The effects of PstR, a PadR family transcriptional regulatory factor, in Plesiomonas shigelloides are revealed by transcriptomics

doi:10.21203/rs.3.rs-4962630/v1

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The effects of PstR, a PadR family transcriptional regulatory factor, in Plesiomonas shigelloides are revealed by transcriptomics

https://doi.org/10.21203/rs.3.rs-4962630/v1

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Background: Plesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors.

Results: In this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and lux assays. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, RNA-seq, qRT-PCR, and lux assays demonstrated that PstR also positively regulates T3SS expression, which aids in P. shigelloides' capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides.

Conclusions: Overall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria.

Plesiomonas shigelloides

PstR

RNA-seq

motility

T3SS

fatty acid degradation and metabolism

Plesiomonas shigelloides is a gram-negative rod-like pathogen, the only species in the genus Plesiomonas, and belongs to the family Enterobacteriaceae [1]. Widespread in freshwater environments, consumption of their contaminated aquatic products and water can lead to gastrointestinal and extraintestinal diseases, such as cholera-like illness, an invasive shigellosis-like disease, acute secretory gastroenteritis, meningitis, pseudoappendicitis, and bacteremia [2–7]. P. shigelloides is a significant zoonotic opportunistic pathogen that requires adequate attention to health and epidemic prevention. As a result, it is critical to understand its network regulation and pathogenic mechanisms, beginning with unknown virulence regulatory factors.

Phenolic acid decarboxylase (Pad) activity was first characterized in Pediococcus pentosaceus [8]. It was shown that this mechanism involved two proteins that were designated as phenolic acid decarboxylase, or PadA (PadC in Bacillus subtilis), and phenolic acid decarboxylase repressor, or PadR. It was further shown that the deletion of padA led to growth inhibition in the presence of phenolic acids, while deletion of padR led to constitutive overexpression of padA and high resistance to phenolic acids [8–11]. PadR-type regulators contain a highly conserved N-terminal winged helix-turn-helix (wHTH) domain with about 80 to 90 residues, which is responsible for the binding of these regulatory proteins to their target DNA [12]. In addition to the wHTH domain, there is a variable C-terminal domain in PadR-like proteins that is involved in the dimerization of these proteins. Depending on the length of this C-terminal domain, PadR-like proteins have been classified into two subfamilies, subfamily-1 and subfamily-2 [12]. PadR-like proteins of subfamily-1, are approximately 180 amino acid residues in length [13–16]. Subfamily-2 members are shorter, with approximately 110 amino acids [17–20]. To date, fifty-one PadR family member structures have been deposited in the Protein Data Bank (http://www.rcsb.org/) [21]. The PadR-like protein family of transcriptional repressors regulates the expression of enzymes involved in diverse processes, including detoxification of phenolic acid [8–10], antibiotic resistance [20, 22], aromatic compound catabolism [14], and virulence gene expression [23, 24]. Microorganisms generally respond to changes in environmental conditions through the actions of specific systems, which detect physical or chemical changes and develop coordinated cellular responses to adapt to new conditions [8]. Particularly, microorganisms can resist toxic compounds through various responses that are activated upon exposure to stress. Most of the time, detoxification involves either active efflux of the toxic compound from the cell by highly specific systems [25, 26] or enzymatic conversion of the toxic compound into a less toxic form [27]. Phenolic acids, also called substituted hydroxycinnamic acids, are abundant in the plant kingdom because they are involved in the structure of plant cell walls [28] and are released by hemicellulases produced by several fungi and bacteria [29]. Surprisingly, phenolic acids are not potentially toxic to all microorganisms. Some Pseudomonas strains [30, 31], as well as Acinetobacter calcoaceticus [32], are able to use them as the sole source of carbon for growth. At present, besides the founding member, only a few of the PadR-like proteins have been or are currently under investigation. These include AphA, which activates virulence gene expression in Vibrio cholerae [33], LmrR and LadR, which are repressors of genes encoding a multi-drug resistance pump in Lactococcus lactis and Listeria monocytogenesis, respectively [15, 33], and Pex, a regulator of circadian rhythms in Synechococcus elongates [34]. In addition, a novel phenolic acid decarboxylase regulator (PadR)-like regulator, PrhP, was demonstrated to positively regulate the expression of genes encoding the type III secretion system (T3SS) and many virulence-related genes, such as the flagella and type IV pili [35]. To date, only a few members of the PadR family have been known to play a major role in the biology of their host bacteria. In P. shigelloides, we discovered a transcriptional regulator, PstR, of the PadR family, whose function is rarely reported.

