The Quorum-Sensing Regulator SdiA Activates npsA Expression and Modulates Cytotoxicity in Klebsiella oxytoca

Abstract

Toxigenic Klebsiella oxytoca strains linked to antibiotic-associated hemorrhagic colitis produce the cytotoxins tilimycin and tilivalline, which contribute to intestinal epithelial damage during infection. Tilimycin and tilivalline are synthesized by enzymes encoded within the nonribosomal peptide synthetase (NRPS) operon, yet the regulatory mechanisms controlling operon expression remain poorly understood. SdiA, an orphan LuxR-type quorum-sensing regulator, detects exogenous N-acyl homoserine lactones (AHLs) produced by neighboring bacterial species and modulates gene expression in response to interspecies communication. Although SdiA has been implicated in virulence regulation in several enteric pathogens, its role in K. oxytoca remains unclear. This study demonstrates that SdiA positively regulates npsA, the first gene in the NRPS operon, and that this regulatory effect is enhanced in the presence of exogenous AHL. Electrophoretic mobility shift assays indicate that SdiA directly binds to the upstream regulatory region of npsA, supporting a direct interaction consistent with positive transcriptional regulation. Furthermore, deletion of sdiA significantly reduces cytotoxicity toward HeLa cells under the conditions tested. Collectively, these findings identify SdiA as a quorum-sensing-responsive activator of npsA expression and support its role in modulating cytotoxicity in toxigenic K. oxytoca strains. These results provide new insight into the influence of interspecies quorum-sensing signals on virulence-associated regulatory pathways in K. oxytoca.

1. Introduction

Klebsiella oxytoca is a Gram-negative bacterium that forms part of the human gut microbiota. It colonizes approximately 2–10% of healthy individuals [1,2]. Although typically regarded as a commensal organism, K. oxytoca can behave as an opportunistic pathogen and is associated with a range of healthcare-related infections. Notably, it exhibits intrinsic resistance to β-lactam antibiotics due to chromosomally encoded β-lactamases [3]. Perturbation of the intestinal microbiota during antibiotic treatment can lead to dysbiosis. This reduces colonization resistance and facilitates the expansion of pathobionts such as K. oxytoca [4].

A subset of K. oxytoca strains produces enterotoxins responsible for antibiotic-associated hemorrhagic colitis (AAHC). This condition is identified by abdominal pain and bloody diarrhea [5]. The enterotoxins tilimycin (TM) and tilivalline (TV) are synthesized by a nonribosomal peptide synthetase (NRPS) system encoded by the NRPS operon within the til pathogenicity island and specifically constituted by the npsA, thdA, and npsB genes [6]. TM induces DNA damage and arrests the cell cycle at the G1/S transition, while TV stabilizes microtubules and blocks progression at the G2/M phase. Both toxins compromise epithelial barrier integrity by altering tight junction proteins, such as claudin-1, leading to apoptosis and intestinal damage [4,7]. Although the effects of TM and TV are well characterized, the regulatory mechanisms controlling NRPS operon expression remain poorly understood.

Quorum-sensing (QS) is a bacterial communication system that enables coordinated regulation of gene expression in response to population density. QS depends on the production, release, accumulation, and detection of diffusible signaling molecules called autoinducers. As bacterial cell density increases, the concentration of autoinducers in the environment also rises, thereby modulating gene expression at the population level. In Gram-negative bacteria, QS is mostly mediated by N-acyl homoserine lactones (AHLs), which are small, diffusible molecules synthesized by LuxI-type enzymes. These signals freely diffuse across the cell membrane and, at sufficiently high concentrations, are recognized by cognate LuxR-type transcriptional regulators. In many LuxR-type systems, AHL binding induces conformational changes that can modulate DNA binding and transcriptional regulation of target genes [8,9,10].

The detection of AHLs in the gut has been debated due to the methods and the chemical instability of these molecules [11]. Initial studies with LuxR biosensors reported low or undetectable AHL levels. Later, mass spectrometry confirmed the presence of a range of AHLs in intestinal samples from both healthy and dysbiotic individuals [12]. Also, the detection of LuxI/LuxR-like genes in gut bacteria suggests endogenous AHL production [11].

AHL availability within the gut is likely heterogeneous. It is influenced by factors such as pH, enzymatic degradation, and the spatial distribution of microbial populations [11]. In this complex environment, some bacteria lack AHL synthases but possess LuxR-type receptors, such as SdiA. These are found in Escherichia, Enterobacter, and Klebsiella. They detect external AHL signals and modulate gene expression [13].

Detection of signals from nearby bacteria is significant in the gut microbiota, where many species coexist. Acyl-homoserine lactones (AHLs) act as signals between different kingdoms. They affect the host by maintaining intestinal balance and controlling inflammation [13,14]. Here, SdiA may act as a central sensor. It detects AHLs and regulates genes, including those in virulence pathways such as npsA, the first gene of the NRPS operon in K. oxytoca.

