GlnK Regulates the Type III Secretion System by Modulating NtrB-NtrC Homeostasis in Pseudomonas aeruginosa

Abstract

Bacterial pathogens exploit host-derived nutrients to coordinate metabolism and virulence determinants to optimize fitness in vivo. In Pseudomonas aeruginosa, GlnK is a central regulator of nitrogen metabolism. It senses the intracellular nitrogen status by integrating 2-oxoglutarate (2-OG) and glutamine signals, which in turn triggers its uridylylation and conformational changes. This reversible post-translational modification modulates its interaction with target proteins, thereby precisely regulating carbon-nitrogen metabolic homeostasis and enabling adaptive nitrogen metabolism in response to host-derived nutrient cues. In this study, we found that glnK is upregulated during infection in a mouse pneumonia model. By growing bacteria in mouse bronchoalveolar lavage fluid (BALF), we demonstrated that the expression of glnK is activated by the NtrB-NtrC two-component regulatory system in response to the host nutrient environment. Mutation of glnK impairs bacterial virulence. Transcriptomic analysis revealed downregulation of the type III secretion system (T3SS) genes in the glnK mutant. Further studies revealed a role of GlnK in maintaining the homeostasis of the NtrB-NtrC system through a negative feedback mechanism, which is required for the expression of the T3SS genes. Collectively, these findings reveal a role of GlnK in interconnecting carbon–nitrogen balance and the T3SS in response to the host environment.

1. Introduction

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that poses substantial threats to human health, particularly in immunocompromised populations, individuals with cystic fibrosis and severe burns, and those suffering from chronic wounds [1,2,3]. A key driver of its pathogenicity lies in its ability to survive and proliferate within hosts. This exceptional adaptability is attributed to its capacity to sense and integrate environmental cues, such as fluctuations in nutrient availability, which in turn allows it to optimize metabolic pathways and virulence-associated traits [4,5].

The type III secretion system (T3SS) is one of the key virulence factors of P. aeruginosa [6]. This specialized syringe-like apparatus directly delivers effector proteins into host cells to disrupt immune responses and promote colonization [7]. Transcription of the T3SS genes is controlled by the master regulator ExsA [8]. The exsA gene is located in the exsCEBA operon and driven by the P_exsC promoter and an adjacent P_exsA promoter that is located in the intergenic region between exsB and exsA. The P_exsA promoter is primarily regulated by cAMP in partnership with the cAMP receptor protein (CRP) Vfr [9]. Under T3SS-non-inducing conditions, ExsA is sequestered by ExsD and ExsC binds to ExsE. Under T3SS-inducing conditions, e.g., Ca²⁺ depletion or contact with host cells, the intracellular cAMP level is increased, resulting in activation of exsA transcription from the P_exsA promoter [10,11]. Meanwhile, ExsE is secreted through the T3SS machinery, releasing ExsC to sequester ExsD, resulting in free ExsA that directly activates the expression of T3SS machinery and effector genes [12]. The P_exsC promoter is also activated by ExsA, thus forming a positive feedback regulation [13].

Previous studies have demonstrated that during lung infection, long-chain fatty acids (LCFAs) and mucin are major carbon sources of P. aeruginosa [14,15]. We found that a regulator, PvrA, senses the LCFA metabolic intermediate molecule palmitoyl coenzyme A and regulates genes involved in LCFA metabolism and the PQS quorum sensing system [16].

Besides carbon sources, nitrogen is indispensable for microbial survival. Bacteria predominantly utilize reactive forms of nitrogen such as ammonium (NH₄⁺) and nitrate (NO₃⁻) [17]. NO₃⁻ is usually converted into the preferred ammonium (NH₄⁺), and ammonium assimilation is mediated by two specialized pathways. The high-affinity GS-GOGAT pathway proceeds in two steps: glutamine synthetase (GS) first assimilates ammonium into glutamine (GLN), and GLN then condenses with α-ketogluterate (α-KG) via glutamate synthase (GOGAT) to generate glutamate. In contrast, the glutamate dehydrogenase (GDH) pathway relies on GDH to directly catalyze the reductive amination of α-KG with ammonium into glutamate. This pathway functions efficiently only when ammonium is abundant [18]. Together, glutamate and glutamine act as versatile nitrogen donors in transamination and transamidation reactions, supporting microbial metabolism and growth.

