inhibition of YdiV by H-NS. As a result, YdiV is expressed in large quantities. YdiV binds to FlhDC to shut-off the transcription of secondary and tertiary flagellar genes, and flagellum biogenesis is terminated. Although ∆ydiV has better mobility than WT under acidic conditions, it still has motility defect compared with mobility under pH 7.0. We speculate that it could be a second mechanism that also affects motility at acidic stress, which is independent of YdiV. In summary, our results indicate an acid-related flagellum control mechanism that is mainly mediated by CadC and YdiV. This regulation allows Salmonella to rapidly sense the host condition and turn off flagellar biogenesis to escape recognition by the immune system. Salmonella employs a complex mechanism to control flagellar synthesis. Under acidic conditions, the number of flagella in CadC knockout Salmonella was much greater than that of wild type Salmonella, but less than that of ydiV knockout Salmonella, suggesting that although CadC acts in the flagellar control of Salmonella, it might not be the only acid-mediated regulator. The results of our DNA pull-down experiment indicated that other transcription factors in addition to CadC may bind to the promotor of the ydiV gene to modulate expression of ydiV, suggesting that there is likely greater complexity of this regulatory network, which is worthy of further study.

Figure 9.

Model of acid-mediated flagellar control in Salmonella.

Figure 9.

Before Salmonella enters host cells, repressor H-NS inhibits the transcription of ydiV by binding to the promoter region to promote flagellar biogenesis. When Salmonella enters macrophage phagosomes and is subjected to acid stress signal, the membrane-bound transcriptional regulator CadC is activated by proteolytic cleavage. Binding of active CadC to the ydiV promoter releases the H-NS molecules, so CadC thereby functions as a transcriptional activator of ydiV. Increased levels of YdiV protein led to negative regulation of the flagellar master transcriptional activator complex, FlhD4C2, and subsequent downregulation of flagellar biogenesis.

Open in a new tab

Material and methods

Bacterial strains and growth conditions

Salmonella typhimurium ATCC 14028s was used for functional studies. Bacteria were routinely grown at 37°C in LB medium or Vogel and Bonner E medium with a final concentration of 0.4% glucose.63 When required, ampicillin was added to the culture medium to a final concentration of 100 μg/mL. For acid stress experiments, the bacteria were grown to OD600 = 0.4 in E glucose medium (pH 7.0), and then cultured under acidic condition (pH 5.0, 10 mM L-lysine). Unless otherwise specified, the acid used in acid stress experiments is acetic acid.

Construction of CadC knock-out strain

The CadC knock-out strain was constructed with the one-step gene inactivation method as described previously.64

Swimming motility

Swimming motility of bacteria strains was determined on semisolid E glucose medium containing 0.25% agar. Briefly, bacterial cells were cultured to OD600 = 0.4–0.6 and then adjusted to OD600 = 10 in PBS. The bacteria were inoculated on dry soft agar plates at different pH, after adjustment with acetic acid. The bacteria swimming diameters were measured after incubation at 30°C for 24 h.

Negative-stain electron microscopy

To detect the effects of environmental pH changes on the number of flagella, the strains used in the motility assay were further analyzed by transmission electron microscopy. Cells were cultured at 37°C to early-logarithmic growth phase (OD600 = 0.4) in E glucose medium, followed by acid stress for 2 h. The cells were centrifuged at 5000 × g for 5 min at room temperature, and then suspended in 2.5% (w/v) glutaraldehyde. After dipping with carbon nets and then drying for 5 min in a natural state, samples were negatively stained with 1% phosphotungstic acid for 3 min. The samples were observed with a JEM-1200EX transmission electron microscope.

RNA-sequencing analysis

The Salmonella wild-type strain was cultured overnight in LB medium. Cultures were diluted 1:100 in fresh E glucose medium (pH 7.0) and allowed to continue growth to OD600 = 0.4. Subsequently, half of the culture was treated with acid stress at pH 5.0 for 2 h and the remaining bacteria were further cultured. Next, 3 mL bacterial cells were obtained by centrifugation (3000 rpm, 5 min) and freezing treatment in liquid nitrogen. RNA isolation, library construction, and sequencing were performed by Novogene Co. Ltd. Total RNA samples were isolated from the obtained bacteria using TRIzol reagent according to the reagent manufacturer’s instructions. The quantification and qualification of total RNA was measured using 1% agarose gels and Agilent 2100 Bioanalyzer. The RNA was purified by removal of rRNA and random fragmentation before synthesis of cDNA. After adenylation of 3’ ends of DNA fragments, adaptors were ligated to prepare for hybridization. Finally, the library fragments were purified by AMPure XP system to preferentially select cDNA fragments 370–420 bp in length. PCR products were purified and library quality was assessed on the Agilent Bioanalyzer 2100 system. The libraries were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.