Although interspecies signaling is increasingly recognized in bacterial pathogenesis, the regulatory connection between quorum sensing and toxin biosynthesis in K. oxytoca remains unresolved. To address this gap, this study demonstrates that the orphan LuxR-type regulator SdiA positively regulates npsA expression and directly binds its upstream regulatory region. Genetic, transcriptional, and biochemical analyses reveal that SdiA positively regulates npsA by binding directly to its upstream regulatory region, a process significantly potentiated by the presence of AHL.

These findings suggest that K. oxytoca integrates quorum-sensing signals from neighboring microbes to regulate npsA expression and associated cytotoxic phenotypes, with SdiA acting as the interface that links these microbial signals to harmful effects in hosts. This provides insight into regulatory mechanisms potentially associated with AAHC and highlights interspecies quorum-sensing as a potential target for managing toxin-mediated disease.

2. Materials and Methods

2.1. Bacterial Strains

Experimental procedures were conducted using the toxigenic wild-type (WT) strain K. oxytoca MIT 09-7231, a clinical isolate from mouse tumor abscess. This WT strain served as the parental strain for the isogenic ∆sdiA mutant and its trans-complemented counterpart (∆sdiA pT3-SdiA), both of which were included in the study. The WT strain was previously characterized by API 20E profiling (bioMérieux, Marcy l’Etoile, France), amplification of the polygalacturonase-encoding pehX gene, and 16S rRNA gene sequencing [15].

Table 1. Bacterial strains and plasmids used in this study.

Strains	Description *	References
K. oxytoca MIT 09-7231	Wild-type Klebsiella oxytoca strain MIT 09-7231, AmpR	[15]
K. oxytoca ∆sdiA	K. oxytoca ∆sdiA::Kan, KanR	This study
K. oxytoca ∆sdiA pT3-SdiA	K. oxytoca ∆sdiA::Kan pT3-SdiA, KanR, TetR (trans-complemented)	This study
K. oxytoca ∆npsA	K. oxytoca ΔnpsA::FRT	[16]
E. coli MC4100	Cloning strain F− araD139∆(argF-lac) U169 rspL150 relA1 flbB5301 fruA25 deoC1 ptsF25	[17]
E. coli BL21 (DE3)	F− ompT hsdSB(rB−, mB−) gal dcm (DE3)	Invitrogen
Plasmids	Description *	References
pKD119	pINT-ts derivative containing the λ-Red recombinase system under an arabinose-inducible promoter, TetR	[18]
pKD4	pANTsy derivative template plasmid containing the kanamycin cassette for λ-Red recombination, AmpR Plasmid that shows temperature-sensitive	[18]
pMPM-T3	p15A derivative low-copy-number expression vector, lac promoter, TetR	[19]
pT3-SdiA	pMPM-T3 derivative expressing sdiA from the lac promoter, TetR	This study
pMPM-T6	p15A derivative expression vector, pBAD (ara) promoter, TetR	[19]
pT6-SdiA	pMPM-T6 derivative expressing N-terminal His6-SdiA from the pBAD(ara) promoter, TetR	This study

* Amp^R, ampicillin resistance; Kan^R, kanamycin resistance; Tet^R, tetracycline resistance.

summarizes the characteristics of all strains and plasmids.

2.2. Growth Condition

Cells were routinely grown in tryptic soy broth (TSB; Difco, Beirut, Lebanon) at 37 °C under aerobic conditions. Growth media contained ampicillin (100 µg/mL), kanamycin (50 µg/mL), or tetracycline (10 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA) as needed. For all assays, K. oxytoca cells were harvested at late stationary phase with an OD₆₀₀ of about 1.6.

In selected experiments, cultures were supplemented with N-(3-oxo-octanoyl)-L-homoserine lactone (AHL) to evaluate its effects. AHL was dissolved in chloroform and added to the culture medium before bacterial inoculation, at the start of subculture from the overnight culture, to achieve a final concentration of 10 µM. This specific AHL and concentration were selected based on previous research indicates that SdiA homologs preferentially recognize medium-chain oxo-substituted AHLs, especially N-(3-oxo-octanoyl)-L-homoserine lactone, which serves as one of the most effective ligands for SdiA-mediated activation in Salmonella enterica [20]. Thus, these selections ensure the experimental conditions effectively probe the relevant signaling pathway.

In the same study, dose–response analyses demonstrated that SdiA-dependent activation is concentration-dependent and approaches saturation near 10 µM for several AHL molecules [20]. Consistent with these findings, micromolar concentrations of AHLs are routinely used in in vitro quorum-sensing assays, ensuring sufficient receptor occupancy under controlled conditions [21,22,23,24,25]. To control vehicle effects, samples received an equivalent volume of chloroform, resulting in a final concentration of 0.1% (v/v). This concentration was chosen based on prior research indicating that 0.1% chloroform does not induce cytotoxicity in epithelial cells [26]. Inclusion of this solvent control revealed no significant effect of chloroform on bacterial growth, HeLa cell viability, or baseline LDH release.

