T. mobilis YJ3 was cultured in 2216E marine broth (MB, Becton Dickinson, Franklin Lakes, NJ, United States) at 28°C. The AHL biosensor Agrobacterium tumefaciens A136 (pCF218) (pCF372) was maintained on Luria Bertani (LB) agar and grown in AT minimal medium containing 0.5% (wt/vol) glucose for detecting AHLs (C₆ to C₁₄) by the liquid X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) assay (Tang et al., 2013). The AHL biosensors Chromobacterium violaceum CV026 and VIR24 (Latifi et al., 1995) were used for detecting short-chain (C₄ to C₈) and long-chain (C₈ to C₁₄) AHLs, respectively, by culturing them on LB agar plates and incubating at 28°C. Escherichia coli BL21 (DE3) was cultured in LB medium supplemented with kanamycin (50 μg/mL) and incubated at 37°C, it served as the host for expressing proteins whose encoding genes were constructed into the pET28a(+); P. aeruginosa PAO1 was cultured in nutrient broth (NB) medium at 37°C.

C₆-HSL, 3-oxo-C₆-HSL, C₈-HSL, and C₁₄-HSL were purchased from Cayman Biologicals (Ann Arbor, MI, USA); 3-oxo-C₈-HSL, C₁₀-HSL, 3-oxo-C₁₀-HSL, C₁₂-HSL, 3-oxo-C₁₂-HSL, 3-oxo-C₁₄-HSL, Azocasein, Orcinol (3,5-dihydroxytoluene) were purchased from Sigma Chemical (St. Louis, MO, USA); all AHLs were diluted by dimethyl sulfoxide (DMSO) into 10–500 mM and stored at −20°C. Enterokinase was purchased from Sangon Biotech (Shanghai) Co., Ltd. Chloroform was purchased from Sinopharm Chemical Reagent Co., Ltd.

Experimentally validated protein sequences of active QQ enzymes were retrieved from the UniProt online database¹ for the purpose of constructing a local database. The putative lactonase gene rmmLII from T. mobilis YJ3 was searched from the whole-genome sequence of YJ3 (Genbank accession No. CP181340-CP181345) through local BLASTP analysis against some known QQ enzyme sequences. The amino acid sequences of verified AHL lactonases were systematically compared to RmmLII by using the sequence analysis software MEGA11, while the Neighbor-Joining (NJ) method was employed to construct a phylogenetic tree. The results derived from the multiple sequence alignment were presented through ESPript, along with a detailed analysis of the active site residues.

The genomic DNA of T. mobilis YJ3 was extracted by the phenol-chloroform method, and rmmLII was amplified by the PCR method using a pair of primers: rmmLII-F: 5′-CGC GGA TCC GAC GAC GAC AAG ATG GTG TCG GTG GCG-3′ and rmmLII-R: 5′-CCC AAG CTT CTA GCG GCG CTG GTA AAC-3′, and then cloned into pET28a(+) to construct a recombinant plasmid pET28a(+)-rmmLII. The restriction enzyme sites BamH I and Hind III were incorporated into the forward and reverse primers, respectively, and an EK enzyme site was inserted to remove the His tag from the purified protein. PCR was conducted using the following conditions: denaturation at 98°C for 30 s, followed by 35 cycles of denaturation at 98°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 90 s.