RNA preparation and real-time quantitative PCR

Total RNA was extracted with TRIzol reagent and reverse transcribed using the RevertAid cDNA Synthesis Kit according to the manufacturer’s instructions. Real-time quantitative PCR assay was performed using iTaq Universal SYBR Green SuperMix and corresponding primers with an Applied Biosystems 7500 Sequence Detection system. The relative mRNA expression of target genes was normalized to the expression level of GAPDH and the quantification result was calculated using the 2−ΔΔct method.

Western blot

Bacterial total proteins were heated at 95°C for 10 min and separated by SDS-PAGE. The proteins were transferred from gel onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% milk in phosphate-buffered saline plus 0.1% Tween (PBST) at 25°C for 1 h, the membranes were incubated with primary antibodies in PBST overnight at 4°C. The membrane was then washed three times in PBST and incubated with secondary antibodies at 25°C for 1 h. After three washes with PBST, proteins on membranes were visualized with a chemiluminescent substrate and detected using a FluorChem imager. The protein levels were detected and quantified using ImageJ software. Primary antibodies against DnaK (Abcom, 1:10,000 dilution), FliC (Dia-An Biotech, 1:5,000 dilution), and YdiV (Dia-An Biotech, 1:2,000 dilution) were used. The secondary antibodies were anti-rabbit and anti-mouse IgG antibodies (Abcom, 1:10,000 dilution).

Cell culture and bacterial infection experiments

The murine RAW264.7 macrophage-like cell line (ATCC) was cultured at 37°C with 5% CO2 in DMEM (Dulbecco's Modified Eagle Medium) medium supplemented with 10% fetal bovine serum (Gibco). The cells were seeded in triplicate in 100-mm tissue culture dishes at a cell density of 1 × 107 cells per dish. Salmonella wild-type strain was cultured in LB medium at 37°C until reaching log-phase. The bacterial cells were then diluted in DMEM medium and seeded on RAW264.7 cells at a multiplicity of infection (MOI) of 10. After infection for 1 h, the cells were washed twice with PBS. Fresh DMEM medium with 100 µg/mL gentamicin was added to kill any remaining extracellular bacteria. At each time point post-infection, cells were washed and lysed in TRIzol reagent. The samples were stored at −70°C before RNA extraction. For the intracellular survival assays of bacteria strain within RAW264.7 cells, the cells were lysed with 1% Triton X-100, and the number of intracellular bacteria was detected by the colony-forming units (CFU) counts of viable colonies.

Biotin-streptavidin pull-down

Proteins that bind the ydiV promoter were detected by biotin-streptavidin pull-down assay as previously described.65–67 Briefly, the DNA sequence of ydiV promoter was amplified by PCR and labeled with biotin using Biotin DecaLabel DNA Labeling Kit. Next, 5 ug biotin-labeled ydiV promoter DNA probes and 500 ug nuclear protein extracts from bacteria cells were mixed and incubated on ice. After washing with PBS buffer, 100 µL Streptavidin-agarose G beads were added into the DNA-nuclear protein mixture and incubated for 1 h at 4°C. Beads were then collected by centrifugation and washed three times with PBS buffer. The protein-DNA-streptavidin-agarose complexes were identified by Western blotting and mass spectrometry.

The “bacterial extract” used in the above experiment is prepared as follows. Salmonella typhimurium ATCC 14028s were grown at 37°C in E glucose medium (pH 7.0) to OD600 = 0.4. Subsequently, half of the culture was treated with acid stress at pH 5.0 for 2 h and the remaining bacteria were further cultured. Cells were then resuspended in PBS buffer and disrupted by high pressure systems at 4°C. After centrifugation at 14,000 rpm for 60 min to remove debris, the supernatant used as the bacterial extract.