According to earlier research, P. shigelloides possessed two distinct gene clusters: one was only involved in the biosynthesis of the polar flagella, while the other contained genes involved in the lateral flagella [36]. Compared to the regions found in E. coli or S. typhimurium, the polar flagella gene regions of P. shigelloides have more similarities to those found in V. parahemolyticus and A. hydrophila [36, 37]. Currently, some studies have reported that transcriptional regulators affect the motility and the flagella synthesis of P. shigelloides, such as RpoN (σ⁵⁴), the pyruvate dehydrogenase complex regulator PdhR, and the Arc two-component signal transduction system response regulator ArcA [38–40]. In this study, we also found that PstR is able to positively regulate P. shigelloides motility as well as flagellar synthesis.

Gram-negative microorganisms use the Type III secretion system (T3SS), which is responsible for delivering effectors from the bacterial cytoplasm to a target eukaryotic cell via a needle-like structure, to further their pathogenicity [41]. T3SS utilizes an injectisome, which is composed of more than 200 subunits of up to 20 different proteins, for the secretion of bacterial effector proteins into eukaryotic host cells [42]. Furthermore, the regulatory mechanisms of gene expression, assembly, and secretion, as well as secreted effectors, differ amongst bacteria and even within the same bacteria, depending on pathogen requirements [43]. In this study, we also confirmed that PstR positively influences both the expression of P. shigelloides' T3SS gene cluster and its ability to infect Caco-2 cells, which suggests that PstR is a potential virulence regulator.

In bacteria, fad-related metabolic genes are responsible for catalyzing the catalytic fatty acid degradation pathway and are negatively regulated by FadR as well as PsrA [44]. The fadL-encoded specific outer membrane transporter transports long-chain fatty acids through the periplasmic space and inner membrane (IM) into the cytoplasm [45], followed by the fadD-encoded acyl CoA synthetase, which converts the fatty acid into a form that can enter the β-oxidized lipoyl CoA [46]. While FadE encodes a lipoyl CoA dehydrogenase that completes the first oxidation step of the cycle to form the enoyl CoA, which can undergo further oxidation and decarbonization under the catalysis of FadBA or FadIJ [47]. Furthermore, we suggested that PstR positively influences psrA expression while negatively regulating the expression of fad-related genes, hence negatively regulating fatty acid degradation metabolism.

In a word, we focused on the PstR of the PadR family transcriptional regulatory factor in P. shigelloides, and the regulatory effects of the PstR on motility, virulence, and physiological metabolism were revealed for the first time by RNA-seq as well as related experiments in this work.

2.1 Bacterial strains, plasmids and growth conditions

The bacterial strains and plasmids utilized in this study are listed in Table S1. Luria-Bertani (LB) liquid, solid, and semi-solid medium, and Dulbecco's Modified Eagle's Media (DMEM) supplemented with 20% fetal bovine serum (FBS), were used to cultivate bacteria at 30°C or at 37°C. If needed, supplements of ampicillin (25 µg/mL), kanamycin (50 µg/mL), or chloramphenicol (25 µg/mL) were added to the media.

2.2 Construction of gene deletion strains and complementation

To explore the function of PstR in P. shigelloides, the pstR gene was deleted using the suicide plasmid pRE112 [48]. In brief, the genome of P. shigelloides was used as the PCR amplification template to amplify the upstream and downstream gene fragments of pstR, respectively, which were then linked together via PCR amplification. The successfully connected DNA fragment and the pRE112 plasmid were digested by restriction endonuclease and linked with DNA ligase to form a new recombinant vector, and then introduced into E. coli S17-1 λpir for homologous recombination with WT. The positive clones were screened and identified based on the sucrose-sensitive lethal features of the pRE112 plasmid and the appropriate antibiotic concentration. Furthermore, we used the aforementioned suicide vector, pRE112 mediated homologous recombination, to similarly generate the pstR complementation strain. The correct deletion mutations and complementation strains were confirmed using agarose gel electrophoresis and DNA sequencing of PCR products. All primers used in this study are listed in Table S2.