2.3. Generation of the ΔsdiA Mutant Strain

The sdiA gene in K. oxytoca was deleted using the λ-Red recombinase system, following a previously described one-step mutagenesis protocol [18]. A PCR fragment was generated using gene-specific primers (Table S1). This fragment contained a kanamycin resistance cassette flanked by sequences homologous to the upstream and downstream regions of the sdiA locus. The purified DNA products were introduced by electroporation into competent K. oxytoca cells carrying the λ-Red recombinase helper plasmid pKD119. Recombinase expression was induced by adding 1% (w/v) L-(+)-arabinose (Sigma-Aldrich, St. Louis, MO, USA). Successful replacement of the target gene and chromosomal integration of the resistance marker were confirmed by PCR analysis and DNA sequencing. Once mutant strains were established, plasmids were constructed for complementation and protein expression studies.

2.4. Plasmid Construction

Complementation of the ΔsdiA mutant strain used the pT3-SdiA expression plasmid. The sdiA coding sequence from K. oxytoca was amplified by PCR using the primers listed in Supplementary Table S1. After purification, the amplicon was digested with KpnI and BamHI and ligated into the pMPM-T3 vector. For recombinant SdiA protein production and purification, a modified sdiA variant with an N-terminal hexahistidine tag (His₆-tag) was engineered. This His₆-sdiA fragment was cloned into the pMPM-T6 vector using NcoI and HindIII restriction sites. The resulting construct was pT6-SdiA. Assembly and sequence fidelity of all plasmids were verified by DNA sequencing. With these plasmids generated, molecular and protein assays were pursued.

2.5. RNA Extraction and Reverse Transcription–Quantitative PCR Analysis

Total RNA was isolated from K. oxytoca cells harvested at the stationary growth phase (OD₆₀₀ = 1.6) using the hot phenol method [27]. Contaminating genomic DNA was removed by digestion with the TURBO DNA-free Kit (Invitrogen, Waltham, MA, USA). RNA yield and purity were quantified with a NanoDrop ONE spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Sample integrity was checked by visualization on 1.5% bleach-denaturing agarose gels [28]. This RNA served as the template for downstream Reverse Transcription-quantitative PCR (RT-qPCR).

First-strand complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Negative controls lacking reverse transcriptase were included in each assay. These cDNA preparations were used for Quantitative PCR (qPCR).

The qPCR was performed on a LightCycler 480 instrument (Roche Diagnostics, Basel, Switzerland) using SYBR Green I Master Mix (Roche Diagnostics, Basel, Switzerland). Amplification mixtures (10 µL total volume) contained 5 µL of 2X SYBR Green I Master Mix, 2.5 µL of synthesized cDNA (approximately 25 ng), 1.5 µL of nuclease-free water, and 0.5 µL each of forward and reverse primers (20 µM; see Supplementary Table S1). The results from these reactions were analyzed for methodological rigor and reliability.

qPCR assays were performed in technical triplicate. Samples from three independent biological replicates were used. The rrsH transcript served as the endogenous reference for normalization of gene expression. Cycling conditions were an initial denaturation at 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s, 59 °C for 10 s, and 72 °C for 10 s. Fluorescence was measured at the end of each cycle. Melt-curve analysis confirmed specificity, using 10 s at 95 °C, a ramp from 65 °C to 97 °C, and a final 10 s at 40 °C.

Methodological rigor was ensured by including no-template and no-reverse transcriptase controls. Relative fold changes in gene expression were calculated using the 2^−∆∆Ct method [29,30]. Data represent the average values from three independent biological assays.

2.6. Purification of Recombinant His₆-SdiA Protein

For recombinant protein production used in DNA-protein interaction assays with the npsA gene promoter, the pT6-SdiA expression plasmid was introduced into chemically competent E. coli BL21 (DE3) cells. Transformants were selected on Lysogeny Broth (LB) agar containing the appropriate antibiotic. Individual colonies were inoculated into LB with tetracycline (10 µg/mL) and incubated overnight at 37 °C. Subsequently, 2 mL of this culture was transferred into 200 mL of Terrific Broth with the same antibiotic and incubated at 37 °C with shaking until OD₆₀₀ reached 0.4. Expression of His₆-SdiA was induced by adding L(+)-arabinose (Sigma-Aldrich, St. Louis, MO, USA) to 0.1% (w/v) at 37 °C for 5 h. Induced cells were then harvested for protein extraction and purification.