Electrophoretic mobility shift assay

EMSA was performed by the method described.19 Briefly, 5 µM DNA was incubated with different ratios of proteins in reaction buffer (20 mM Tris–HCl, 100 mM NaCl, 1 mM MgCl2, 1 mM ZnCl2 and 4%(v/v) glycerol, pH 8.0) for 10 min at room temperature. Samples were separated on native 5% polyacrylamide gel and then stained using GelRed and Coomassie brilliant blue.

For EMSA competition assay, 5 µM ydiV promoter DNA was incubated with 30 µM H-NS for 30 min at 4°C. Then, increasing amounts of the CadCc protein (3.75 to 150 μM) was added. After incubation at 4°C for 30 minutes, samples were separated on native 5% polyacrylamide gel and then stained using GelRed and Coomassie brilliant blue.

DNase I footprinting assay

DNase I footprinting assays were performed similarly to the method described by Wang et al.68 For preparation of fluorescent FAM labeled probes, the ydiV promoter region was PCR amplified using primers of ydiV promoter-5 (FAM) and ydiV promoter-3. After purification and quantification, 300 ng FAM-labeled probes were incubated with different amounts of proteins (CadCc or H-NS) for 30 min at 30°C in a total volume of 40 µL. The protein-probe mixture was then added 10 µL reaction buffer, which contained about 0.015 unit DNase I and 100 nmoL freshly prepared CaCl2 solution, and then incubated at 37°C for 1 min. The reaction was stopped by the addition of 140 µL DNase I stop buffer (200 mM unbuffered sodium acetate, 30 mM EDTA, and 0.15% SDS). Samples were extracted with phenol/chloroform and then precipitated with ethanol. The obtained pellets were dissolved in 30 µL Milli-Q water and used for DNA ladder preparation and electrophoresis. The obtained data were analyzed using Peak Scanner software v1.0.

Biolayer interferometry assay

Biolayer interferometry (BLI) assay was performed using an Octet RED96 instrument at 25°C.69 To examine the interaction between CadCc and the overlapping DNA fragment, the DNA was biotin-conjugated at the C-terminus and diluted to 1 μM in buffer A (PBS buffer containing 1 mM ZnCl2). Following an initial baseline step in buffer A, the streptavidin sensor captured biotin-conjugated DNA at a ligand density of ~1 nm. Following equilibration in buffer A for 60 s, the ligand-coated sensor was transferred to buffer B (buffer A containing 50 µg/mL CadCc) for 180 s followed by buffer A for 180 s to determine the binding/association signal. The unloaded DNA fragment sensor was used as the control group. The interaction between H-NS and the overlapping DNA fragment was detected by the same method, and the buffer A used in the process was PBS buffer.

Plasmid construction

DNA fragments of CadCc and H-NS genes were amplified by PCR from genomic DNA from E. coli strain K-12 substrain MG1655. After digestion with restriction endonuclease BamH I and Xho I, the PCR fragments were inserted into the pGL01 vector, the resulting recombinant plasmids pGL01-CadCc or pGL01-H-NS, which has 6 × His-tag at the N terminal of the protein. CadCc mutants (R7A, R32A, H42A, H43A, R60A, and W106A) were constructed using a site-directed mutagenesis system. Briefly, the DNA fragments of CadCc mutants gene were amplifified by PCR from recombinant plasmid pGL01-CadCc. After digestion with Dpn I, the linear PCR fragments were ligated and cyclized to generate plasmids pGL01-CadCmut. All plasmids and primers used in this study are listed in Table S2 and S3.

Protein expression and purification

E. coli BL21 (DE3) was used to express the CadCc and H-NS proteins from the corresponding plasmids. E. coli cells were grown in LB media containing 100 μg/mL ampicillin at 37°C and protein expression was induced overnight by addition of 0.6 mmol/L isopropyl β-D-1- thiogalactopyranoside (IPTG) at 16°C. Cells were then harvested and disrupted. Proteins were purified by Ni2+ His-Trap HP, and determined by SDS-PAGE followed by coomassie blue staining. All protein purification steps were performed at 4°C.

