Downregulation of a novel flagellar synthesis regulator AsiR promotes intracellular replication and systemic pathogenicity of Salmonella Typhimurium

ABSTRACT The intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) exploits host macrophage as a crucial survival and replicative niche. To minimize host immune response stimulated by flagellin, the expression of flagellar genes is downregulated during S. Typhimurium growth within host macrophages. However, the underlying mechanisms are largely unknown. In this study, we show that STM14_1285 (named AsiR), a putative RpiR-family transcriptional regulator, which is downregulated within macrophages as previously reported and also confirmed here, positively regulates the expression of flagellar genes by directly binding to the promoter of flhDC. By generating an asiR mutant strain and a strain that persistently expresses asiR gene within macrophages, we confirmed that the downregulation of asiR contributes positively to S. Typhimurium replication in macrophages and systemic infection in mice, which could be attributed to decreased flagellar gene expression and therefore reduced flagellin-stimulated secretion of pro-inflammatory cytokines IL-1β and TNF-α. Furthermore, the acidic pH in macrophages is identified as a signal for the downregulation of asiR and therefore flagellar genes. Collectively, our results reveal a novel acidic pH signal-mediated regulatory pathway that is utilized by S. Typhimurium to promote intracellular replication and systemic pathogenesis by repressing flagellar gene expression.


Introduction
Salmonella is recognized as a major foodborne pathogen, causing self-limiting gastroenteritis or life-threatening systemic disease in both humans and a broad range of animals [1]. Infection by Salmonella represents a considerable burden worldwide, with more than 90 million human cases of gastroenteritis and 20 million human cases of systemic disease being reported each year [2,3]. Of the 2600+ serovars identified so far, Salmonella enterica serovar Typhimurium (S. Typhimurium) represents a primary pathogenic serovar [4]. Infection by S. Typhimurium results in gastroenteritis in humans but leads to a typhoid-like systemic infection in mice [5]. The mouse model of S. Typhimurium has been extensively used to investigate the human systemic disease and the mechanisms of Salmonella virulence [6].
Salmonella infection generally begins with the consumption of contaminated water or food [7]. After it travels along the host's gastrointestinal tract, Salmonella invades the intestinal epithelium and ultimately spreads to the liver and spleen to induce systemic infection [8].
Invasion of intestine epithelial cells and replication inside host macrophages are two hallmarks of Salmonella pathogenesis [1], which are necessary for gastroenteritis and systemic disease, respectively.
During the intracellular phase, Salmonella resides and replicates within a unique vacuolar compartment termed Salmonella-containing vacuole (SCV) [9]. Salmonella encounters several stresses in the SCV, including low magnesium (Mg 2+ ) and phosphate (P i ) concentration, mildly acidic pH, and reactive oxygen species [10]. To resist these intracellular antibacterial factors, Salmonella species have developed a variety of virulence regulatory mechanisms [11,12]. Several regulatory proteins have been previously identified to be involved in the regulatory network and have been reported to contribute to Salmonella intracellular replication in response to various environmental stimuli. PagR, a LacI family transcriptional regulator, is induced by the low Mg 2+ and low P i stimuli in host macrophages, and it activates the expression of Salmonella pathogenicity island (SPI)-2 genes, which are essential for Salmonella intracellular replication [13]. The two-component regulatory systems, PhoP/Q and EnvZ/ OmpR, also activate the expression of SPI-2 genes in response to low Mg 2+ , acidic pH, and the presence of antibacterial peptides inside host macrophages (PhoP/ Q) [14] or acidic pH (EnvZ/OmpR) [15]. The putative LysR-type transcriptional regulator Hrg was found to repress the uvrA and katG genes to reduce ROS production in macrophages and thus favor intracellular survival of Salmonella [16,17].
The flagella contribute to S. Typhimurium pathogenesis by promoting bacterial adherence to and invasion of host epithelial cells, providing a survival advantage during the early stages of S. Typhimurium infection [18,19]. However, during the systemic phase of infection, flagellin can be easily detected by the host immune system, which triggers an elevated innate immune response, including secretion of IL-1β and TNF-α, resulting in bacterial clearance from the host [20]. Flagella overexpression in S. Typhimurium leads to a reduction of bacterial growth in murine macrophages and to attenuation of bacterial virulence in mice [21]. The transcriptome and proteome landscape of intracellular S. Typhimurium has revealed that the expression of most flagellar genes is significantly downregulated after the entry of S. Typhimurium into murine macrophages [22][23][24]. The deletion of STM1697, which encodes an EAL-like protein, resulted in the upregulation of flagellar gene expression, leading to the attenuated ability of S. Typhimurium to colonize the host organs [25]. These results implicate that the downregulation of flagellar genes in macrophages contributes to the successful intracellular replication and full virulence of S. Typhimurium. Several studies have focused on the molecular mechanisms associated with the downregulation of flagellar genes in response to the stimuli within macrophages. Flagellar gene expression can be repressed by PhoP/Q in response to acidic pH [26]. A leader mRNA originated from the mgtCBR virulence operon, which is highly induced by low Mg 2 + , binds to the coding region of the flagellin gene fljB to induce fljB mRNA degradation and thus repress flagellin gene expression [27]. STM14_1285, herein named AsiR (Acid signalinduced regulator), is predicted to be an RpiR family transcriptional regulator in S. Typhimurium and comprises a predicted DNA˗binding helix˗turn˗helix (HTH) motif at its N terminus (https://blast.ncbi.nlm. nih.gov/Blast.cgi). The regulatory function of AsiR in S. Typhimurium is currently unknown. The regulatory functions of RpiR-family regulators are mostly investigated in Lactococcus lactis and are generally known to be involved in the regulation of carbon metabolism [28]. However, IdoR, an RpiR-family regulator in S.
Typhimurium, has been shown to repress the expression of SPI-2 effector gene srfJ [29], implying that members in this family also have other functions including the regulation of virulence genes.
The expression of asiR is downregulated after S. Typhimurium entry into murine macrophages [10], but its implication in S. Typhimurium virulence remains elusive. Here, we demonstrated that the downregulation of asiR expression facilitates S. Typhimurium replication in murine macrophages and systemic pathogenesis in mice. Further investigation revealed that AsiR positively regulates the expression of flagellar genes and that the downregulation of AsiR leads to a decrease in flagellar gene expression and proinflammatory cytokine secretion. Finally, we found that the mildly acidic pH in macrophages is a trigger for the downregulation of asiR.