Bacterial PII signal transduction proteins play an essential role in sustaining intracellular pools of glutamate and glutamine as well as maintaining cellular carbon–nitrogen balance in response to fluctuating nitrogen levels [19,20]. In E.coli, GlnK modulates GS activity to coordinate nitrogen uptake and assimilation in a unified regulatory network. Under nitrogen-replete conditions, GlnK undergoes deuridylylation, adopting a conformation that binds to the bifunctional enzyme adenylyl-transferase/adenylyl-removase (AT/AR) and promotes its adenylyl-transferase activity, leading to GS inactivation via adenylylation (GS~AMP) to prevent excessive nitrogen assimilation. Conversely, under nitrogen-limitation conditions, uridylylated GlnK (GlnK~UMP) reduces the affinity for AT/AR, allowing AT/AR to switch to unadenylylase activity and reactivate GS through deadenylylation [21]. GlnK also regulates AmtB, a high-affinity ammonium transporter responsible for NH₄⁺ uptake in a nitrogen-dependent manner. Under nitrogen repletion, deuridylylated GlnK binds to AmtB at the cytoplasmic interface, inducing a conformational change that blocks its transport channel and shuts down NH₄⁺ uptake. Under nitrogen limitation, GlnK~UMP dissociates from AmtB, restoring the activity of the transporter to scavenge scarce environmental NH₄⁺ [22,23]. This dual regulation of GS and AmtB by GlnK ensures tight coupling of nitrogen acquisition and utilization, optimizing resource allocation and avoiding energy waste on superfluous nitrogen processes. Notably, these interactions are governed by the modification and demodification events of GlnK, a process regulated by the signal-transducing bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) GlnD. GlnD switches its dual catalytic activity in response to metabolic signals of 2-oxoglutarate (2-OG) and glutamine, with these metabolites also directly influencing GlnK’s conformational dynamics and its subsequent protein–protein interaction affinity. Under nitrogen-replete conditions with high glutamine and low 2-OG, GlnD exerts uridylyl-removing (UR) activity to mediate the deuridylylation of GlnK, whereas under nitrogen-limiting conditions characterized by low glutamine and high 2-OG, it triggers uridylyltransferase (UTase) activity to drive the covalent uridylylation of GlnK. Thus, the GlnD-catalyzed reversible modification of GlnK, together with its direct metabolic modulation, alters its conformational state and ultimately governs the coordinated regulation of glutamine synthetase (GS) activity and ammonium uptake to maintain cellular carbon-nitrogen homeostasis.

In addition to its well-characterized role in regulating GS and AmtB, GlnK also interacts with and modulates the activity of a diverse set of proteins, including various enzymes, transcriptional regulators, and nutrient transporters [24,25]. Most of these interacting proteins participate in the homeostasis of metabolism, highlighting the function of GlnK as a major metabolic hub that coordinates nitrogen metabolism and downstream physiological processes. It has been demonstrated that bacterial metabolism is tightly coupled to pathogenicity [26]. However, whether GlnK is involved in bacterial response to the host environment and the regulation of virulence factors in P. aeruginosa remains largely unknown, leaving a critical knowledge gap that warrants further investigation.

In this study, we utilized a murine pneumonia model to investigate the role of GlnK in the pathogenesis of P. aeruginosa. We found that the expression of GlnK is increased in response to in vivo nitrogen-limiting conditions. In addition, GlnK contributes to bacterial virulence through the homeostatic regulation of the NtrB-NtrC two-component system, which is required for T3SS activation. These findings reveal that GlnK integrates nitrogen signals with virulence regulation, highlighting nutrient–virulence cross-talk in P. aeruginosa.