2.3 RNA isolation and Transcriptome sequencing

The WT and ΔpstR mutant strains were inoculated into 20 mL of LB liquid medium at a ratio of 1:100 and shaken at 37°C for an overnight culture. The bacteria were then transferred to fresh medium the next day, grew to OD₆₀₀ = 0.8, and were harvested using centrifugation at 8,000 × g for 2 min. TRIzol® Reagent (Invitrogen, Waltham, MA, USA) was used to isolate total RNA from WT and ΔpstR mutant strains, and the RNA was then treated with RNase-free DNase and dissolved in RNase-free water. Subsequently, a NanoDrop-2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA) was used to measure the purity of the RNA, and 1% agarose gels were used to assess RNA degradation and contamination. The cDNA library was sequenced using an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) to generate 150 bp paired-end reads after testing and qualifying. HTSeq was used to quantify gene expression levels, and the Fragments Per Kilobase of transcript sequence per Million base pairs sequenced (FPKM) value of each gene was determined based on its length and the number of reads mapped to it. The criteria for differentially expressed genes (DEGs) were set as |log2 fold change| ≥1 and adjusted P-value (padj) ≤ 0.05.

2.4 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

In order to confirm the RNA-Seq data and investigate the regulatory link between PstR and downstream genes, qRT-PCR was conducted in the WT, ΔpstR, and ΔpstR/pstR⁺ strains. The extraction, quantification, and detection of total RNA were carried out according to the aforementioned methods. The PrimeScript™ RT reagent Kit (Takara Bio, Shiga, Japan) was used to synthesize cDNA. Using an Applied Biosystems ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA), qRT-PCR analysis was performed. The relative target gene expression levels were calculated as fold changes using the cycle threshold approach (2^−△△CT) [49], with the P. shigelloides gyrB gene serving as a reference control. Each experiment was conducted thrice.

2.5 Motility assay and transmission electron microscopy (TEM) of flagella

The flagella gene cluster of P. shigelloides is positively regulated by PstR, as demonstrated by RNA-seq data. We confirmed this association with motility assays [50] that measured the motility of the WT, ΔpstR, and ΔpstR/pstR⁺ strains. In short, WT, ΔpstR, and ΔpstR/pstR⁺ strains were inoculated into 20 mL of LB liquid medium and cultured overnight at 37°C. The next day, the strains were transferred to fresh medium and cultured to OD₆₀₀ = 0.8. Subsequently, 1 µL of the fresh bacterial solution was absorbed into the center of the swimming agar plates. After 12 h at 30°C in an incubator with swimming agar plates containing the injected bacterial solution, the bacteria's swimming distance was measured and photographed. Three time points were used for the trials, with six repeats each. Additionally, TEM and negative staining were used to visualize the flagella of the WT, ΔpstR, and ΔpstR/pstR⁺ strains, as previously described [51].

2.6 Luminescence screening assay

The luminescence screening assay was carried out as previously described [52]. Briefly, the amplification products of the appropriate promoter regions were digested and cloned into the XhoI-BamHI site of the plasmid pMS402. The fusion reporter plasmids used in this study (Table S1) were transformed into the WT and ΔpstR strains and grown in LB medium at 37°C till reaching the mid-logarithmic phase. Promoter activity was evaluated at OD₆₀₀ with a Synergy 2 plate reader (Agilent BioTek, Santa Clara, CA, USA). Each experiment was repeated three times, with six replicates each.

2.7 Growth assay

Growth assays were performed as described previously [39]. In brief, the WT, ΔpstR, and ΔpstR/pstR⁺ strains were cultured overnight at 37°C with shaking in sterile LB medium. Subsequently, the bacterial solution was added to five wells of a 96-well cell plate containing 200 µL of LB or DMEM at a ratio of 1:200 per well. As a control, fresh LB or DMEM was added to the wells around it. In order to conduct the dynamic growth experiment, the constructed 96-well cell plate was finally put in a Molecular Devices Spectramax 190 full-wavelength microplate reader (Molecular Devices LLC, San Jose, CA, USA). Three time points were used for the trials, with six repeats each.