Bacterial strains, plasmids, and growth conditions
S. Typhimurium strain 14028s (wild-type, WT) and its derivative strains, as well as plasmids used in this study, are described in Table 1. Primers used are described in Supplementary Table 1. Bacteria were routinely cultured in conventional Luria-Bertani (LB) medium containing tryptone 10 g·L −1 , yeast extract 5 g·L −1 , and NaCl 10 g·L −1 . For quantitative real-time PCR (qRT-PCR) experiments, overnight bacterial cultures in LB were diluted at 1:100 into fresh LB or N-minimal medium [30] and then incubated in a shaking incubator (37°C, 180 rpm) to reach stationary phase. Antibiotics were added into the culture medium to achieve the following final concentrations: kanamycin (Km), 50; chloramphenicol (Cm), 25; blasticidin (Bs), 100; gentamicin (Gm): 20 or 100 μg·mL −1 .

Construction of plasmids and strains
The mutant strains (ΔasiR, ∆flhDC, and ∆asiR∆flhDC) were constructed with the λ Red recombinase system [31]. The transcriptional fusions asiR-lux, ssrA-lux, and flhDC-lux were generated as described previously [13]. The amplification products of respective promoter regions were digested and cloned into the XhoI-BamHI site upstream of the lux genes in the plasmid pMS402. To generate a complemented strain, the amplification products (including the ORF and the upstream promoter sequence) of the corresponding genes were digested and cloned into the EcoRI-BamHI site of plasmid pBluescript and transformed the recombinant plasmids to the mutant strains.
To generate the strain asiR on (a strain persistently expresses asiR gene within macrophages), we replaced the promoter of asiR with the promoter of the SPI-2-encoded regulator gene ssrA by homologous recombination. First, the promoter region of ssrA was amplified from the genomic DNA of WT with primers ssrApromoter-F and ssrA-promoter-R. The chloramphenicol resistance gene was amplified from the plasmid pKD3 with primers Cm-F and Cm-R. Subsequently, the above two PCR products were purified and mixed for the overlap PCR splicing with primers ssrApromoter-F and Cm-R. The resulting spliced PCR products were electroporated into WT harboring the plasmid pSim17. The promoter replaced bacteria were selected by their resistance to Cm, and verified by PCR amplification and sequencing.
To generate a construct expressing AsiR protein, the asiR gene was amplified from the genomic DNA of S. Typhimurium WT and inserted into the EcoRI and BamHI restriction sites of plasmid pET-28a to generate the plasmid pET-asiR. The pET-asiR plasmid was then transformed into Escherichia coli BL21 (DE3) cells to generate strain BL21+ pET-asiR, for the expression and purification of 6× His-tagged AsiR protein (his 6 -AsiR).