2. Materials and Methods

2.1. Bacterial Strains, Plasmids, and Culture Media

The bacterial strains and plasmids employed in this study, along with their descriptions and sources, are summarized in Table S3 [27]. For the cultivation of bacterial cells, Luria–Bertani (LB) medium (5 g/L yeast extract, 10 g/L tryptone, and 5 g/L NaCl, pH 7.4) and M9 minimal medium (22.0 mM KH₂PO₄, 2.3 mM Na₂HPO_4, 8.6 mM NaCl, 1 mM MgSO₄, 0.1 mM CaCl₂, 18.7 mM NH₄Cl, and 22.2 mM glucose) were used. Bronchoalveolar lavage fluid (BALF) was collected from healthy 6-week-old female BALB/c mice as previously described [28]. The trachea was exposed and intubated with an 18-GN polyethylene catheter (BD Angiocath™ IV Catheter), followed by instillation with 1 mL sterile 0.9% NaCl. Then the lavage fluid was gently aspirated. The instillation and aspiration was performed twice. The collected BALF samples were sterilized by filtering through a 0.22 μm filter and pooled for further experiments. To ensure experimental consistency, the concentration of each batch of BALF was normalized based on total protein concentration, which was quantified using a BCA Protein Assay Kit (Beyotime, Haimen, China).

2.2. Ethics Statement

All animal studies were conducted in accordance with national regulations and the ethical guidelines for animal research formulated by Nankai University. The protocol was approved by the institutional animal care and use committee of the College of Life Sciences of Nankai University (approval code: NK-04-2012; approval date: 12 March 2012).

Specific pathogen-free (SPF) female BALB/c mice, aged 6 weeks and weighing 16–20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were randomly housed in a pathogen-free facility with a maximum of 5 mice per cage. All animals were maintained under a 12 h light and dark cycle at a controlled temperature (20–25 °C).

2.3. Mouse Pneumonia Model and Analysis

Indicated bacterial cells were initially cultured in LB at 37 °C overnight. Subsequently, the bacteria were transferred to LB and grown until OD₆₀₀ reached 1.0 (~5 × 10⁸ CFU/mL). The bacterial cells were harvested, washed once with sterile 0.9% NaCl, and then diluted 1:2.5 with sterile 0.9% NaCl adjusted to the concentration of 2 × 10⁸ CFU/mL or maintained at 5 × 10⁸ CFU/mL. For intranasal inoculation, each SPF female BALB/c mouse was anesthetized until loss of the pedal reflex, then placed in a supine position with its head slightly elevated to maintain a patent airway. A 20 μL aliquot of the bacterial suspension was subsequently instilled slowly dropwise into the bilateral nasal cavities (10 μL per nostril), resulting in a final inoculum dose of 4 × 10⁶ CFU or 1 × 10⁷ CFU per mouse. At 12 or 6 h post-infection (hpi), the mice were euthanized via carbon dioxide (CO₂) inhalation. For bacterial load quantification at 12 hpi, lungs were immediately isolated and homogenized in 1% proteose peptone (Solarbio, Tongzhou, China), and bacterial loads were determined by serial dilution and plating. For histopathological analysis, hematoxylin and eosin (H&E) staining was performed as previously described [29]. Briefly, the lungs were fixed in 4% paraformaldehyde overnight, dehydrated through a graded series, and embedded in paraffin prior to sectioning and H&E staining. For in vivo gene expression analysis at 6 hpi, BALF was collected immediately after euthanasia using an 18-GN polyethylene catheter, via two rounds of sterile 0.9% NaCl injection and aspiration. Following centrifugation, the bacterial pellets were harvested for subsequent RNA extraction.

2.4. RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)

Total RNA was isolated using the TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China) following the manufacturer’s protocol. Prior to washing with the kit’s Wash Buffer, DNase I digestion was performed using RNase-free DNase I (Cat. No. GD201, TransGen Biotech, Beijing, China) to eliminate genomic DNA (gDNA) contamination. After RNA extraction, the concentration of each RNA sample was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), with the A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios used to evaluate RNA purity. Subsequently, complementary DNA (cDNA) was synthesized from 1 μg of total RNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). RT-qPCR was performed using Super Multiple Probe qPCR PreMix (Vazyme, Nanjing, China), alongside no-reverse transcription (no-RT) controls to exclude gDNA contamination. The 30S ribosomal protein gene rpsL was employed as the internal reference.