2.8 Expression and purification of proteins and Electrophoretic mobility shift assays (EMSAs)

PstR and PsrA, the proteins used in this study's EMSAs, were cloned into pMAL-c5X, expressed in E. coli BL21 (DE3), and purified using amylose resin (New England Bio Labs, Ipswich, MA, USA) affinity chromatography. To conduct EMSAs, a mixture of each probe (60 ng) and increasing concentrations of PstR-maltose-binding protein (MBP) fusion protein was incubated at 30°C for 45 min in a 20-µL reaction volume containing 20 mM Tris-HCl (pH 7.4), 150 mM KCl, 10 mM MgCl₂, 10% glycerol, 1 mM DTT, and 1 µg Poly (dI.dC). Additionally, the EMSAs of PsrA and each of the purified promoter fragments were performed by adding increasing concentrations of PsrA-maltose-binding protein (MBP) fusion protein in binding buffer (20 mM HEPES (pH 7.6), 1 mM EDTA, 10 mM (NH₄)₂SO₄, 2 mM DTT, 100 mM KCl, and 10% glycerol) at room temperature for 30 min [53]. A 6% PAGE in 0.5×TBE buffer was used to separate the DNA-protein complexes for 2.5 h at 120 V. Gels were scanned using a gel imaging system (GE Healthcare, Chicago, IL, USA) after being stained with GelRed for 5 min.

2.9 Invasion assay

The invasion assay was performed with minor modifications to the previously reported protocol [54]. Briefly, the WT, ΔpstR, and ΔpstR/pstR⁺ strains were cultured overnight in LB, then transferred to new LB at a 1:100 inoculation ratio until the bacteria reached OD₆₀₀ = 0.6. In 24-well plates, confluent monolayers of roughly 1 × 10⁵ Caco-2 cells per well were overlaid with approximately 5 × 10⁷ WT, ΔpstR, and ΔpstR/pstR⁺ bacterial cells. Caco-2 and bacterial cells were co-cultured at 37°C in 5% CO₂ for 1 hour to induce invasion. To kill extracellular bacteria, 100 µg/mL of gentamicin was added to the cell culture medium 1 h after infection and incubated for 1 h. Following 1 h of incubation, the cells were lysed for 10 minutes using 0.1% Triton X-100 after the monolayer had been twice washed with PBS. The invasion rate was calculated as the ratio of the number of recovered bacteria to the total number of bacterial cells used for infection. And the invasion assay was performed three times, with six repetitions each time.

2.10 Statistical analysis

The data was statistically analyzed using GraphPad Prism v7.0 software (GraphPad Inc., La Jolla, CAS, USA), and all data are expressed as means ± standard deviation (SD) [53]. Furthermore, the independent-samples t-test or the Mann-Whitney U test were used to determine differences between groups. A probability value (P) ≤ 0.05 was considered statistically significant (*** p ≤ .001; ** p ≤ .01; * p ≤ .05; ns indicates not significant).

3.1 Transcriptome sequencing identified PstR-related gene expression in P. shigelloides

In this study, transcriptome sequencing demonstrated the function of PstR in P. shigelloides, and RNA-seq data revealed that PstR influenced the expression of 337 differential genes, accounting for 9.83% of the overall transcriptome, including 146 upregulated genes and 191 downregulated genes (Fig. 1A and Table S3). Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis of RNA-seq data showed that the downregulated genes mainly participate in flagellar assembly, bacterial chemotaxis, bacterial secretion system (T3SS cluster), two-component system, glycolysis/gluconeogenesis, microbial metabolism in diverse environments, the biosynthesis of cofactors, and sulfur relay system (Fig. 1B). Upregulated genes mainly participate in oxidative phosphorylation, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, fatty acid degradation and metabolism, and the citrate cycle (Fig. 1C). Furthermore, in the WT, ΔpstR, and ΔpstR/pstR⁺ strains, qRT-PCR was used to validate 8 upregulated and 8 downregulated DEGs in the transcriptome profiles (Fig. 1D), respectively. The reliability of the RNA-seq was demonstrated by the consistency of the qRT-PCR results with the RNA-seq analysis (Fig. 1E).

3.2 Phylogenetic analysis of PstR

To further understand the function of PstR, we created a phylogenetic tree, which consists of 16 species of bacteria based on the amino acid sequence of PstR in P. shigelloides, and used the neighbor-joining method in MEGA software. The results showed that PstR was conserved in the same genus, although PstR in P. shigelloides was far from the homology of other strains (Fig. 2). PstR differs between strains, its function may alter. Simultaneously, the domain of the PadR protein was shown to be present in PstR based on the amino acid sequence (Fig. S1). Furthermore, this study focused on revealing the function of PstR in P. shigelloides, and the construction of a PstR phylogenetic tree will lay the groundwork for the functional investigation of PstR in various strains.