CadCc–DNA molecular modeling

Docking simulations between CadCc and DNA were performed and measured using Mode Tec, Inc. (Sichuan, China) based on the CadC structure and molecular docking. The structure of the E.coli CadC DNA binding domain (PDB code: 5JU7) was used to model the Salmonella CadC structure by homology modeling.70

Supplementary Material

Supplemental Material
Click here for additional data file. (850.8KB, pdf)

Funding Statement

National Natural Science Foundation of China [81902038 to W.W.W., 32170034 to B.Q.L, 31800054 to Y.Y.Y]. the Special fund for Taishan Scholars Project (B.Q.L.). Natural Science Foundation of Shandong Province [ZR2019PH038 to W.W.W., ZR202102230675 to F.Y.Z.]. National Science Foundation for Post-doctoral Scientists of China [2021M701996 to F.Y.Z.]. the Academic Promotion Programme of Shandong First Medical University [2019LJ001 to B.Q.L.]. Youth Innovation Talent Development Plan of Shandong Provincial University (B.Q.L.).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2022.2146979

Data Availability Statement

The data that support the findings of this study are available from Bingqing Li (bingqingsdu@163.com), upon reasonable request. Original data is available in Mendeley Dataset (http://doi.org/ 10.17632/r733bshdnv.1) and GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE210570).