2.5. β-Galactosidase Activity Assay

The β-galactosidase activity assay was performed as previously described with minor modifications [29,30]. Bacteria were grown in LB at 37 °C. When the culture reached an OD₆₀₀ of 0.8, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce NtrC expression in the ΔntrC strain, with parallel identical IPTG induction in the strains of PA14/pMMB67EH and ΔntrC/pMMB67EH. The induction was continued until the OD₆₀₀ reached 1.0. The bacteria were harvested by centrifugation. The cell pellet was resuspended in a buffer containing 0.04 M NaH₂PO₄, 0.06 M Na₂HPO₄, 0.001 M MgSO₄, 0.01 M KCl, and 0.05 M β-mercaptoethanol (added freshly prior to use). The OD₆₀₀ of the resuspended bacterial suspension was measured. Subsequently, 10 μL 0.1% (w/v) sodium dodecyl sulfate (SDS) and 10 μL chloroform were added to 500 μL of the bacterial suspension, followed by vortexing for 10 s to lyse the cells. After adding 100 μL of o-nitrophenyl-β-D-galactopyranoside (ONPG) solution, the mixture was immediately incubated at 37 °C. Once the solution turned pale yellow, the enzymatic reaction was terminated by adding 500 μL of 1M Na₂CO₃. The reaction mixture was then centrifuged at 16,000× g for 5 min to eliminate cell debris, and the absorbance of the resulting supernatant was measured at OD₄₂₀. The OD₆₀₀ value of each sample was used as the internal control. The calculation formula for Miller units is as follows, where T denotes the reaction duration (min):

$$\mathrm{M}\mathrm{i}\mathrm{l}\mathrm{l}\mathrm{e}\mathrm{r} \mathrm{u}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{s}={\displaystyle \frac{1000\times {\mathrm{O}\mathrm{D}}_{420}}{500\times \mathrm{T}\times {\mathrm{O}\mathrm{D}}_{600}}}$$

2.6. Electrophoretic Mobility Shift Assay (EMSA)

The electrophoretic mobility shift assay (EMSA) was performed as previously described with minor modifications [15]. The 59 bp DNA probe was incubated with purified NtrC protein at the indicated concentrations in a 20 μL buffer (100 mM Tris–HCl, pH 7.4, 500 mM KCl, 5 mM EDTA, 35 mM MgCl₂, and 5 mM DTT). Following incubation, samples were loaded onto a 12% native polyacrylamide gel and electrophoresed at 10 mA for 60 min on ice in 0.5 × TBE (Tris-borate EDTA) buffer (44.5 mM Tris base, 44.5 mM boric acid, and 1 mM EDTA, pH 8.0).

2.7. RNA-Seq and Data Analysis

Wild-type PA14 and the ΔglnK mutant were cultured in LB at 37 °C to the log phase (OD₆₀₀ = 1.0), followed by RNA purification. The concentration and quality of total RNA from each sample were determined using Agilent 2100/2200 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), NanoDrop (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 1% agrose gel electrophoresis. The RNA-seq was performed by Azenta (Suzhou, China). Briefly, starting with 1 μg of total RNA, library construction was completed through rRNA depletion, RNA fragmentation, reverse transcription, end repair, dA-tailing, and adaptor ligation. Subsequently, adaptor-ligated DNA was size-selected to recover ~400 bp fragments and the RNA strand was digested. Libraries were amplified by PCR with indexed P5/P7 primers. The samples were validated by Qsep100 and quantified using Qubit3.0.

Multiplexed libraries were sequenced on an Illumina HiSeq/Novaseq instrument (Illumina, San Diego, CA, USA) employing a 2 × 150 paired-end (PE) mode in accordance with the manufacturer’s instructions. Sequence reads were mapped to the PA14 reference genome (GCF_000014625.1). The DESeq2 Bioconductor package was used for differential expression analysis, with differentially expressed genes identified by a Benjamini–Hochberg-adjusted p-value (P_adj) < 0.05 to control the false discovery rate.

2.8. Cytotoxicity Assay

Lactate dehydrogenase (LDH) release assays were employed to assess cellular cytotoxicity. A549 cells were seeded into 24-well plates (2  ×  10⁵ cells per well) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 ℃ with 5% CO₂. Indicated bacterial strains were cultured overnight in LB. On the following day, the bacteria were subcultured into fresh LB and grown to log phase (OD₆₀₀ = 1.0), followed by washing once and resuspension in RPMI 1640 medium. A549 cells were infected with bacteria at a multiplicity of infection (MOI) of 50, followed by centrifugation at 500× g for 5 min to synchronize bacterial cell contact. Addition of 1640 medium to parallel wells was used as a blank control. For bacterial strains requiring inducible expression, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture. After 2.5 or 4.5 h of infection, LDH was measured using an LDH Cytotoxicity Assay Kit (Solarbio, Tongzhou, China) following the manufacturer’s instructions. Cells incubated with the kit-supplied LDH release buffer served as the total LDH control group and the cytotoxicity rate was calculated by the following formula:

$$\mathrm{C}\mathrm{y}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y} \mathrm{R}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)=\left[1-{\displaystyle \frac{{\mathrm{A}}_{\mathrm{i}\mathrm{n}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{d}}-{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}}{{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}}}\right] \times 100$$