3.3 PstR positively regulates the motility and flagellar synthesis of P. shigelloides

Among the 191 downregulated differential genes in the transcriptome, we found that genes involved in flagellar assembly and bacterial chemotaxis accounted for the largest proportion. Subsequently, polar and lateral flagellar genes with different down-regulated levels in RNA-seq were selected for qRT-PCR, and promoter-lux fusion was constructed for verification. Consistent results for RNA-seq, qRT-PCR, and lux assays suggest that PstR positively regulates flagellar gene expression in P. shigelloides (Fig. 3A to 3F). To further visualize the effect of PstR on P. shigelloides motility, the migration of the WT, ΔpstR, and ΔpstR/pstR⁺ strains in swimming agar plates was observed, and the results suggested that deletion of the pstR gene leads to loss of P. shigelloides motility, while complementation of the pstR gene restores the loss of motility in the ΔpstR strain (Fig. 3G). Moreover, TEM observations of the flagella generated by the WT, ΔpstR, and ΔpstR/pstR⁺ strains revealed that PstR deficiency affects flagellar production in P. shigelloides (Fig. 3H), consistent with the motility assay. The aforementioned results demonstrated that PstR influences motility and flagellar synthesis by positively regulating flagellar gene expression in P. shigelloides.

3.4 PstR positively regulates the expression of P. shigelloides' T3SS gene cluster as well as its ability to infect Caco-2 cells

In addition to occupying the majority of flagella-associated genes, differentially expressed genes downregulated in the transcriptome also included T3SS gene clusters (Fig. 4A and Table S3) that were not counted by KEGG. Simultaneously, 11 down-regulated T3SS genes from RNA-seq were selected for qRT-PCR validation (Fig. 4B), and promoter-lux fusions of 8 T3SS genes were constructed to further verify the regulatory effect of PstR on the T3SS gene cluster (Fig. 4C). The results indicated that PstR positively regulates P. shigelloides' T3SS gene cluster, which simultaneously implies that PstR has the potential to affect the virulence of P. shigelloides. In the investigation, we also evaluated whether the absence of pstR had an effect on the growth of P. shigelloides and found that there was no significant difference in the growth of ΔpstR and WT in LB and DMEM media (Fig. 4D and 4E). Subsequently, the invasion assay with WT, ΔpstR, and ΔpstR/pstR⁺ strains was conducted, and the results showed that compared with WT and ΔpstR/pstR⁺, the infection ability of ΔpstR to Caco-2 cells was significantly reduced (Fig. 4F). This result suggests that PstR affects the virulence of P. shigelloides by positively regulating the expression of T3SS.

3.5 PstR negatively regulates fatty acid degradation and metabolism in P. shigelloides

This study's transcriptome results also revealed several up-regulated gene clusters, including those involved in fatty acid degradation and metabolism (Fig. 1C). PsrA regulators are known to regulate genes involved in fatty acid degradation and metabolism, and psrA is down-regulated in the transcriptome, implying that the loss of PstR affects psrA transcription. As a result, we wanted to understand whether PstR regulates the expression of fatty acid degradation and metabolism related genes by directly regulating psrA. Subsequently, RNA-seq data and qRT-PCR demonstrated that PstR indirectly positively regulates psrA expression while indirectly negatively regulating the expression of genes involved in fatty acid degradation and metabolism (Fig. 5A and 5B). However, EMSA demonstrates that PstR protein does not bind to the psrA promoter (Fig. 5C), implying that PstR does not directly regulate psrA to affect the expression of downstream genes of PsrA, and the specific regulatory mechanism requires further study. At present, the downstream genes of fatty acid degradation and metabolism regulated by PsrA were also not reported in P. shigelloides, and we intended to identify the downstream genes regulated by PsrA in order to better reveal the regulatory network of PstR in P. shigelloides in the future. The results reveal that in P. shigelloides, PsrA can directly bind to the promoters of fadB, fadD, and fadI (Fig. 5D to 5F), but not to the promoters of fadE and fadE1 (Fig. 5G), while also repressing the expression of these genes (Fig. 5H).

3.6 The primary metabolic pathways in P. shigelloides that PstR regulates

In this study, we summarized the RNA-seq data and performed associated experiments to validate it, ultimately identifying the key metabolic pathways regulated by PstR in P. shigelloides (Fig. 6) with specific regulatory mechanisms that will be gradually revealed in the near future.