Cell culture
The RAW264.7 macrophage cell line was obtained from the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured routinely in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and were incubated at 37°C in a humidified atmosphere containing 5% CO 2 /95% air. Cells were seeded into sterile 12-well tissue culture plates at a concentration of 1 × 10 5 cells/well and maintained as differentiated monolayers for 48 h before infection.

Macrophage replication assays
Macrophage replication assays were performed as previously described [30]. Briefly, S. Typhimurium strains (WT, ΔasiR, asiR on , and cAsiR) were incubated in LB medium overnight at 37°C with shaking (180 rpm) for 16 h. The overnight bacterial cultures were diluted and opsonized for 20 min in 10% normal mice serum (Gibco). The bacteria were added to RAW264.7 cells at a multiplicity of infection (MOI) of 10. After incubated for 45 min, the infected cells were washed with PBS for three times. Then, the cells were incubated in fresh RPMI-1640 medium supplemented with 100 μg·mL −1 Gentamycin for 2 h followed by incubation in RPMI-1640 supplemented with 20 μg·mL −1 Gentamycin for another 14 h. At 2 and 16 h postinfection (hpi), the intracellular bacteria were collected, serially diluted in PBS, and then spread onto LB agar for counting of bacterial colony-forming units (CFUs). Fold intracellular replication was calculated as a ratio of the intracellular CFUs at 16 hpi relative to that at 2 hpi.

Mouse infection
All animal experiments performed here were approved by the Institutional Animal Care Committee at Nankai University (Tianjin, China). BALB/c mice (female, 6-8 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). In order to determine the survival rate, the mice were inoculated intraperitoneally (i.p.) with 2 × 10 4 CFUs of WT (n = 14), ΔasiR (n = 15), asiR on (n = 14) and cAsiR (n = 14) and were monitored and recorded daily for a period of 20 days. To enumerate the bacterial burdens in mice organs, four groups of mice (n = 7/group) were inoculated i.p. with 5 × 10 4 CFUs of WT, ΔasiR, asiR on , or cAsiR, respectively, and were euthanized after 3 days. The collected liver and spleen were weighed, homogenized and diluted in PBS, and spread onto LB agar without or with appropriate antibiotics for enumeration of CFUs. For competitive infection assays, six mice were co-infected i.p. with a 1:1 mix totaling ~5 × 10 4 CFUs of ∆flhDC and ∆asiR∆flhDC and euthanized on day 3 postinfection to enumerate the bacterial burden in the liver and spleen. The competitive index (CI) was calculated as follows: (single mutant strain CFUs recovered/ ∆asiR∆flhDC CFUs recovered)/(single mutant strain CFUs inoculated/∆asiR∆flhDC CFUs inoculated).

RNA preparation
The protocol of RNA preparation was conducted as previously described [13]. To obtain the RNA of intracellular S. Typhimurium for qRT-PCR assays, we performed the macrophage replication assays as described above. The infected macrophages were lysed on ice for 30 min in 0.1% SDS, 1% acidic phenol, and 19% ethanol in water. The cell lysates were centrifugated to isolate and harvest the intracellular bacteria. The harvested bacteria were immediately frozen in liquid nitrogen and late used to extract and purificate the total RNA.

qRT-PCR
The RN43-EASY spin Plus Rapid Extraction Kit (Aidlab) was used for the extraction and purification of total RNA, following the manufacturer's instructions. cDNA synthesis was performed with the PrimeScript RT Reagent kit (TaKaRa). qRT-PCR reaction was carried out in the QuantStudio 5 Real-Time PCR system (Applied Biosystems). The 16S rRNA was used for normalization in gene expression analysis. The 2 -ΔΔCt method was used to calculate the fold change in tested gene expression [32].