References

  • 1.Smith SI, Seriki A, Ajayi A.. Typhoidal and non-typhoidal Salmonella infections in Africa. Eur J Clin Microbiol Infect Dis. 2016;35(12):1913–17. doi: 10.1007/s10096-016-2760-3. [DOI] [PubMed] [Google Scholar]
  • 2.Galan JE. Salmonella Typhimurium and inflammation: a pathogen-centric affair. Nat Rev Microbiol. 2021;19(11):716–725. doi: 10.1038/s41579-021-00561-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Whiley H, Gardner MG, Ross K. A review of salmonella and squamates (Lizards, snakes and amphisbians): implications for public health. Pathogens. 2017;6(3):38. doi: 10.3390/pathogens6030038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Moffatt CR, Musto J, Pingault N, Miller M, Stafford R, Gregory J, Polkinghorne BG, Kirk MD. Salmonella typhimurium and outbreaks of egg-associated disease in Australia, 2001 to 2011. Foodborne Pathog Dis. 2016;13(7):379–385. doi: 10.1089/fpd.2015.2110. [DOI] [PubMed] [Google Scholar]
  • 5.Buckle GC, Walker CL, Black RE. Typhoid fever and paratyphoid fever: systematic review to estimate global morbidity and mortality for 2010. J Glob Health. 2012;2(1):010401. doi: 10.7189/jogh.01.010401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Muvhali M, Smith AM, Rakgantso AM, Keddy KH. Investigation of salmonella enteritidis outbreaks in South Africa using multi-locus variable-number tandem-repeats analysis, 2013-2015. BMC Infect Dis. 2017;17(1):661. doi: 10.1186/s12879-017-2751-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Haiko J, Westerlund-Wikstrom B. The role of the bacterial flagellum in adhesion and virulence. Biology (Basel). 2013;2(4):1242–1267. doi: 10.3390/biology2041242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Rossez Y, Wolfson EB, Holmes A, Gally DL, Holden NJ, Bliska JB. Bacterial flagella: twist and stick, or dodge across the kingdoms. PLoS Pathog. 2015;11(1):e1004483. doi: 10.1371/journal.ppat.1004483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Gut AM, Vasiljevic T, Yeager T, Donkor ON. Salmonella infection - prevention and treatment by antibiotics and probiotic yeasts: a review. Microbiology (Reading). 2018;164(11):1327–1344. doi: 10.1099/mic.0.000709. [DOI] [PubMed] [Google Scholar]
  • 10.Frye J, Karlinsey JE, Felise HR, Marzolf B, Dowidar N, McClelland M, Hughes KT. Identification of new flagellar genes of Salmonella enterica serovar typhimurium. J Bacteriol. 2006;188(6):2233–2243. doi: 10.1128/JB.188.6.2233-2243.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chilcott GS, Hughes KT. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiol Mol Biol Rev. 2000;64(4):694–708. doi: 10.1128/MMBR.64.4.694-708.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Yanagihara S, Iyoda S, Ohnishi K, Iino T, Kutsukake K. Structure and transcriptional control of the flagellar master operon of Salmonella typhimurium. Genes Genet Syst. 1999;74(3):105–111. doi: 10.1266/ggs.74.105. [DOI] [PubMed] [Google Scholar]
  • 13.Kutsukake K, Ohya Y, Iino T. Transcriptional analysis of the flagellar regulon of Salmonella typhimurium. J Bacteriol. 1990;172(2):741–747. doi: 10.1128/jb.172.2.741-747.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ide N, Ikebe T, Kutsukake K. Reevaluation of the promoter structure of the class 3 flagellar operons of Escherichia coli and Salmonella. Genes Genet Syst. 1999;74(3):113–116. doi: 10.1266/ggs.74.113. [DOI] [PubMed] [Google Scholar]
  • 15.Karlinsey JE, Tanaka S, Bettenworth V, Yamaguchi S, Boos W, Aizawa SI, Hughes KT. Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol Microbiol. 2000;37(5):1220–1231. doi: 10.1046/j.1365-2958.2000.02081.x. [DOI] [PubMed] [Google Scholar]
  • 16.Tomoyasu T, Takaya A, Isogai E, Yamamoto T. Turnover of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease. Mol Microbiol. 2003;48(2):443–452. doi: 10.1046/j.1365-2958.2003.03437.x. [DOI] [PubMed] [Google Scholar]
  • 17.Liu Y, Yu K, Zhou F, Ding T, Yang Y, Hu M, Liu X. Quantitative proteomics charts the landscape of Salmonella carbon metabolism within host epithelial cells. J Proteome Res. 2017;16(2):788–797. doi: 10.1021/acs.jproteome.6b00793. [DOI] [PubMed] [Google Scholar]