After many years in the family Vibrionaceae, the genus Plesiomonas, represented by a single species, P. shigelloides, currently resides in the family Enterobacteriaceae, although its most appropriate phylogenetic position may yet be determined [1]. The bacterium has been associated with acute bacterial gastrointestinal disease [47, 55] and, in rare cases, extraintestinal infections [56–58]. A variety of virulence factors have been associated with the pathogenesis of this microorganism, including β-hemolysins [59–61], enterotoxins [62], cholera-like toxins [63], and possible endotoxins [64]. A review summarizing the geographic locations, human infections, disease syndromes, and pathogenicity [1]. In this study, we mainly revealed the PstR of the PadR family in P. shigelloides, its function, and potential regulatory pathways through transcriptome sequencing. The RNA-seq results revealed that PstR regulates 9.83% of the transcriptome, including 191 downregulated and 146 upregulated DEGs. The downregulated DEGs were mainly involved in flagellar assembly, bacterial chemotaxis, T3SS, glycolysis/gluconeogenesis, sulfur relay system, periplasmic nitrate reductase, ABC-type transporter, pyrimidine metabolism, and hemolysin. The upregulated DEGs were mainly involved in fatty acid degradation and metabolism, NADH-quinone oxidoreductase, citrate cycle, dipeptide transport system, ubiquinol-cytochrome c reductase, and Cbb3-type cytochrome c oxidase. In this study, based on the results of RNA-seq, we primarily focused on the effect of PstR on motility, virulence, and fatty acid degradation and metabolism in P. shigelloides.

A previous study reported the draft genome sequences of 12 P. shigelloides strains and indicated that the pan-genome of P. shigelloides contained two gene clusters coding two types of flagellum systems, including a lateral flagellum gene cluster and a polar flagellum gene cluster. Two strains (G5880 and G5890) lacked a lateral flagellum gene cluster; however, two types of flagellum systems were present in the rest of the strains [65]. Yin et al. also suggested that the lateral flagellum loci of Photobacterium sanguinicancri ME15 were most homologous to the lateral flagellum gene cluster, and the polar flagellum loci of Vibrio parahaemolyticus ATCC 17802 exhibited the highest homology to the polar flagellum gene cluster [64]. In this study, transcriptome profiles revealed a substantial number of downregulated polar and lateral flagellar genes, and qRT-PCR and lux assays validated the RNA-seq results. Motility assays and TEM of WT, ΔpstR, and ΔpstR/pstR⁺ strains show that PstR deficiency reduces motility and flagellar synthesis; these results suggest that PstR is a positive regulator of motility in P. shigelloides. Currently, the transcriptional regulation network of polar and lateral flagella has not been documented. According to the previously reported results, FlaK, FlaM, FliA, and RpoN should be responsible for the transcriptional regulation of the polar flagella gene region in P. shigelloides, whereas LafK as well as FliA_L should be responsible for the transcriptional regulation of the lateral flagella gene region. However, RNA-seq, qRT-PCR, and lux assay data revealed that the transcript levels of flaK and flaM did not change significantly, demonstrating that PstR does not influence the expression of downstream flagellar genes via the regulatory factors FlaK and FlaM. It is likely that PstR influences the expression of flagellar gene clusters by controlling other regulators or that PstR directly regulates flagellar gene clusters; however, this hypothesis needs to be further proved in subsequent experiments.

Many clinically important Gram-negative pathogens, such as Salmonella enterica, Shigella spp., enteropathogenic and enterohemorrhagic Escherichia coli, and their murine equivalent, Citrobacter rodentium, rely on the Type III secretion system as a key virulence component [66]. The T3SS's structural component is the needle apparatus, which is assembled from approximately 20 different proteins and comprises a base, an extracellular needle, a tip, and a translocon [67]. T3SS can sense contact with the host cell membrane and translocate effector proteins into the host cell cytoplasm, where they modulate the host system and enhance bacterial localization and infection [68]. Our work indicated that the ΔpstR mutant's transcriptome profile showed a downregulation of the T3SS cluster, which was confirmed by qRT-PCR and lux assay. Meanwhile, the invasion assay revealed that ΔpstR infection of Caco-2 cells was dramatically reduced compared to WT and ΔpstR/pstR⁺. This finding suggests that PstR acts as a virulence regulator by positively regulating the expression of P. shigelloides' T3SS gene cluster. Interestingly, we previously reported that the study of PdhR [40] and PstR reported in this study both positively regulate the expression of the flagellar gene cluster and T3SS gene cluster in P. shigelloides, thereby affecting the virulence and motility of P. shigelloides. However, PstR is an unreported transcriptional regulatory factor in the PadR family, and it is not surprising that motility and virulence are often interrelated in most studies of pathogenic bacteria. At present, we did not find evidence that PstR directly regulates T3SS or that PstR regulates T3SS via other mechanisms. At the same time, the regulatory network and mechanism of T3SS in P. shigelloides have not been reported; therefore, more work remains to be done in the future to expose the virulence mechanism of PstR regulating P. shigelloides as well as the whole T3SS regulation network.