Motility assays
The protocol of motility assays was conducted as previously described [33]. Overnight cultures of WT and ΔasiR were adjusted to an OD 600 of 1.0. One microliter of each adjusted strain was spotted on 0.3% LB agar medium. After incubation at 37°C for 8 h, the motility of the bacteria was observed by measuring the diameter of the swimming zone surrounding the inoculation site.

Statistical analysis
All data in this study were representative of three separate experiments unless otherwise stated and displayed as mean ± SD. GraphPad Prism v7.0 software was used for the statistical analysis. Student's unpaired t-test, one-way ANOVA test, Log-rank (Mantel-Cox) test, and Mann Whitney U-test were performed to assess the statistical significance. P-values of less than 0.05 were considered statistically significant.

asiR transcription is downregulated in murine macrophages
To verify whether the expression of asiR was downregulated in host macrophages, as indicated by previously published RNA-seq data [10], murine RAW264.7 cells were infected with S. Typhimurium WT for a period of 24 h, and the expression of asiR was determined. In comparison with the bacteria cultured in RPMI-1640 medium, the expression of asiR was significantly decreased at 2, 8, 16, and 24 hpi, as revealed by both qRT-PCR and the bioluminescent reporter assays using an asiR promoter-lux fusion (asiR-lux) (Figure 1(a, b)), confirming the downregulation of asiR in host macrophages.

asiR contributes negatively to S. Typhimurium replication in murine macrophages
To explore the implication of asiR downregulation in S. Typhimurium growth within macrophages, we constructed an asiR gene knock-out strain (ΔasiR), a complemented strain (cAsiR), and a strain that expresses asiR persistently during its growth in RAW264.7 cells (asiR on ). We confirmed that the expression of asiR was upregulated in asiR on within RAW264.7 cells, in contrast to the downregulation of asiR expression in WT, and was maintained above the WT level during the 24 h infection period (Figure 1(b) and Figure S1). Both ΔasiR and asiR on grew equally well as the WT in LB and RPMI-1640 medium ( Figure  S2(a) and Figure S2(b)). However, when we determined the growth of those strains in RAW264.7 cells, it was found that the replication rate of ΔasiR was significantly increased compared to that of WT at 16 hpi and reduced to the WT level by complementation with asiR (cAsiR) (Figure 1(c)). On the contrary, the replication rate of asiR on in RAW264.7 cells was significantly decreased compared to that of WT (Figure 1 (c)). We further used immunofluorescence microscopy to quantify the number of intracellular S. Typhimurium per RAW264.7 cell. At 2 hpi, the average number of WT, ΔasiR, and asiR on cells per RAW264.7 cell was similar, with one to four bacteria per RAW264.7 cell (Figure 1(d)). However, at 16 hpi, the average number of ΔasiR cells per RAW264.7 cell (~22 bacteria/macrophage) was significantly higher than that of WT (~16 bacteria/macrophage), while in asiR on the number was significantly lower than that of WT (~8 bacteria/macrophage) (Figure 1(d)). These results indicate that AsiR contributes negatively to the growth of S. Typhimurium in host macrophages, and thus, downregulation of AsiR promotes the intracellular growth of S. Typhimurium.