Biomass was harvested by centrifugation at 12,000× g for 10 min. For extraction under denaturing conditions, the cell pellet was resuspended in 1X Phosphate-Buffered Saline (PBS) (pH 8.0) containing 8 M urea and 0.3 M NaCl. Cells were disrupted by sonication on ice for 20 min, with 1 min pulses separated by 1 min of cooling. The crude lysate was clarified by centrifugation at 16,000× g for 20 min at 4 °C. The resulting supernatant was filtered through a 0.2 µm filter. This extract was then subjected to affinity chromatography to isolate the His₆-SdiA protein.

For affinity chromatography, the clarified extract was loaded onto a pre-equilibrated nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen, Hilden, Germany). Contaminants were removed by sequential washing steps (10 mL each) with the base buffer (1X PBS, 8 M urea, 0.3 M NaCl; Sigma-Aldrich, St. Louis, MO, USA) containing increasing imidazole concentrations: 5, 10, 15, and 25 mM. The recombinant protein was then eluted using 6 mL of the same buffer with 250 mM imidazole. Eluted fractions were analyzed and prepared for further use.

Collected fractions were analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue R-250 (Bio-Rad Protein Assay, Hercules, CA, USA). Pooled eluates were dialyzed at 4 °C with gentle agitation using a MWCO 12 kDa cellulose membrane (Sigma-Aldrich, St. Louis, MO, USA) to remove urea and imidazole, thereby facilitating protein refolding. Renaturation was achieved by dialyzing for 12 h against 1 L of buffer containing 20 mM Tris-HCl at pH 8.0, 50 mM KCl, 1 mM DTT, and 10% v/v glycerol (Sigma-Aldrich, St. Louis, MO, USA). Urea concentrations were sequentially reduced from 6 M to 0 M (6 M, 4 M, 2 M, 1 M, 0.5 M, 0 M), with buffer exchanged every 2 h to match the decreasing urea concentration [31,32]. This buffer composition closely approximates physiological ionic conditions, which are critical for SdiA folding. The addition of glycerol further improved protein stability and minimized aggregation during renaturation. The dialyzed and refolded protein was subsequently prepared for quantification and storage.

After dialysis, samples were centrifuged at 12,000× g for 10 min at 4 °C to remove insoluble aggregates. The supernatant containing soluble, renatured His₆-SdiA was collected. Protein concentration was determined using the Bradford colorimetric method (Bio-Rad Protein Assay, Hercules, CA, USA) with bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) as the standard. For long-term storage, purified protein was aliquoted and stored at −70 °C in stabilization buffer (50% v/v glycerol, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4; Sigma-Aldrich, St. Louis, MO, USA). The purified protein was then used in subsequent functional interaction studies.

2.7. Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays (EMSA) were performed to assess the binding of recombinant His₆-SdiA protein to the regulatory region of the npsA gene. DNA probes covering the regulatory regions of K. oxytoca npsA and E. coli ftsQ (positive control) [33] and an internal fragment of K. oxytoca pehX (negative control) were amplified by PCR from genomic DNA. Purified DNA probes were standardized to 300 fmol per reaction. These preparations enabled a detailed investigation of SdiA-DNA binding parameters.

Binding reactions were performed in 20 µL volumes containing 1X binding buffer (20 mM Tris-HCl, pH 7.5; 50 mM KCl; 2.0 mM MgCl₂; 0.1 mM EDTA; 1.0 mM DTT; 0.01% v/v Tween-20; 2% v/v glycerol; 0.1 mg/mL BSA; Sigma-Aldrich, St. Louis, MO, USA). N-(3-Oxo-octanoyl)-L-homoserine lactone (AHL; Sigma-Aldrich, St. Louis, MO, USA) was added at a final concentration of 10 µM. AHL was included based on previous reports indicating that LuxR-family regulators can respond to exogenous AHL signals during transcriptional regulation. Purified His₆-SdiA was added at increasing concentrations (0.00, 0.25, 0.30, 0.35, and 0.40 µM). Each sample was then incubated with the DNA probe for 20 min at room temperature.

Samples were resolved by electrophoresis on 6% non-denaturing polyacrylamide gels in 0.5X Tris-borate-EDTA (TBE) buffer at 120 V. Temperature was controlled to maintain complex stability. Gels were stained with ethidium bromide (0.5 µg/mL), rinsed with distilled water, and visualized by UV transillumination. SdiA-DNA complexes appeared as retarded bands, while free DNA probe bands migrated more rapidly. These results supported a direct regulatory role for SdiA on the npsA gene. Further validation of binding sites was conducted using an in silico analysis as described below.

2.8. Identification of a Promoter and SdiA-Binding Motif in the npsA Regulatory Region

Promoter prediction within the npsA regulatory region was performed by analyzing 500 nucleotides upstream of the start codon. The K. oxytoca genomic sequence (GenBank accession no. GCF_001078175.1) was used for this analysis. The Neural Network Promoter Prediction web tool (https://fruitfly.org/seq_tools/promoter.html (accessed on 31 March 2026)) was also employed.