2.9. Western Blotting

The protocol was performed as previously described with minor modifications [29]. Equal amounts of bacteria were collected by centrifugation (10,000× g, 1 min) and resuspended in 1 × SDS loading buffer. After boiling at 99 °C for 10 min, samples were separated by 12% SDS-PAGE, followed by semi-wet transfer onto polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% non-fat milk in PBST for 1 h at room temperature, incubated with primary antibodies (1:2000 dilution; mouse anti-6 × His, rabbit anti-Flag or mouse anti-RNA polymerase α subunit) for 1 h, washed three times with PBST (7 min each), and then incubated with HRP-conjugated secondary antibodies (1:2000 dilution) for 1 h. Protein bands were detected by an Immobilon Western kit (Millipore, Burlington, MA, USA).

2.10. Affinity Chromatography Purification–Mass Spectrometry (AP-MS)

To purify GlnK-His₆ and GST-His₆ recombinant proteins, we utilized the ΔglnK mutant harboring the pMMB67EH plasmid, on which the fusion protein’s expression was driven by the tac promoter. Following growth to OD₆₀₀ 0.5, cells were induced with 1 mM IPTG for 4 h and then harvested by centrifugation (8000× g, 10 min). Subsequently, pellets were resuspended in a lysis buffer (300 mM NaCl, 50 mM Na₂HPO₄, pH 8.0, and 5 mM imidazole). The cells were sonicated and centrifuged (16,000× g, 10 min). The resulting supernatant was incubated with Ni-NTA agarose (QIAGEN, Hilden, Germany) at 4 °C for 2 h with gentle rotation. The resin was washed five column volumes (CVs) with the lysis buffer containing 20 mM imidazole, followed by elution with an elution buffer (pH 8.0) consisting of 300 mM NaCl, 50 mM NaH₂PO₄, and 250 mM imidazole.

After purification, the proteins were quantified using a BCA assay. Following enzymatic digestion, equal amounts of protein samples were desalted with C18 reversed-phase solid-phase extraction (RP-SPE) and subjected to a nano-liquid chromatography quadrupole ion trap/orbitrap high-resolution mass spectrometry system (nanoLC-MS/MS) analysis. Protein identification and quantification were performed by matching the acquired peptide fragment spectra against the PA14 reference proteome (derived from genome accession GCF_000014625.1).

2.11. Co-Purification Assay

The ΔglnK mutant co-expressing GlnK-His₆ (from the plasmid pMMB67EH) and NtrB-Flag (from the plasmid pAK1900), along with control cells expressing only GlnK-His₆ (harboring empty pAK1900 plasmid), were cultured in LB. For co-purification analysis, expression of GlnK-His₆ was induced by IPTG for 4 h. Culture of each strain (25 mL) was harvested by centrifugation (8000× g, 10 min). The bacterial pellets were snap-frozen in liquid nitrogen for 30 s before being subjected to Ni-NTA purification [31]. Purified protein samples were analyzed by Western blotting using appropriate antibodies.

2.12. Statistical Analysis

Values are shown as mean ± standard deviation (SD). Statistical analyses were conducted with GraphPad software v8.0.1 (San Diego, CA, USA), employing either Student’s t-test or ANOVA coupled with Dunnett’s multiple comparison test.

3. Results

3.1. The glnK Gene Is Upregulated in Response to the Mouse Lung Environment

To investigate whether GlnK is involved in bacterial response to the host environment, we determined its expression level in a mouse pneumonia model. RT-qPCR results revealed upregulation of glnK in bacteria isolated from mouse lungs compared to those grown in LB or M9 medium (Figure 1A and Figure S1). To verify that the nutritional environment of the mouse lung induces the expression of glnK, we grew wild-type PA14 in mouse bronchoalveolar lavage fluid (BALF) for 1h, which resulted in upregulation of glnK (Figure 1B).