asiR contributes negatively to S. Typhimurium virulence in mice
Replication of S. Typhimurium within macrophages is essential for systemic infection. To validate the contribution of asiR downregulation in macrophages to S. Typhimurium virulence, mice were infected with WT, ΔasiR, asiR on , or cAsiR via i.p. injection and monitored for survival. All of the mice infected with ΔasiR died within 8 days with a mean survival time of 4 days, while the mice infected with WT and cAsiR died within 13 days with a mean survival time of 5 and 6 days, respectively (Figure 2(a)). The results indicate that the absence of asiR reduces the survival of infected mice. On the contrary, only 36% of the mice infected with asiR on died within 8 days, with 64% surviving throughout the period of our experiment (20 days), indicating that the overexpression of asiR enhances the survival of infected mice. We further verified the virulence phenotype of WT, ΔasiR, asiR on , or cAsiR by determining their colonization abilities in the liver and spleen of infected mice. Three days after infection, ΔasiR showed significantly increased bacterial counts in the liver and spleen of infected mice compared to WT and reduced to the WT level by complementation with asiR ( Figure  2(b)). The results indicate that the absence of asiR increases the colonization of S. Typhimurium in systemic loci. On the contrary, asiR on showed decreased bacterial counts in the liver and spleen of infected mice compared to WT (Figure 2(b)), indicating that the overexpression of asiR reduces the colonization of S.
Typhimurium in systemic loci. Therefore, the expression of asiR contributes negatively to S. Typhimurium virulence in mice, and it is downregulated in murine macrophages to promote S. Typhimurium replication and virulence.

asiR contributes negatively to S. Typhimurium intracellular replication and virulence by positively regulating the expression of flagellar genes
Next, we investigated the mechanism underlying the negative contribution of AsiR to S. Typhimurium intracellular replication and virulence. SPI-2 genes are necessary for S. Typhimurium replication within macrophages and systemic infection in mice. Therefore, we tested whether AsiR influences the expression of SPI-2 genes. qRT-PCR analysis showed no significant change in the expression of three representative SPI-2 genes (ssrA, ssaG, and sifA) in ΔasiR compared to that in WT when bacteria were grown to stationary phase in N-minimal medium ( Figure S3(a)) and during its growth inside RAW264.7 cells ( Figure S3 (b)), indicating SPI-2 gene expression is not affected by AsiR.
Through an in vitro phenotypic assay, the motility assay, we found that ΔasiR exhibited a defect in motility compared to WT and the defect was rescued by complementation with asiR (Figure 3(a)), implying that AsiR  genes was downregulated in ΔasiR compared to that in WT, and restored to the WT level by complementation with asiR (Figure 3(b) and S4(a)), indicating that AsiR positively regulates the expression of flagellar genes. asiR on , which expresses asiR persistently in RAW264.7 cells (Figure 1(b)), showed enhanced motility (Figure 3 (a)) and flagellar gene expression in LB and after infection of RAW264.7 cells (Figure 3(b) and S4(a)). Bioluminescent reporter assays further confirmed that the expression of the flhDC operon was decreased in ΔasiR while increased in asiR on during bacterial growth within RAW264.7 cells (Figure S4(b)). Collectively, these results indicate that AsiR positively regulates the expression of flagellar genes.
The deletion of asiR in macrophages could result in the decrease of flagellar gene expression, leading to the enhanced intracellular replication of S. Typhimurium. To further verify that AsiR represses the intracellular replication and virulence of S. Typhimurium by activating flagellar gene expression. We next constructed asiR/ flhDC double mutant strain (∆asiR∆flhDC) and two complemented strains (∆asiR∆flhDC strain complemented with asiR or flhDC: ∆asiR∆flhDC+P-asiR, ∆asiR∆flhDC+P-flhDC), and performed macrophage replication assay. The growth rate of ∆asiR∆flhDC in RAW264.7 cells was comparable to that of ∆asiR, indicating that mutation of asiR in ∆flhDC did not confer an additional replication advantage (Figure 3(c)). Meanwhile, the growth rate of ∆asiR∆flhDC+P-asiR in RAW264.7 cells was comparable to that of ∆asiR∆flhDC (Figure 3(c)), confirming that AsiR represses S. Typhimurium intracellular replication by activating flagellar gene expression.
We then conducted competitive infection assays to measure the difference in systemic tissue colonization between the single mutant strain (∆flhDC and ∆asiR) and the double mutant (∆asiR∆flhDC). The competitive ability of ∆asiR∆flhDC was comparable with that of ∆flhDC or ∆asiR in both the liver and spleen of the infected mice (Figure 3(d)), indicating that mutation of asiR in ∆flhDC did not provide an additional virulence advantage. Collectively, these results suggest that AsiR contributes negatively to the intracellular replication and virulence of S. Typhimurium by activating the expression of flagellar genes.

asiR positively regulates flagellar gene expression by directly binding to the promoter of flhDC
We next investigated whether AsiR regulates flagellar gene expression by binding to the flhDC promoter, which is a target of a broad range of transcriptional regulators that regulate the expression of flagellar genes [35]. The EMSA results showed that purified Histagged AsiR shifted the flhDC promoter fragments in a concentration-dependent manner, but did not bind to the 16S rRNA DNA (negative control) (Figure 3(e)). Therefore, AsiR directly binds to the flhDC promoter, leading to the activation of flagellar gene expression.