Potential SdiA-binding sites in the npsA gene regulatory region were identified through in silico sequence analysis using the PRODORIC v2.0 web database (https://www.prodoric.de/ (accessed on 31 March 2026)). A 500-nucleotide region upstream of the npsA translational start codon (ATG) was extracted for further analysis. Motif searches and comparisons were performed using the Virtual Footprint tool in PRODORIC. Predictive alignments were based on the established SdiA consensus recognition sequence (5′-AAAAG(N8)GAAAA-3′), previously identified in related Enterobacteriaceae [33,34,35,36]. These analyses supported the experimental EMSA results. They also increased confidence in the identification of the SdiA binding site.

2.9. LDH Cytotoxicity Assays

Cytotoxicity was evaluated by measuring lactate dehydrogenase (LDH) release using the CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen, Waltham, MA, USA), following the manufacturer’s instructions (https://documents.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0018500_CyQUANT-LDH-Cytotoxicity-Assay-Kit_PI.pdf (accessed on 31 March 2026)). HeLa cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 4.5 g/L glucose and 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). Cells were seeded in 96-well flat-bottom plates at a density of 1 × 10⁴ cells. Cell viability and counts were previously determined via trypan blue exclusion using a Neubauer chamber [37].

For the cytotoxicity assay, 90 µL of HeLa cell suspension was treated with 10 µL of cell-free filtered supernatants obtained from the WT, ΔsdiA, and ΔsdiA pT3-SdiA strains. As an additional control, the previously generated ΔnpsA mutant strain from this research group [16], which lacks the ability to produce the TM and TV cytotoxins, as previously demonstrated [6,38], was included. The use of this mutant strain confirmed that the cytotoxic effects observed in the assay were specifically associated with TM and TV production by K. oxytoca. All bacterial strains were pre-cultured in the absence or presence of 10 µM AHL prior to supernatant collection. Plates were subsequently incubated for 48 h at 37 °C in a humidified atmosphere containing 5% CO₂, conditions previously established as optimal for detecting measurable cytotoxic effects [15,16,39,40,41].

Following incubation, 50 µL of the supernatant from each well was transferred to a fresh 96-well plate, and LDH activity was quantified by adding the respective kit reagents. Absorbance was recorded at 490 nm and 680 nm using a Multiskan Ascent microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The final LDH release was calculated as the difference between these two optical density (OD) values. Phosphate-buffered saline PBS and tryptic soy broth TSB were used as negative controls, whereas the provided lysis buffer served as the positive control. All assays were performed in triplicate across three independent biological replicates.

2.10. Statistical Analyses

All statistical analyses were conducted using GraphPad Prism software, version 10.5.0 (GraphPad Software, San Diego, CA, USA). Differences between experimental groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison post hoc test. Statistical significance was set at p < 0.05.

3. Results

3.1. SdiA Activates the Expression of the npsA Gene

The effect of SdiA on npsA gene expression was analyzed via RT-qPCR in WT, ΔsdiA mutant, and ΔsdiApT3-SdiA complemented K. oxytoca strains. In the WT strain, addition of AHL led to a distinct increase in npsA expression, resulting in a 3-fold induction compared to the vehicle control (Figure 1). This identifies AHL-dependent activation of npsA as a key result in the WT background.

Deletion of sdiA resulted in a substantial reduction in npsA expression, as the ΔsdiA mutant exhibited approximately a 2-fold decrease compared to the WT strain under basal conditions. Notably, npsA expression in the ΔsdiA mutant did not change following AHL supplementation, indicating that SdiA is required for AHL responsiveness.

Thus, the disparity in npsA expression between WT and ΔsdiA mutant is more pronounced under AHL-treated conditions, reflecting induction exclusively in the WT strain (Figure 1). Together, these results indicate that SdiA mediates AHL-responsive activation of npsA transcription.

Moreover, complementation of the ΔsdiA mutant with pT3-SdiA restored npsA expression to WT levels under both AHL-supplemented and vehicle control conditions (Figure 1), confirming that the observed changes in gene expression are specifically due to the loss of sdiA. These findings indicate that SdiA positively contributes to both basal and AHL-induced npsA expression, establishing its central role in signal-dependent regulation.

No significant differences in bacterial growth were detected among the strains or experimental conditions. The reference gene rrsH exhibited stable expression across all conditions (data not shown). These findings support the reliability of RT-qPCR normalization and validate the observed differences in npsA expression.

3.2. Identification of Putative Promoter and SdiA-Binding Site in the npsA Regulatory Region

The cytotoxin biosynthetic genes are organized within the NRPS (nonribosomal peptide synthetase) operon, which comprises npsA, thdA, and npsB [6,38]. In silico analyses examined the regulatory architecture of this operon, with particular attention to potential promoter elements and SdiA-binding sites located upstream of npsA.

A putative promoter was predicted 329 bp upstream of the npsA coding sequence. Additionally, a candidate SdiA-binding box was identified within the same regulatory region, located 71 bp upstream of the predicted transcription start site (+1) (Figure 2).