Then we explored the regulatory mechanism of glnK. In Pseudomonas putida KT2440, the transcription of glnK is directly activated by NtrC in response to nitrogen limitation [32]. Sequence alignment revealed >90% sequence identity between the NtrC ortholog from P. putida KT2440 and P. aeruginosa PA14 (Figure S2A). Additionally, the P. putida NtrC-binding motifs (Motif 1 and Motif 2) are conserved in the glnK promoter regions in strains of the two species (Figure S2B) [32]. To investigate the role of NtrC in regulating glnK in P. aeruginosa, we constructed a glnK promoter–lacZ transcriptional fusion. Mutation of ntrC reduced LacZ expression, which was restored to wild-type level by complementation with an ntrC gene (Figure 2A). Consistently, the glnK mRNA level was downregulated in the ΔntrC mutant, which was restored to wild-type level by complementation with an ntrC gene (Figure 2B). EMSA results demonstrated the binding of NtrC to the glnK promoter region (Figure 2C and Figure S3). In addition, deletion of ntrC in wild-type PA14 reduced glnK expression in mouse lungs and BALF (Figure 2D). These results demonstrate that P. aeruginosa NtrC directly regulates glnK in response to the in vivo environment.

The NtrB-NtrC two-component system is activated by nitrogen limitation [33]. Besides glnK, the expression of ntrB, ntrC, and nitrogen assimilation genes regulated by the NtrB-NtrC system was increased in mouse lungs and BALF (Figure 2E). Supplementation of NH₄Cl in the BALF reduced the expression of these genes and glnK (Figure 2F). Collectively, these results demonstrate activation of the NtrB-NtrC system in mouse lungs, which upregulates the expression of glnK.

3.2. Glnk Regulates Bacterial Virulence

The upregulation of glnK in mouse lungs and BALF indicated a potential role of GlnK in bacterial virulence. We thus infected mice with a glnK transposon insertion mutant (glnK::Tn) from the PA14 mutant library. Compared to wild-type PA14, the bacterial loads of the glnK::Tn mutant were reduced, which were restored by complementation with a glnK gene (Figure S4). To verify the role of GlnK in bacterial virulence, we constructed a glnK deletion mutant in wild-type PA14 (ΔglnK), which displayed reduced colonization in mouse lungs (Figure 3A). Hematoxylin and eosin (HE) staining revealed alleviated alveolar damage and inflammatory cell infiltration in the lung of the ΔglnK-infected mouse (Figure S5). These results demonstrate that GlnK contributes to bacterial virulence during lung infection.

3.3. GlnK Is Required for the Expression of the T3SS

To elucidate the mechanism of GlnK-mediated regulation on P. aeruginosa virulence, we performed transcriptomic analysis. A total of 735 genes were differentially expressed (>2-fold change) in wild-type PA14 and the ΔglnK mutant (Figure 3B). Of note, the T3SS genes were downregulated in the ΔglnK mutant (

Table 1. Downregulated T3SS genes in the ΔglnK mutant.