  • 18.Srikumar S, Kroger C, Hebrard M, Colgan A, Owen SV, Sivasankaran SK, Cameron ADS, Hokamp K, Hinton JCD. RNA-seq brings new insights to the intra-macrophage transcriptome of Salmonella typhimurium. PLoS Pathog. 2015;11(11):e1005262. doi: 10.1371/journal.ppat.1005262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Li B, Li N, Wang F, Guo L, Huang Y, Liu X, Wei T, Zhu D, Liu C, Pan H, et al. Structural insight of a concentration-dependent mechanism by which YdiV inhibits Escherichia coli flagellum biogenesis and motility. Nucleic Acids Res. 2012;40(21):11073–11085. doi: 10.1093/nar/gks869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Wada T, Morizane T, Abo T, Tominaga A, Inoue-Tanaka K, Kutsukake K. EAL domain protein YdiV acts as an Anti-FlhD 4 C 2 factor responsible for nutritional control of the flagellar regulon in Salmonella enterica serovar typhimurium. J Bacteriol. 2011;193(7):1600–1611. doi: 10.1128/JB.01494-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Takaya A, Erhardt M, Karata K, Winterberg K, Yamamoto T, Hughes KT. YdiV: a dual function protein that targets FlhDC for ClpXP-dependent degradation by promoting release of DNA-bound FlhDC complex. Mol Microbiol. 2012;83(6):1268–1284. doi: 10.1111/j.1365-2958.2012.08007.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Li B, Yue Y, Yuan Z, Zhang F, Li P, Song N, Lin W, Liu Y, Yang Y, Li Z, et al. Salmonella STM1697 coordinates flagella biogenesis and virulence by restricting flagellar master protein FlhD4C2 from recruiting RNA polymerase. Nucleic Acids Res. 2017;45(17):9976–9989. doi: 10.1093/nar/gkx656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ma Y, Yue Y, Jia H, Song N, Zhai L, Wang W, Li, C, Li, B. Switching off bacterial flagellar biogenesis by YdiU-mediated UMPylation of FlhDC. mBio 2022:e0024922. doi: 10.1128/mbio.00249-22. [DOI] [PMC free article] [PubMed]
  • 24.LaRock DL, Chaudhary A, Miller SI. Salmonellae interactions with host processes. Nat Rev Microbiol. 2015;13(4):191–205. doi: 10.1038/nrmicro3420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gogoi M, Shreenivas MM, Chakravortty D. Hoodwinking the big-eater to prosper: the Salmonella-macrophage paradigm. J Innate Immun. 2019;11(3):289–299. doi: 10.1159/000490953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.de Jong Hk, Parry CM, van der Poll T, Wiersinga WJ, de Jong HK. Host-pathogen interaction in invasive Salmonellosis. PLoS Pathog. 2012;8(10):e1002933. doi: 10.1371/journal.ppat.1002933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Sholpan A, Lamas A, Cepeda A, Franco CM. Salmonella spp. quorum sensing: an overview from environmental persistence to host cell invasion. AIMS Microbiol. 2021;7(2):238–256. doi: 10.3934/microbiol.2021015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Fang FC, Frawley ER, Tapscott T, Vazquez-Torres A. Bacterial stress responses during host infection. Cell Host Microbe. 2016;20(2):133–143. doi: 10.1016/j.chom.2016.07.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Pradhan D, Devi Negi V. Stress-induced adaptations in Salmonella: a ground for shaping its pathogenesis. Microbiol Res. 2019;229:126311. doi: 10.1016/j.micres.2019.126311. [DOI] [PubMed] [Google Scholar]
  • 30.Haraga A, Ohlson MB, Miller SI. Salmonellae interplay with host cells. Nat Rev Microbiol. 2008;6(1):53–66. doi: 10.1038/nrmicro1788. [DOI] [PubMed] [Google Scholar]
  • 31.Zwir I, Shin D, Kato A, Nishino K, Latifi T, Solomon F, Hare JM, Huang H, Groisman EA. Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica. Proc Natl Acad Sci U S A. 2005;102(8):2862–2867. doi: 10.1073/pnas.0408238102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Tu X, Latifi T, Bougdour A, Gottesman S, Groisman EA. The PhoP/PhoQ two-component system stabilizes the alternative sigma factor RpoS in Salmonella enterica. Proc Natl Acad Sci U S A. 2006;103(36):13503–13508. doi: 10.1073/pnas.0606026103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Garcia-Calderon CB, Casadesus J, Ramos-Morales F. Rcs and PhoPQ regulatory overlap in the control of Salmonella enterica virulence. J Bacteriol. 2007;189(18):6635–6644. doi: 10.1128/JB.00640-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Chakraborty S, Winardhi RS, Morgan LK, Yan J, Kenney LJ. Non-canonical activation of OmpR drives acid and osmotic stress responses in single bacterial cells. Nat Commun. 2017;8(1):1587. doi: 10.1038/s41467-017-02030-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Chakraborty S, Mizusaki H, Kenney LJ, Laub MT. A FRET-based DNA biosensor tracks OmpR-dependent acidification of Salmonella during macrophage infection. PLoS Biol. 2015;13(4):e1002116. doi: 10.1371/journal.pbio.1002116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Erhardt M, Dersch P. Regulatory principles governing Salmonella and Yersinia virulence. Front Microbiol. 2015;6:949. doi: 10.3389/fmicb.2015.00949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Banos RC, Pons JI, Madrid C, Juarez A. A global modulatory role for the Yersinia enterocolitica H-NS protein. Microbiology (Reading). 2008;154(5):1281–1289. doi: 10.1099/mic.0.2007/015610-0. [DOI] [PubMed] [Google Scholar]