The deletion of asiR in macrophages reduces the host inflammatory response
As the flagellin triggers innate immune responses by stimulating the secretion of pro-inflammatory cytokines, such as IL-1β and TNF-α [36], we hypothesized that the deletion of asiR could reduce host inflammatory responses by reducing flagellin synthesis. To test this hypothesis, we infected RAW264.7 cells with WT, ΔasiR, or asiR on , and measured the amount of IL-1β and TNF-α in the culture supernatant at 16 hpi by ELISA. As shown in Figure 4(a, b), RAW264.7 cells infected with ΔasiR showed significantly decreased IL-1β and TNF-α levels than cells infected with WT or asiR on . The results indicate that the expression of asiR contributes positively to the secretion of pro-inflammatory cytokines. Further, the IL-1β and TNF-α levels in ∆asiR∆flhDC-infected RAW264.7 cells were comparable to that of ∆asiR∆flhDC+P-asiR-infected RAW264.7 cells, while the IL-1β and TNF-α levels in ∆asiR∆flhDC+P-flhDCinfected RAW264.7 cells were comparable to that of WTinfected RAW264.7 cells (Figure 4(c, d)), indicating that AsiR promotes cellular inflammatory responses by activating flagellar gene expression. Therefore, asiR is downregulated to evade host detection by reducing flagellin synthesis and thereby reducing flagellin-stimulated inflammatory responses.

The expression of asiR and therefore flagellar genes is downregulated in response to acidic pH in macrophages
To evaluate whether any of the known environmental cues in macrophages, including low Mg 2+ , low P i , low O 2 , and acidic pH, contribute to the downregulation of asiR, we examined the asiR expression of WT grown in vitro under defined conditions. According to qRT-PCR results, the expression of asiR was not significantly altered in the N-minimal medium with low Mg 2+ & low P i conditions compared to that in the medium with high Mg 2+ & high P i conditions ( Figure  S5(a)). Moreover, the expression of asiR is not significantly altered under low O 2 conditions compared to that under high O 2 conditions ( Figure S5(b)). However, the expression of asiR decreased significantly in WT grown in both LB and N-minimal medium with pH 5.0 compared to that in the medium with pH 7.0, as revealed by qRT-PCR analysis ( Figure 5(a)) and confirmed by bioluminescent reporter assays ( Figure S5(c)). These results indicated that the expression of asiR is repressed by mildly acidic pH. Consistent with the downregulation of asiR, the expression of flagellar genes (fliA, fijB, fliC, flhC, and flhD) in WT was also downregulated in N-minimal medium at acidic pH (pH 5.0) ( Figure 5(b)). In contrast, the expression of these flagellar genes was not changed at pH 5.0 in ΔasiR compared to that at pH 7.0 ( Figure 5(c)) but downregulated in cAsiR ( Figure S5 (d)), indicating that asiR is required for the downregulation of flagellar genes at acidic pH. This was further confirmed by comparing the expression of flagellar genes in WT and ΔasiR at pH 5.0 and pH 7.0, respectively ( Figure S5(e)). Therefore, the acidic conditions within macrophages can lead to the repression of asiR expression and its downstream flagellar genes.