Gene ID	Gene Name	Description	log2FC a
PA14_42250	pscL	T3SS stator protein PscL	−1.18
PA14_42260	pscK	T3SS sorting platform protein PscK	−1.35
PA14_42270	pscJ	T3SS inner membrane ring lipoprotein PscJ	−1.06
PA14_42280	pscI	T3SS inner rod subunit PscI	−0.91
PA14_42290	pscH	T3SS polymerization control protein PscH	−1.53
PA14_42300	pscG	T3SS chaperone PscG	−1.68
PA14_42310	pscF	T3SS needle filament protein PscF	−1.15
PA14_42320	pscE	T3SS co-chaperone PscE	−2.54
PA14_42340	pscD	T3SS inner membrane ring subunit PscD	−1.01
PA14_42350	pscC	T3SS outer membrane ring subunit PscC	−1.18
PA14_42360	pscB	T3SS chaperone PscB	−1.51
PA14_42380	exsD	T3SS regulon anti-activator ExsD	−1.18
PA14_42390	exsA	T3SS regulon transcriptional activator ExsA	−1.17
PA14_42400	exsB	T3SS pilotin ExsB	−1.79
PA14_42410	exsE	T3SS regulon translocated regulator ExsE	−2.12
PA14_42430	exsC	T3SS regulatory chaperone ExsC	−1.53
PA14_42440	popD	T3SS translocon subunit PopD	−2.27
PA14_42450	popB	T3SS translocon subunit PopB	−2.49
PA14_42460	pcrH	T3SS chaperone PcrH	−3.08
PA14_42470	pcrV	T3SS needle tip protein PcrV	−2.06
PA14_42480	pcrG	T3SS chaperone PcrG	−3.12
PA14_42490	pcrR	T3SS chaperone PcrR	−1.41
PA14_42500	pcrD	T3SS export apparatus subunit PcrD	−0.68
PA14_42510	pcr4	T3SS chaperone	−2.09
PA14_42530	pcr2	T3SS protein	−1.68
PA14_42540	pcr1	T3SS gatekeeper subunit Pcr1	−1.36
PA14_42550	popN	T3SS gatekeeper subunit PopN	−1.64
PA14_42570	pscN	T3SS ATPase PscN	−1.34
PA14_42580	pscO	T3SS central stalk protein PscO	−1.48
PA14_42600	pscP	T3SS needle length determinant PscP	−0.80
PA14_42610	pscQ	T3SS cytoplasmic ring protein PscQ	−0.67
PA14_42620	pscR	T3SS export apparatus subunit PscR	−0.80
PA14_42630	pscS	T3SS export apparatus subunit PscS	−3.32
PA14_42640	pscT	T3SS export apparatus subunit PscT	−0.78
PA14_42660	pscU	T3SS export apparatus subunit PscU	−1.03
PA14_51530	exoU	T3SS effector cytotoxin ExoU	−1.19
PA14_00560	exoT	T3SS effector bifunctional cytotoxin exoenzyme T	−1.77

^a FC, fold change (ΔglnK vs. wild-type PA14).

). RT-qPCR verified downregulation of the T3SS regulatory genes exsA and exsC, structural gene pcrV, and the effector protein gene exoU in the ΔglnK mutant (Figure 4A). By using a 6 × His-tagged exoU driven by its native promoter (P_exoU-exoU-His), we further verified the defective expression of ExoU in the ΔglnK mutant (Figure 4B). In agreement with the downregulation of T3SS genes, the cytotoxicity was reduced in the ΔglnK mutant (Figure 3C). Chromosomal complementation with a glnK gene driven by its native promoter restored the expression of the T3SS genes and bacterial cytotoxicity (Figure 3C and Figure 4). To investigate whether GlnK controls bacterial virulence through the T3SS, we overexpressed the T3SS master regulator gene exsA in the ΔglnK mutant, which increased the expression of the T3SS gene as well as the bacterial cytotoxicity and virulence (Figure 5). Altogether, these results demonstrate a role of GlnK in regulating the T3SS.

3.4. GlnK Controls the T3SS by Regulating the NtrB/NtrC Two-Component System Through a Negative Feedback Mechanism

Since GlnK exerts its regulatory function by binding to target proteins, we performed affinity chromatography using a C-terminal 6 × His-tagged GlnK (GlnK-His₆) to decipher its regulatory mechanism on the T3SS. A C-terminal 6 × His-tagged GST (GST-His₆) was used as a control (Figure 6A). The purified proteins were subjected to mass spectrometry (MS) analyses. Compared to the control group, the known GlnK-interacting proteins, including ArgB, RelA, Rho, TrkA, GlnD, and NtrB were enriched (Table S1) [24]. However, no T3SS-related proteins were identified. A previous study demonstrated that the NtrB-NtrC system is involved in the regulation of bacterial virulence factors [34]. In addition, carbon–nitrogen balance has been shown to regulate the T3SS [35,36]. Given the role of GlnK in maintaining carbon–nitrogen balance through regulating NtrB-NtrC and glutamine synthetase activities, we suspected that GlnK might regulate the T3SS by modulating the NtrB-NtrC system.