The spatial arrangement of these elements suggests a regulatory mechanism in which SdiA binds to the DNA sequence immediately upstream of the promoter region. The predicted regulatory arrangement supports a potential role for SdiA in the positive regulation of npsA expression. Further experimental studies are required to clarify the precise molecular mechanism by which SdiA modulates promoter activity, such as through site-directed mutagenesis of the putative SdiA-binding site within the npsA regulatory region.

3.3. SdiA Directly Interacts with npsA Regulatory Region

To assess direct interaction between SdiA and the npsA regulatory region, recombinant His₆-tagged SdiA was purified and subjected to EMSA. The assays revealed a clear, reproducible, and concentration-dependent DNA mobility retardation, indicating stable SdiA–DNA complex formation. This mobility shift was observed in the absence (Figure 3A) and presence (Figure 3B) of AHL, demonstrating that SdiA binds the npsA upstream regulatory region in vitro under both conditions.

While SdiA-mediated activation of npsA expression increases in the presence of AHL during bacterial growth, EMSA experiments indicate that SdiA binds the npsA regulatory region in vitro regardless of exogenous AHL.

The specificity of the SdiA–DNA interaction was validated using both positive and negative controls. The regulatory region of ftsQ from E. coli, a well-characterized SdiA target, exhibited the expected mobility shift (Figure 3C). Conversely, a DNA fragment from the coding region of pehX in K. oxytoca, used as a negative control, did not display a shift under identical conditions (Figure 3D), confirming sequence-specific interaction. Collectively, these results demonstrate that SdiA directly and specifically binds the npsA promoter region.

3.4. SdiA Enhances the Cytotoxic Activity of K. oxytoca on Epithelial Cells

The impact of SdiA on K. oxytoca cytotoxicity was assessed using cell-free supernatants from the toxigenic WT, the ∆sdiA mutant, and the complemented ∆sdiA pT3-SdiA strain. Cytotoxicity was measured by lactate dehydrogenase (LDH) release in HeLa cells. Supernatants from the WT strain induced substantial cytotoxicity, resulting in pronounced HeLa cell death. This effect was significantly enhanced when bacteria were cultured in the presence of AHL (Figure 4), indicating that quorum-sensing signals potentiate toxin-associated activity.

Supernatants from the ∆sdiA mutant exhibited a marked reduction in cytotoxic activity under both tested conditions, regardless of AHL presence. The absence of sdiA significantly reduced the cytotoxic phenotype observed under the tested conditions. In contrast, cytotoxicity in the complemented ∆sdiA pT3-SdiA strain was restored to WT levels, confirming that the phenotype is specifically associated with the loss of SdiA (Figure 4). The solvent control did not significantly affect bacterial growth, HeLa cell viability, or basal LDH release under the experimental conditions evaluated (data not shown).

To further confirm the association between npsA and the cytotoxic phenotype observed in this study, supernatants from the ∆npsA mutant strain were analyzed as a negative control. Under the experimental conditions tested, these supernatants did not induce detectable HeLa cell death, consistent with previous reports of reduced epithelial cytotoxicity in npsA-deficient strains [6,38]. Collectively, these findings indicate that SdiA modulates the cytotoxic phenotype in K. oxytoca.

4. Discussion

The quorum-sensing regulator SdiA acts as a positive regulator of npsA, the initial gene in the NRPS operon, and exerts a significant influence on the cytotoxic phenotype of K. oxytoca. Transcriptional, in silico, biochemical, and functional analyses collectively support a model in which SdiA enhances npsA expression in response to AHL signals. Nevertheless, these findings require cautious interpretation. As direct quantification of TM and TV by LC-MS, HPLC, or related analytical methods was not conducted, the data do not provide definitive evidence that SdiA directly or quantitatively regulates TM/TV biosynthesis. Instead, the results indicate that SdiA modulates the cytotoxicity under the specific experimental conditions tested in K. oxytoca.

Transcriptional analyses demonstrated that deletion of sdiA significantly reduced npsA expression, indicating that SdiA is a positive regulator of npsA, the first gene of the NRPS operon. Supplementation with AHL increased npsA transcription in the WT strain but failed to affect the ∆sdiA mutant, demonstrating that SdiA mediates the transcriptional response to quorum-sensing signals. Importantly, AHL supplementation did not reduce npsA expression in the ∆sdiA mutant relative to its corresponding untreated condition. Rather, the apparent increase in the expression difference between WT and ∆sdiA strains under AHL-supplemented conditions resulted from the selective induction of npsA expression in the WT strain.

This behavior is consistent with the established role of LuxR-type regulators, which detect exogenous AHL molecules and modulate gene expression in response to population density and microbial community composition [42,43]. Notably, K. oxytoca lacks a LuxI homolog, suggesting that SdiA enables the organism to detect AHL signals produced by neighboring bacterial species and integrate interspecies communication into its regulatory network.