Discussion
In this study, we revealed a novel regulatory mechanism mediated by AsiR for the downregulation of flagellar genes within host macrophages. AsiR positively regulates the expression of flagellar genes by binding to the flhDC promoter. The expression of asiR and therefore flagellar genes is downregulated within macrophages, and the acidic pH was identified as a host cue for this downregulation. The downregulation of flagellar genes mediated by AsiR contributes significantly to S. Typhimurium systemic pathogenesis, as indicated by the significant attenuated ability of S. Typhimurium to replicate in both macrophages and systemic organs when asiR was persistently expressed. We propose that asiR is downregulated in response to acidic pH signal of macrophages, followed by repression of the flagellar gene expression to increase the intracellular replication and pathogenicity of S. Typhimurium ( Figure 6).
Flagellin is a ligand detected by the cell surfacelocalized Toll-like receptor 5 (TLR5) and also cytosolic NOD-like receptor 4 (NLRC4) [37,38]. The activation of the TLR5 signaling pathway induces the secretion of pro-inflammatory cytokines, including TNF-α [39]. The activation of the NLRC4 inflammasome elicits the caspase-1-dependent processes, which also promotes pro-inflammatory cytokines (e.g., IL-1β) production [40]. In agreement with the regulatory role of AsiR in flagellar gene expression, the production of IL-1β and TNF-α in the macrophages is significantly reduced due to the repression of AsiR. This result implicates that downregulation of flagellar gene expression by AsiR is an important mechanism utilized by S. Typhimurium to reduce the production of pro-inflammatory cytokines and replicate successfully within macrophages.
The assembly of flagella is regulated by complex mechanisms, including transcriptional, translational, and posttranslational regulation [35]. The flhDC flagellar master operon in S. Typhimurium activates transcription of other flagellar genes [41]. Several regulators have been reported to modulate flagellar gene expression by acting on FlhDC. The LysR-family protein HdfR represses flagellar gene expression through directly binding flhDC promoter to reduce flhDC transcription [42]. The global regulatory protein CsrA activates flagellar gene expression by protecting the translation of flhDC mRNA from RNase E-mediated cleavage [43]. STM1697 represses flagellar gene expression by binding to FlhD protein to repress the recruitment of RNA polymerase [25]. In this study, we found that AsiR activates flhDC and downstream flagellar gene expression at the transcriptional level by directly binding to the promoter of flhDC. In addition to promoting intracellular replication, AsiR may also contribute to Salmonella pathogenesis by affecting adherence and invasion, as flagella have been previously demonstrated to be required for the full adhesive and invasive potential of S. Typhimurium to intestinal epithelial cells [44,45]. Our preliminary investigation showed that ΔasiR exhibited a decreased ability to adhere to and invade HeLa cells ( Figure S6). Whether AsiR has a physiological role in promoting adherence and invasion needs to be further investigated.
There are six potential transcriptional start-sites (TSSs) within the flhDC promoter region, to which many transcriptional regulators bind, including PhoP [46]. Several transcriptional regulators exert their control of flhDC expression at different stages of the bacterial growth phase through activating the transcription of two functional TSSs [46]. We demonstrate that AsiR binds directly to the flhDC promoter region. However, the exact AsiR binding sequence and its regulatory function in the growth phase are not identified in this study and will be the subject of future studies.
Inside the SCV of macrophages, the pH value has been estimated to be around 5 [47]. It is known that SCV acidification promotes the intracellular replication of S. Typhimurium by inducing the expression of SPI-2 genes [48]. Although acidic pH can repress flagellar gene expression as revealed in minimal E glucose (minimal EG) medium [49], the underlying regulatory mechanisms are not clear. Here, we found that the expression of flagellar genes is downregulated in response to acidic pH via a novel regulator, AsiR, and confirmed that acidic pH also contributes to pathogenesis by repressing flagellar gene expression. Several regulatory systems are known to be responsive to the acidic signal, including OmpR/ EnvZ and PhoP/Q [13,50]. Our preliminary investigation revealed that the deletion of phoP increased asiR expression in N-minimal medium at pH 5.0 based on qRT-PCR analysis, but the deletion of ompR did not influence asiR expression ( Figure S7), suggesting that PhoP is a negative regulator of asiR. Whether PhoP/Q and/or other undiscovered systems are involved in acidic signaling-mediated downregulation of asiR and flagellar genes is a subject of future studies.
In summary, this work describes a novel reciprocal feedback between S. Typhimurium and host cells, in which AsiR responds to the acidic signal within macrophages and in return represses the immune reaction of host macrophages to promote bacterial intracellular replication.