The recombinant plasmid pET28a(+)-rmmLII was then transformed into E. coli BL21 (DE3) to construct the recombinant strain E. coli BL21 (DE3)/pET28a(+)-rmmLII for protein expression. The bacterial strain was cultured in LB liquid medium added to 30 μg/mL Kan at 37°C with shaking at 170 rpm until the OD₆₀₀ of the culture was 0.4–0.6. Subsequently, 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the culture induce protein expression, and the culture was incubated at 16°C with moderate shaking (150 rpm) for 15 h. The induced culture was subjected to high-speed centrifugation at 4°C to pellet the cells, which were then washed once with PBS buffer (50 mM, pH 7.0). The cells were resuspended with 10 times the original volume of binding buffer (Tris–HCl, 10 mM; imidazole, 10 mM; NaCl, 0.5 M). The cell suspension was sonicated on an ice bath, and the supernatant was collected via centrifugation at 13,000 rpm for 10 min at 4°C and filtered through a 0.22-μm PVDF membrane. The sample was loaded onto the NTA-Ni (Qiagen) column for purification, following the manufacturer’s instructions, to obtain the recombinant RmmLII-His fusion protein. In order to remove the His tag from purified protein, RmmLII-His was incubated overnight at 4°C with enterokinase, and then purified again by NTA-Ni columns. The purified RmmLII was analyzed by SDS-PAGE and stored in Tris–HCl buffer containing 10% glycerol at −20°C.

The degradation activity of RmmLII on AHLs was tested under the A136 liquid X-Gal method described previously (Tang and Zhang, 2018). Briefly, the A136 reporter strain was cultured in LB broth (containing spectinomycin 50 μg/mL, tetracycline 4.5 μg/mL) at 28°C until the OD₆₀₀ reached approximately 2.0. RmmLII, 1,4-piperazinediethanesulfonic acid (PIPES) buffer (1 mM, pH 6.7), and various AHL signal molecules (1 mM) were mixed in a volume ratio of 10:1:0.1 and incubated at 28°C for 24 h. Tris–HCl was used as a substitute for RmmLII in the negative control. A 1% solution of A136 was added to the AT medium (containing spectinomycin 50 μg/mL, tetracycline 4.5 μg/mL) with a final concentration of 250 μg/mL of X-Gal. The mixture was thoroughly mixed to obtain the detection solution. Then 10 μL of the reaction supernatant was combined with 190 μL of the detection solution in a 96-well plate and incubated at 28°C for 24 h. The OD₄₉₂ and OD₆₃₀ were measured, and the β-galactosidase activity values were determined, which were inversely correlated with the RmmLII enzymatic activity. Based on Equation (1), the calculation was performed accordingly.

To further confirm the degradation activity of RmmLII, a plate detection method was employed. C. violaceum CV026 and VIR24 were inoculated into LB broth and incubated overnight at 28°C. Then, the cultures were introduced into the semi-solid LB medium consisting of 0.8% (wt/vol) agar at approximately 45°C with an inoculum amount of 3%. After the medium solidified, the wells were created, and the reaction solution was added to each well. The plates were incubated at 28°C for 24 h to observe the formation of blue or purple halos and size.

To determine the chemical structure of the degradation products resulting from AHL signaling molecule catalyzed by RmmLII, 10 μL of RmmLII (1 mg/mL) was mixed with 100 μL of PIPES buffer (1 M, pH 6.7), 200 μL of C₁₀-HSL (10 mM), and deionized water was added up to a final volume of 1 mL. The mixture was thoroughly mixed and incubated at 28°C for 24 h, and was extracted thrice by ethyl acetate. The ethyl acetate solution was then evaporated to dryness by a nitrogen blower and analyzed by high-performance liquid chromatography (HPLC) using a SunFire C18 reversed-phase column (3.5 μm, 4.6 by 50 mm) using a mobile phase of acetonitrile-water (0.01% trifluoroacetic acid; a linear gradient [v/v] of acetonitrile from 5 to 95% over 15 min, held for 1 min, and then reduced to 5%; flow rate of 0.5 mL/min). The resulting fractions were analyzed by electrospray ionization mass spectrometry (ESI-MS).