To verify this hypothesis, we first confirmed the interaction between GlnK and NtrB by affinity chromatography (Figure 6B). Then we found that mutation of glnK resulted in upregulation of ntrB, ntrC, and nitrogen assimilation genes (Figure 6C), indicating a negative regulation of the NtrB-NtrC system by GlnK. Overexpression of ntrC reduced the expression of the T3SS genes and bacterial cytotoxicity (Figure 7A,B), demonstrating a negative regulatory role of NtrC on the T3SS. Deletion of ntrB or ntrC in the ΔglnK mutant restored the expression levels of exsA, exsC, exoU, and pcrV, as well as bacterial cytotoxicity (Figure 7C,D). Overexpression of ntrC in the ΔglnKΔntrC double mutant reduced the bacterial cytotoxicity to a similar level to the ΔglnK mutant (Figure 7D). Collectively, these results reveal a role of GlnK in regulating the T3SS by modulating the NtrB-NtrC system.

4. Discussion

In this study, we demonstrated that GlnK modulates the T3SS by regulating the NtrB-NtrC system through a negative feedback mechanism. During colonization in mouse lung or growth in BALF, the NtrB-NtrC system is activated. Supplementation of NH₄Cl in the BALF reduced the expression of ntrB, ntrC, and nitrogen assimilation genes, indicating a nitrogen-limiting environment in vivo. Unlike most typical two-component regulatory systems, the sensor NtrB is an intracellular protein [37]. It is a bifunctional enzyme with both kinase and phosphatase activities which is regulated by GlnK. Under nitrogen-replete conditions, unmodified GlnK binds to NtrB, favoring its phosphatase activity, thus leading to dephosphorylation of NtrC. Conversely, under nitrogen-limitation conditions, GlnK undergoes uridylylation to form GlnK~UMP. GlnK~UMP loses its ability to bind NtrB. As a result, NtrB switches to its kinase activity, driving increased phosphorylation of NtrC (NtrC~P) [38,39].

Besides the nitrogen acquisition genes, NtrC~P upregulates the glnK gene, thereby forming a negative feedback loop. Specially, deletion of glnK might keep NtrB in its kinase-active state and subsequently promote the formation of NtrC~P, which traps the bacteria in a nitrogen starvation response state. It has been demonstrated that bacteria may prioritize survival processes over energy-costly virulence traits in response to nutrient starvation. For instance, culture of Salmonella typhimurium in minimal media reduced its ability to invade epithelial cells [40].

As for P. aeruginosa, growth in mannitol resulted in lower T3SS gene expression than those grown in the preferred carbon source succinate, which also suggests a role of catabolite repression in regulating the T3SS [41]. Indeed, the catabolite repressor Crc is required for the expression of T3SS genes [35]. Crc in partnership with the RNA chaperone Hfq controls gene expression at the post-transcriptional level [42,43]. The activity of Crc is antagonized by a small RNA CrcZ, whose expression is regulated by the CbrA-CbrB two-component system in response to different carbon sources [44]. Besides carbon sources, the CbrA-CbrB two-component system might also be involved in responding to carbon–nitrogen imbalance [45]. A previous study demonstrated that perturbation of histidine uptake and catabolism lead to defective T3SS, likely due to carbon–nitrogen imbalance [36]. Given the role of GlnK in maintaining carbon–nitrogen balance, further studies are warranted to investigate the interconnection between the NtrB-NtrC and CbrA-CbrB two-component systems and their roles in regulating the T3SS.

Since our data revealed activation of the NtrB-NtrC two-component system in the glnK mutant, it is possible that NtrB-NtrC upregulates a negative regulator of the T3SS. The regulator might repress the transcription of the T3SS genes and the activity of ExsA or other T3SS positive regulators. Another possibility is that NtrB-NtrC upregulates a small RNA that represses the T3SS. Currently, efforts are being made to elucidate the regulatory pathway.

Collectively, the GlnK-NtrB-NtrC-T3SS regulatory axis identified in this study adds critical depth to our understanding of how P. aeruginosa integrates metabolic cues with virulence.

5. Conclusions

In this work, we demonstrate that GlnK, a central regulator of nitrogen metabolism, functions as a critical link between nutrient sensing and virulence regulation. Our results reveal a GlnK–NtrB–NtrC–T3SS regulatory axis that allows P. aeruginosa to adapt to the pulmonary nutrient condition and fine-tune the expression of the virulence factor type III secretion system. These findings bridge metabolic adaptation and pathogenicity by elucidating a “nitrogen-sensing–virulence” signaling pathway that is essential for bacterial pathogenesis. This work sheds light on the coordination of bacterial metabolism and virulence during infection.