A 10 µM AHL concentration was chosen based on studies showing SdiA-dependent responses in S. enterica [20]. The observed changes in npsA expression and cytotoxicity indicate activation of the SdiA pathway in K. oxytoca. However, this concentration serves only as an in vitro parameter and does not reflect physiological intestinal AHL levels. Micromolar AHL concentrations are commonly used in vitro in quorum-sensing studies with LuxR-type regulators, including SdiA systems, to ensure receptor activation and reproducible transcriptional responses [21,22,23,24,25].

The gastrointestinal tract is a complex and heterogeneous environment in which AHL availability may vary substantially due to microbial composition, spatial organization, diffusion dynamics, oxygen levels, and host-derived factors [11,12,13,14]. Therefore, future dose–response studies using lower AHL concentrations and more physiologically relevant models are required to better define the biological relevance and sensitivity of SdiA-mediated signaling in K. oxytoca.

In silico analysis identified a putative promoter upstream of npsA and a predicted SdiA-binding site located 71 bp upstream of the putative transcription start site. This regulatory arrangement suggests a role for SdiA in the positive regulation of npsA expression. EMSA experiments further supported a direct interaction between purified His₆-SdiA and the npsA upstream regulatory region. Increasing concentrations of purified His₆-SdiA generated concentration-dependent retardation of the DNA fragment, indicating formation of stable nucleoprotein complexes. The specificity of this interaction was supported by the absence of binding to the negative-control fragment and by strong binding to the ftsQ promoter, a previously characterized SdiA target [33].

Collectively, these findings support a direct interaction between SdiA and the npsA regulatory region. However, the specific nucleotides required for SdiA-mediated promoter regulation remain unidentified. The lack of mutational validation of the predicted SdiA-binding motif is a significant limitation of this study. Future research should employ site-directed mutagenesis, footprinting analyses, quantitative binding assays, and promoter-reporter systems to confirm the functional binding motif and elucidate how SdiA binding influences npsA promoter activity.

EMSA results showed that SdiA binds the npsA regulatory region in vitro, both in the presence and absence of exogenous AHL. Comparable results have been reported for other SdiA-regulated promoters [24,33,35,44,45], indicating that exogenous AHL is not strictly required for DNA recognition under in vitro conditions. Although similar DNA retardation patterns were observed in EMSAs, this technique does not provide the quantitative resolution needed to detect subtle differences in binding affinity, protein conformation, or complex stability.

In contrast, RT-qPCR analyses demonstrated increased npsA expression in the presence of AHL during bacterial growth, suggesting that AHL may influence SdiA regulatory activity through mechanisms not fully captured by standard EMSA analysis. Further quantitative binding studies are necessary to determine whether AHL affects the affinity, stability, or regulatory properties of the SdiA–DNA interaction.

Functionally, SdiA-mediated regulation significantly influenced the cytotoxic phenotype observed under the tested experimental conditions. Supernatants from the WT strain induced substantial epithelial cell damage, and this effect increased when bacteria were cultured in the presence of AHL, indicating that quorum-sensing signals modulate cytotoxicity-associated activity. In contrast, the ∆sdiA mutant exhibited markedly reduced cytotoxicity under both experimental conditions, whereas complementation restored the phenotype in the ∆sdiA pT3-SdiA strain.

Importantly, previous studies have demonstrated that K. oxytoca strains lacking npsA fail to produce TM and TV and concomitantly lose cytotoxic activity toward epithelial cells [6,7,38,46]. For this reason, ∆npsA strains are commonly used as controls in studies evaluating the cytotoxicity linked to this genomic region. Consistent with these previous observations, the ∆npsA mutant analyzed in the present study exhibited markedly reduced cytotoxicity under the experimental conditions tested.

The cytotoxic phenotype observed in this study is biologically associated with the npsA-linked NRPS locus. However, despite this established relationship, definitive conclusions regarding the exclusive or quantitative dependence of cytotoxic effects on TM and TV production cannot be made, as direct toxin quantification was not performed. Consequently, these findings should be interpreted as evidence that SdiA contributes to a cytotoxic phenotype, rather than as definitive proof of direct quantitative regulation of TM/TV biosynthesis.

The potential in vivo relevance of SdiA-dependent regulation should be interpreted with caution. While antibiotic-associated hemorrhagic colitis is an intestinal disease, this study utilized in vitro bacterial cultures and HeLa cell cytotoxicity assays. The gastrointestinal tract is a dynamic and heterogeneous ecosystem in which numerous environmental and microbial factors can influence quorum-sensing responses and npsA gene expression. Microbiota-derived AHL molecules can vary greatly in concentration, chemical structure, spatial distribution, and diffusion properties, potentially leading to variable SdiA activation during intestinal colonization [11,12,13,14].