To investigate the effect of specific amino acids on the activity of RmmLII, site-directed mutagenesis was performed using the Fast Mutagenesis System kit (TransGen, China) according to the manufacturer’s instructions. Briefly, the pET28a(+)-rmmLII plasmid was used as the template for mutagenesis. The primers used to obtain mutants are listed in Table 1. The mutated plasmids were sent to Sangon Biotech for sequencing to confirm the accuracy of the desired mutation sites and then transformed into E. coli BL21 (DE3) competent cells. The expression and purification of each mutated protein were conducted as described above, and the activities of mutated proteins were tested by the A136 liquid X-Gal method and compared to that of purified RmmLII.

The optimal temperature, thermostability, pH stability and the effects of various metal ions on the activity of RmmLII (1 mg/mL) were measured by the A136 liquid X-Gal method using C₆-HSL as the substrate. Each experimental group was performed with a minimum of three independent replicates to ensure reproducibility and statistical reliability.

The reaction solution was prepared according to the method described in Section 2.3. For the optimal temperature, the prepared reaction solution was incubated at temperatures of 10°C, 20°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for a duration of 24 h. Following incubation, OD₄₉₂ and OD₆₃₀ were measured, and β-galactosidase activity was calculated using Equation (1), with the activity of RmmLII incubated at 28°C used as the control. The relative activity under other conditions was calculated with this as 100%. To determine the thermostability of RmmLII, purified RmmLII was preincubated at temperatures of 20°C, 40°C, 60°C, 80°C, and 100°C for 30 min, after which the residual activity was evaluated. The β-galactosidase activity values of untreated RmmLII reactions incubated at 28°C were employed as the control values. Similarly, the pH stability was tested after the purified RmmLII was preincubated at pH levels ranging from 2 to 11 at 4°C for 3 h. To evaluate the impact of different metal ions, 1 mM of EDTA was combined with 0.1 mM of metal ions (Zn²⁺, Mg²⁺, Fe²⁺, Ca²⁺, Cu²⁺, Mn²⁺) and added to purified RmmLII, which was then incubated at 4°C for 1 h. Subsequently, metal ions were added to the EDTA-treated RmmLII at a concentration of 1.1 mM, and the mixture was incubated at 4°C for an additional 1 h. The residual activity of RmmLII was subsequently evaluated, with untreated RmmLII serving as the control.

To evaluate the impact of RmmLII on the production of virulence factors in P. aeruginosa PAO1, PAO1 was cultured overnight together with various concentrations of RmmLII [low dose group (1 × 10⁻³ mg/mL), medium does group (3.75 × 10⁻³ mg/mL), high dose group (7.5 × 10⁻³ mg/mL)] in LB or PB medium at 37°C, PAO1 culture without RmmLII was used as the negative control. The secretion levels of virulence factors, including extracellular protease, pyocyanin, and rhamnolipid, were assessed. Each experiment was performed in triplicate.

To assess the inhibitory effect of RmmLII on biofilm formation by P. aeruginosa PAO1, a method adapted from Alam et al. (2020) was used. Briefly, P. aeruginosa PAO1 was inoculated into LB broth at a 2% concentration and grown to an OD₆₀₀ of 0.3. The culture was then transferred to 24-well plates containing sterile slides, and varying concentrations of RmmLII were added to the same. The plates were then incubated statically at 37°C for 24 h. The samples were then washed twice with sterile PBS buffer, fixed in methanol for 15 min, air-dried, and stained with 0.1% crystal violet for 30 min. Excess dye was removed, and the biofilm was treated with 33% (w/v) acetic acid solution, and mixed well before measuring OD₅₅₀. Biofilm morphology was examined by a Sharp Corporation JSM-7500F scanning electron microscope (SEM) after fixing, dehydrating, drying, and gold coating the samples.