  • 38.Lucchini S, Rowley G, Goldberg MD, Hurd D, Harrison M, Hinton JC, Isberg RR. H-NS mediates the silencing of laterally acquired genes in bacteria. PLoS Pathog. 2006;2(8):e81. doi: 10.1371/journal.ppat.0020081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Krin E, Danchin A, Soutourina O. Decrypting the H-NS-dependent regulatory cascade of acid stress resistance in Escherichia coli. BMC Microbiol. 2010;10(1):273. doi: 10.1186/1471-2180-10-273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Stoebel DM, Free A, Dorman CJ. Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria. Microbiology (Reading). 2008;154(9):2533–2545. doi: 10.1099/mic.0.2008/020693-0. [DOI] [PubMed] [Google Scholar]
  • 41.Navarre WW, McClelland M, Libby SJ, Fang FC. Silencing of xenogeneic DNA by H-NS-facilitation of lateral gene transfer in bacteria by a defense system that recognizes foreign DNA. Genes Dev. 2007;21(12):1456–1471. doi: 10.1101/gad.1543107. [DOI] [PubMed] [Google Scholar]
  • 42.Viala JP, Meresse S, Pocachard B, Guilhon AA, Aussel L, Barras F, Chakravortty D. Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella. PLoS One. 2011;6(7):e22397. doi: 10.1371/journal.pone.0022397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Lee YH, Kim JH, Bang IS, Park YK. The membrane-bound transcriptional regulator CadC is activated by proteolytic cleavage in response to acid stress. J Bacteriol. 2008;190(14):5120–5126. doi: 10.1128/JB.00012-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Kieboom J, Abee T. Arginine-dependent acid resistance in Salmonella enterica serovar typhimurium. J Bacteriol. 2006;188(15):5650–5653. doi: 10.1128/JB.00323-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Lee YH, Kim BH, Kim JH, Yoon WS, Bang SH, Park YK. CadC has a global translational effect during acid adaptation in Salmonella enterica serovar typhimurium. J Bacteriol. 2007;189(6):2417–2425. doi: 10.1128/JB.01277-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Fritz G, Koller C, Burdack K, Tetsch L, Haneburger I, Jung K, Gerland U. Induction kinetics of a conditional pH stress response system in Escherichia coli. J Mol Biol. 2009;393(2):272–286. doi: 10.1016/j.jmb.2009.08.037. [DOI] [PubMed] [Google Scholar]
  • 47.Park YK, Bearson B, Bang SH, Bang IS, Foster JW. Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium. Mol Microbiol. 1996;20(3):605–611. doi: 10.1046/j.1365-2958.1996.5441070.x. [DOI] [PubMed] [Google Scholar]
  • 48.Chaban B, Hughes HV, Beeby M. The flagellum in bacterial pathogens: for motility and a whole lot more. Semin Cell Dev Biol. 2015;46:91–103. doi: 10.1016/j.semcdb.2015.10.032. [DOI] [PubMed] [Google Scholar]
  • 49.Dibb-Fuller MP, Allen-Vercoe E, Thorns CJ, Woodward MJ. Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis. Microbiology (Reading). 1999;145(Pt 5):1023–1031. doi: 10.1099/13500872-145-5-1023. [DOI] [PubMed] [Google Scholar]
  • 50.Horstmann JA, Lunelli M, Cazzola H, Heidemann J, Kuhne C, Steffen P, Szefs S, Rossi C, Lokareddy RK, Wang C, et al. Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion. Nat Commun. 2020;11(1):2013. doi: 10.1038/s41467-020-15738-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Stecher B, Hapfelmeier S, Muller C, Kremer M, Stallmach T, Hardt WD. Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar typhimurium colitis in streptomycin-pretreated mice. Infect Immun. 2004;72(7):4138–4150. doi: 10.1128/IAI.72.7.4138-4150.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Miao EA, Alpuche-Aranda CM, Dors M, Clark AE, Bader MW, Miller SI, Aderem A. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf. Nat Immunol. 2006;7(6):569–575. doi: 10.1038/ni1344. [DOI] [PubMed] [Google Scholar]