In addition to focusing on the highly downregulated flagellar and T3SS genes in the PstR transcriptome, this work also identified the upregulated associated genes involved in fatty acid degradation and metabolism. Fatty acids are a significant component of cell membranes and a source of metabolic energy for living organisms, with long-chain fatty acid oxidation being the primary mode of energy acquisition in mammals, many protists, and some bacteria. Four regulatory factors govern fatty acid degradation genes: FadR, PsrA, FadP, and LiuR's direct homologs, which are responsible for the transport (fadL), activation (fadD), and degradation (fadABEFHIJ) of exogenous fatty acids [44, 69]. P. shigelloides contains two regulators of fatty acid degradation, FadR and PsrA. Interestingly, fadR expression did not significantly change in the transcriptome, although PsrA was significantly downregulated. Meanwhile, we found that PstR did not regulate the expression of fatty acid degradation genes by directly regulating PsrA. These results did not reveal the specific mechanism by which PstR regulates fatty acid degradation in P. shigelloides, and to further reveal the regulatory pathway of PstR, we studied the fatty acid degradation and metabolism genes regulated by PsrA. Previous reports in V. cholerae have demonstrated that PsrA represses fatty acid degradation and metabolism gene expression and is able to bind directly to the promoter of psrA itself, as well as to promoters of fadB and fadI [46]. We found that PsrA plays a conserved regulatory role in P. shigelloides, repressing downstream gene expression and directly binding to the promoters of fadB, fadI, and fadD. We also discovered that PsrA can bind to its own promoter (Fig. S2); however, whether PsrA can also inhibit its own expression needs to be confirmed.

In this study, we revealed for the first time the transcriptome of PstR, a PadR family transcriptional regulatory factor, as well as the function of PstR in P. shigelloides. We confirmed that PstR indirectly positively regulates the expression of the flagellar gene clusters as well as the T3SS gene cluster, thereby affecting the motility of P. shigelloides and the ability to infect Caco-2. Furthermore, we showed that PstR negatively regulates the expression of fatty acid degradation and metabolism-related genes and revealed the downstream regulatory genes of PsrA, a regulator of fatty acid degradation and metabolism. In a word, this study established the foundation for future research into the intricate regulatory network of PstR in bacteria by revealing the PstR-controlled pathways that underlie PstR's critical regulatory role in P. shigelloides.

Author Contributions

Junxiang Yan: Investigation, Conceptualization, Project administration, Methodology, Writing - original draft. Zixu Zhang: Project administration, Methodology. Hongdan Shi: Data curation, Formal analysis. Xinke Xue: Project administration, Methodology. Ang Li: Methodology, Formal analysis. Fenxia Liu: Software, Visualization. Peng Ding: Software, Visualization. Xi Guo: Investigation, Conceptualization, Writing-original draft, Writing - review & editing. Boyang Cao: Investigation, Conceptualization, Writing-original draft, Funding acquisition, Supervision, Writing - review & editing.

Funding Statement

This work was funded by the National Key Programs for Infectious Diseases of China (funding numbers 2017ZX10303405-001, 2017ZX10104002-001-006, 2018ZX1 0712001-017).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are presented within the manuscript and the Supplementary Materials. The RNA sequencing data generated in this study are available in the NCBI SRA database (accession numbers: SRR25867280 and SRR25867281).

Conflicts of Interest

The authors declare no conflicts of interest.

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The effects of PstR, a PadR family transcriptional regulatory factor, in Plesiomonas shigelloides are revealed by transcriptomics

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