The motility of swimming and swarming of PAO1 was analyzed according to the previous report (Wang et al., 2021). The swimming assay was tested in a medium containing 10 g/L tryptone (Difco), 5 g/L NaCl, and 3 g/L agarose (GIBCO/BRL), which was covered with cling film to prevent desiccation. The swarming assay medium comprised 0.5% (wt/vol) Difco Bacto agar, 8 g/L NB, and 5 g/L glucose (Ling et al., 2010). The sterilized media for both assays was mixed with RmmLII at approximately 45°C. Once solidified, 2 μL of the 1% PAO1 culture was inoculated onto the agar plates. After the medium had fully absorbed, the plates were incubated at 30°C for 12–14 h to observe bacterial motility by mearsuring the diameter of the microcolony. Each experiment was performed in triplicate and then the statistical analysis of the data was performed.

A total of 40 female BALB/c mice, approximately 2 months old and weighing approximately 30 g, were obtained from Qingdao Daren Fucheng Animal Husbandry Co. Ltd. The mice were housed with free access to food and water in a room maintained at 20–22°C. After a 1-week acclimatization period, the experiments were initiated. All animal procedures adhered to the guidelines established by the Animal Ethics Committee of Qingdao Agricultural University (approval number: QAU2023122502).

Data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was performed using GraphPad Prism 8 software. Comparisons between two groups were made using the Student’s t-test, while multiple comparisons were conducted using one-way ANOVA. Statistical significance was indicated as follows: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

A local BLASTP search against a known QQ enzyme database identified the QQ enzyme gene Peg3631 in YJ3. This gene encodes a protein of 348 amino acids with high similarity to known AHL lactonases. Phylogenetic analysis of validated AHL lactonases and Peg3631 protein sequences revealed that Peg3631 clusters with the phosphotriesterase family of GKL, demonstrating 58% homology (Supplementary Figure S1). Sequence analysis further indicated that Peg3631 contains a highly conserved “HXHXDH~100AA ~ H” domain characteristic of lactonases (Supplementary Figure S1), suggesting that Peg3631 may represent a novel AHL lactonase and it was named RmmLII.

The recombinant plasmid pET28a(+)-rmmLII was successfully constructed and transformed into E. coli BL21 (DE3) cells for expression. Although more than half of the recombinant proteins were expressed in a soluble form, inclusion bodies were also observed (Supplementary Figure S2). Both purified RmmLII-His and RmmLII proteins displayed a single band corresponding to their predicted molecular weights (Figure 1a). The A136 assay and plate assay results demonstrated that the purified RmmLII exhibits degradation activity toward both short- and long-chain AHLs (ranging from C₆ to C₁₄), with particularly high efficiency against long-chain AHLs containing an oxo-group substitution at the C-3 position (Figures 1b,c). RmmLII showed significantly higher degradation efficiency toward C₆-HSL, C₁₀-HSL, C₁₂-HSL, 3-oxo-C₈-HSL, 3-oxo-C₁₀-HSL, 3-oxo-C₁₂-HSL and 3-oxo-C₁₄-HSL compared to C₈-HSL, C₁₄-HSL and 3-oxo-C₆-HSL.

To gain a deeper understanding of the degradation mechanism of RmmLII, the final degradation products from the reaction between RmmLII and the most effective substrate C₁₂-HSL were analyzed using HPLC-MS (Figure 2). The degradation products of C₁₂-HSL by RmmLII were detected in liquid chromatography, showing retention times of 10.48 min and 10.52 min. Mass spectral analysis revealed a prominent excimer (M-H) ion at a mass-to-charge ratio (m/z) of 302.23, indicating that the interaction between RmmLII and C₁₂-HSL (M-H ion m/z of 284.22) resulted in a mass increase of 18. This increase corresponds precisely to the addition of one water molecule. AHL lactonases are known for their ability to selectively hydrolyze the lactone bond of the signaling molecule (Shen et al., 2021), which aligns with the observed degradation mechanism. Therefore, the peak at a retention time of 10.48 min in the LC represents the degradation product C₁₂-HS, confirming RmmLII as an AHL lactonase.