  • 53.Kinchen JM, Ravichandran KS. Phagosome maturation: going through the acid test. Nat Rev Mol Cell Biol. 2008;9(10):781–795. doi: 10.1038/nrm2515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Polen T, Rittmann D, Wendisch VF, Sahm H. DNA microarray analyses of the long-term adaptive response of Escherichia coli to acetate and propionate. Appl Environ Microbiol. 2003;69(3):1759–1774. doi: 10.1128/AEM.69.3.1759-1774.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Gu D, Wang K, Lu T, Li L, Jiao X. Vibrio parahaemolyticus CadC regulates acid tolerance response to enhance bacterial motility and cytotoxicity. J Fish Dis. 2021;44(8):1155–1168. doi: 10.1111/jfd.13376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Maurer LM, Yohannes E, Bondurant SS, Radmacher M, Slonczewski JL. pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12. J Bacteriol. 2005;187(1):304–319. doi: 10.1128/JB.187.1.304-319.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Adams P, Fowler R, Kinsella N, Howell G, Farris M, Coote P, O’Connor CD. Proteomic detection of PhoPQ- and acid-mediated repression of Salmonella motility. Proteomics. 2001;1(4):597–607. doi:. [DOI] [PubMed] [Google Scholar]
  • 58.Ryan D, Pati NB, Ojha UK, Padhi C, Ray S, Jaiswal S, Singh GP, Mannala GK, Schultze T, Chakraborty T, et al. Global transcriptome and mutagenic analyses of the acid tolerance response of Salmonella enterica serovar Typhimurium. Appl Environ Microbiol. 2015;81(23):8054–8065. doi: 10.1128/AEM.02172-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Lee YH, Kim JH. Direct interaction between the transcription factors CadC and OmpR involved in the acid stress response of Salmonella enterica. J Microbiol. 2017;55(12):966–972. doi: 10.1007/s12275-017-7410-7. [DOI] [PubMed] [Google Scholar]
  • 60.Ibrahim GF, Fleet GH, Lyons MJ, Walker RA. Method for the isolation of highly purified Salmonella flagellins. J Clin Microbiol. 1985;22(6):1040–1044. doi: 10.1128/jcm.22.6.1040-1044.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Zhang F, Li B, Dong H, Chen M, Yao S, Li J, Zhang H, Liu X, Wang H, Song N, et al. YdiV regulates Escherichia coli ferric uptake by manipulating the DNA-binding ability of fur in a SlyD-dependent manner. Nucleic Acids Res. 2020;48(17):9571–9588. doi: 10.1093/nar/gkaa696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Hatamoto Y, Wada T, Kutsukake K. Anti-FlhD (4) C (2) protein YdiV plays a central role in H-NS-dependent regulation of flagellar synthesis in Escherichia coli. Genes & genetic systems: genetics soc Japan national inst genetics yata 1111, Mishima, Shizuoka-Ken … , 2011:408. [Google Scholar]
  • 63.Maloy SR, Roth JR. Regulation of proline utilization in Salmonella typhimurium: characterization of put::Mu d(Ap,lac) operon fusions. J Bacteriol. 1983;154(2):561–568. doi: 10.1128/jb.154.2.561-568.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America. 2000;97:6640–6645. doi: 10.1073/pnas.120163297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Chowdhury RP, Gupta S, Chatterji D. Identification and characterization of the dps promoter of mycobacterium smegmatis: promoter recognition by stress-specific extracytoplasmic function sigma factors σ H and σ F. J Bacteriol. 2007;189(24):8973–8981. doi: 10.1128/JB.01222-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Deng WG, Jayachandran G, Wu G, Xu K, Roth JA, Ji L. Tumor-specific activation of human telomerase reverses transcriptase promoter activity by activating enhancer-binding protein-2beta in human lung cancer cells. J Biol Chem. 2007;282(36):26460–26470. doi: 10.1074/jbc.M610579200. [DOI] [PubMed] [Google Scholar]
  • 67.Wu KK. Analysis of protein-DNA binding by streptavidin-agarose pulldown. Methods Mol Biol. 2006;338:281–290. [DOI] [PubMed] [Google Scholar]
  • 68.Wang Y, Cen XF, Zhao GP, Wang J. Characterization of a new glnr binding box in the promoter of amtB in Streptomyces coelicolor inferred a phop/glnr competitive binding mechanism for transcriptional regulation of amtB. J Bacteriol. 2012;194(19):5237–5244. doi: 10.1128/JB.00989-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Sultana A, Lee JE. Measuring protein-protein and protein-nucleic acid interactions by biolayer interferometry. Curr Protoc Protein Sci. 2015;79(1):19 25 1–19 25 6. doi: 10.1002/0471140864.ps1925s79. [DOI] [PubMed] [Google Scholar]
  • 70.Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993;234(3):779–815. doi: 10.1006/jmbi.1993.1626. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
Click here for additional data file. (850.8KB, pdf)

Data Availability Statement

The data that support the findings of this study are available from Bingqing Li (bingqingsdu@163.com), upon reasonable request. Original data is available in Mendeley Dataset (http://doi.org/ 10.17632/r733bshdnv.1) and GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE210570).

Articles from Gut Microbes are provided here courtesy of Taylor & Francis

RESOURCES