Furthermore, oxygen gradients, nutrient availability, microbial competition, host-derived factors, and spatial heterogeneity within the intestinal niche may also affect npsA expression and related cytotoxic phenotypes [47,48]. Thus, the regulatory model proposed here should be viewed as an experimentally supported in vitro framework that requires validation in physiologically relevant systems, such as intestinal colonization and in vivo expression models. Regarding transcriptional regulation, SdiA appears to integrate environmental quorum-sensing signals with the expression of virulence-associated traits in K. oxytoca. The observed increases in cytotoxicity and npsA expression in the presence of AHL suggest that quorum-sensing molecules modulate the regulatory output of the SdiA signaling pathway during bacterial growth. Similar regulatory patterns have been reported for other LuxR-family proteins, which respond to environmental AHLs and regulate genes involved in host interaction and bacterial adaptation [49,50].

Regulation of the NRPS locus is likely multifactorial and may involve the coordinated action of several global regulatory systems. Previous studies have implicated regulators such as CRP and Lrp in controlling this genomic region [16,40]. Within this broader regulatory network, CRP may link carbon metabolism and cellular energetic status to npsA expression, while Lrp may contribute to nutrient-responsive regulation associated with amino acid availability and bacterial physiological adaptation [16,40]. In this context, SdiA may function as an additional quorum-sensing regulatory layer that integrates interspecies signaling information derived from microbiota-associated AHL molecules during intestinal colonization. Rather than acting as an isolated regulator, SdiA likely participates in a broader regulatory framework in which metabolic, environmental, and interspecies signaling pathways converge to modulate npsA expression and associated cytotoxic phenotypes.

Comparative analyses across enteric bacteria reveal both conserved and context-dependent aspects of SdiA-mediated regulation. In S. enterica, the SdiA regulon is relatively limited and includes genes such as the pefI-srgC operon, srgE, and the menFDHBCE operon [51]. Among these, only pefI-srgC has been directly demonstrated to be regulated by SdiA, whereas the remaining loci may be indirectly controlled. These genes are mainly associated with virulence-related functions, including fimbrial regulation and host interaction [42].

In both non-pathogenic and pathogenic E. coli, SdiA regulates functionally analogous systems, including the glutamate-dependent acid resistance island (gad) and, in enterohemorrhagic E. coli, the locus of enterocyte effacement (LEE), the latter through direct regulation of the ler promoter [23,45,52]. Additionally, SdiA represses flagellar gene expression, further supporting its role as a regulator that integrates environmental signals with bacterial adaptation [53,54].

In other Enterobacteriaceae, SdiA-mediated regulation appears more variable and less well defined. In Enterobacter cloacae, the regulon includes genes associated with membrane composition, signal transduction, and metabolism, involving both AHL-dependent and AHL-independent mechanisms [42,55]. Similarly, in K. pneumoniae, SdiA has been implicated in regulating ftsQ, rpoS, and fimA, although the extent of AHL dependence remains unclear [44]. Across these genera, SdiA consistently functions as an interspecies signal sensor that modulates genes associated with host interaction, stress adaptation, and virulence-associated traits.

The regulatory architecture described in this study is consistent with a conserved LuxR-type signaling paradigm. Identification of npsA as an SdiA-responsive gene in K. oxytoca expands the known functional scope of SdiA-mediated regulation while remaining consistent with its broader role as a sensor of interspecies signals and an agent of environmental adaptation.

Although additional regulatory proteins were not directly evaluated, they may cooperate with SdiA to further modulate npsA gene expression in response to environmental factors such as oxygen availability, nutrient status, or host-derived signals. Future studies should identify these potential co-regulators and further characterize the regulatory network governing the npsA gene expression.

In summary, these findings support a model in which SdiA directly interacts with the npsA regulatory region and positively influences npsA expression, thereby contributing to cytotoxicity in K. oxytoca. However, this proposed regulatory framework remains preliminary and should not be regarded as definitive proof of direct quantitative regulation of tilimycin or tilivalline biosynthesis. Future studies should incorporate direct quantification of TM/TV, mutational validation of the predicted SdiA-binding motif, promoter-reporter analyses, dose–response experiments, and physiologically relevant colonization models. Furthermore, the interplay between SdiA and other global regulators, such as CRP and Lrp, should be further investigated to clarify how metabolic, environmental, and quorum-sensing signals are integrated during K. oxytoca colonization and cytotoxicity-associated responses.

5. Conclusions

The findings indicate that SdiA acts as a positive regulator of npsA expression by directly binding to its upstream regulatory region. SdiA thus mediates quorum-sensing-responsive npsA expression and is associated with modulation of the cytotoxic phenotype of K. oxytoca under the tested conditions. Evidence from transcriptional, in silico, biochemical, and functional analyses supports this mechanism. The observed increase in npsA expression and epithelial cytotoxicity in the presence of AHL further substantiates the role of interspecies quorum-sensing signals in modulating cytotoxicity in K. oxytoca.