To investigate the impact of several conserved and functional amino acids of RmmLII, four selected amino acids of RmmLII were subjected to site-directed mutagenesis and their enzymatic activities were evaluated following protein purification (Figure 3). The residue Pro82 of RmmLII was replaced by Histidine, and the mutated protein (P82H) showed decreased activity toward C₆-HSL. When the Ala116 was replaced by Glutamic acid, the mutated protein (A116E) exhibited reduced activity against C₁₀-HSL, C₁₄-HSL, and 3-oxo-C₁₄-HSL. Conversely, the mutated protein E198G (Glu198 was replaced by Glycine) demonstrated enhanced degradation of C₁₄-HSL. The E241D mutant (Glu241 was replaced by Aspartic acid) displayed increased activity toward C₁₀-HSL, reduced activity toward C₁₄-HSL, and no significant changes in activity toward other signaling molecules compared to controls. These findings underscore the crucial roles of residues Pro82, Ala116, and Glu241 in RmmLII activity, affecting its degradation capability. The mutation protein (E198G), which enhances C₁₄-HSL degradation, suggests that substituting glutamate at position 198 with Glycine may improve degradation capability. These results further emphasize the critical role of these sites in the degradation of AHL signaling molecules, although the exact mechanism remains unclear and warrants further investigation into the relationship between protein structure and function.

We assessed the influence of various factors, including temperature, pH, EDTA, and metal ions, on RmmLII activity by A136 liquid X-Gal method, in which the β-galactosidase activity is inversely correlated with the activity of RmmLII. After exposure of RmmLII to various temperatures for 30 min, the RmmLII retained stable enzymatic activity between 20°C and 40°C, showing no significant difference compared to the control group (p > 0.05). However, the enzyme activity decreased significantly at 60°C–100°C (p < 0.0001), culminating in nearly complete inactivation at 100°C (Figure 4a). Following the reaction of RmmLII at various temperatures, high activity was observed within the range of 20–100°C, with enzyme activity peaking at 60°C, identified as the optimal reaction temperature (Figure 4b). After RmmLII was treated with EDTA and various metal ions separately, Zn²⁺ and Cu²⁺ significantly inhibited enzyme activity (p < 0.05), while EDTA alone notably reduced RmmLII activity (p < 0.0001), and Ca²⁺ and Mn²⁺ enhanced its activity. When various metal ions were added to EDTA-treated RmmLII, EDTA+Mn²⁺ significantly enhanced RmmLII activity (p < 0.01), indicating that EDTA forms a stable complex with Mn²⁺, thereby mitigating its partial inhibitory effect on the enzyme (Figure 4c). The enzyme activity of RmmLII significantly decreased within the pH range of 3–6 (p < 0.0001), exhibited minimal variation between pH 7–9, and gradually increased at pH > 10, while peak activity was observed at pH 11 (Figure 4d).

The effect of RmmLII on the secretion of PAO1 virulence factors was evaluated in vitro. Compared to the blank control group, the production of pyocyanin in the three experimental groups treated was significantly reduced (p < 0.0001), suggesting that RmmLII at varying concentrations can diminish pyocyanin expression by attenuating the QS system of P. aeruginosa PAO1. Among these, the high dose group treated with RmmLII exhibited the most significant reduction in pyocyanin expression (Figure 5a). Compared to the blank control group, the low dose group RmmLII had no significant effect on the production of extracellular proteases (p > 0.05), whereas the addition of medium and high does group RmmLII significantly inhibited extracellular protease production (p < 0.01) (Figure 5b). Compared to the blank control group, the rhamnolipid production in the three experimental groups showed varying degrees of reduction, with the high dose group demonstrating the most significant decrease, approximately 20% lower than the control group (Figure 5c). Although the decrease is modest, it is evident that the addition of RmmLII has a measurable impact on reducing rhamnolipid production. As the concentration of RmmLII increased, its inhibitory effect on the secretion of these three PAO1 virulence factors also progressively strengthened, showing a positive correlation between the concentration used and the intensity of inhibition.

To evaluate the impact of varying concentrations of RmmLII on PAO1 biofilm formation (Figure 5d), a significant difference (p < 0.0001) was observed across all experimental groups compared to the blank control. RmmLII exhibited a notable inhibitory effect on biofilm formation in P. aeruginosa PAO1, with the most substantial impact observed at the high dose group. The biofilms formed by the different groups were observed by scanning electron microscopy (SEM). In the control group, the bacteria adhered firmly, forming a network-like structure between strains, with the biofilm densely covering the surface and exhibiting low heterogeneity. In the experimental groups treated with different concentrations of RmmLII, the biofilm was disrupted or cleared to varying extents. Particularly, in the high dose group, only a few bacteria remained, and the networked bacterial structure almost completely disappeared, resulting in near-total destruction of the biofilm structure (Figure 5e).

In the motility assay of PAO1, the swimming and swarming motility ranges were measured under the influence of RmmLII, followed by statistical analysis. RmmLII significantly inhibited both swimming and swarming motilities of PAO1 in the MD and HD groups (Figures 5f,g). Compared to the control group, swimming motility in the HD group was nearly completely abolished, demonstrating full inhibition (p < 0.0001). Similarly, swarming motility was markedly reduced, exhibiting a smaller range of activity and a more pronounced inhibitory effect (p < 0.001).

BALB/c mice were administered 100 μL of RmmLII (1 mg/mL) intranasally 12 h before PAO1 infection, and the 24 h survival rate in the PAO1 + RmmLII group was 100%. In the PAO1 infection group, 4 mice succumbed, resulting in 24 h survival rate of 60%. No fatalities were observed in either the blank control group or the RmmLII group (Figure 6a). These results indicate that RmmLII can significantly reduce the mortality rate in mice following PAO1 infection.

Cytokines such as IL-1β, IL-6, and TNF-α play a central role in the body’s inflammatory response. Changes in their levels can accurately reflect the immune status and degree of inflammation of the body during the infection process of P. aeruginosa PAO1 (Chen et al., 2018; Gaspar et al., 2013). Detection of these cytokines can help us deeply understand the mechanism of the inflammatory response caused by infection and evaluate the intervention effect of RmmLII on the inflammatory response. Moreover, these cytokines are related to various diseases and are helpful for exploring potential therapeutic targets and strategies. ELISA results indicated that the cytokine levels of IL-1β, IL-6, and TNF-α in the serum of mice remained relatively stable at 6 h post-infection. After 12 h, the PAO1 model group showed a significant increase in IL-1β concentration (p > 0.0001), with gradual increases in IL-6 and TNF-α levels. At 24 h, the PAO1 model group exhibited markedly higher IL-1β and IL-6 levels (p > 0.0001), and a significant rise in TNF-α concentration (p < 0.05). No significant changes in cytokine levels were observed in the remaining groups (Figures 6b–d). PAO1 colony units from lung tissue homogenates 24 h post-infection were enumerated. No colonies were detected in the blank control and RmmLII groups, while the PAO1 group showed a significantly higher bacterial colony count compared to the PAO1 + RmmLII group. This finding suggests that RmmLII reduces PAO1 load in the murine lungs (Figure 6e), providing additional evidence for its effectiveness in acute lung infection in mice.

Histopathological examination using HE staining revealed that when compared to the control group, the lungs of the PAO1 infection model group exhibited bruising, hemorrhage, and signs of epithelial cell degeneration and necrotic detachment. Erythrocytes and pale pink fluid were observed in the bronchioles, along with inflammatory cell infiltration in the alveolar lumens. In the PAO1 + RmmLII group, the epithelial lining of the terminal bronchioles remained intact, with minimal foreign body presence. However, some bronchiolar epithelial cells showed degeneration, accompanied by mild inflammatory cell infiltration and slight thickening of the alveolar walls. Overall, RmmLII mitigates the pathological changes induced by PAO1 infection in the murine lungs (Figure 6f).