The Pseudomonas aeruginosa effector protein TesG regulates alternative activation of macrophages through NLRC5

Abstract

Pseudomonas aeruginosa is one of the most common pathogenic bacteria in the clinic. Its large genome and strong genetic plasticity enable it to survive in various environments, posing a significant threat to patient health. We previously identified a key effector protein, TesG, secreted by the type I secretion system, which plays a crucial role in the chronic infection process of P. aeruginosa. However, the underlying mechanism remains incompletely understood. In this study, we newly discovered that TesG can induce alternative activation of macrophages and explored its mechanisms through a series of in vivo and in vitro experiments. We found that TesG promotes the expression of the NLRC5 protein, thereby inducing the polarization of macrophages toward the M2 phenotype. The activation of macrophages induced by TesG primarily occurs through the currently known mechanisms of NLRC5. These findings suggest that P. aeruginosa can regulate the alternative activation of macrophages through the TesG/NLRC5 signaling pathway during chronic infection, significantly aiding its evasion of the host immune system killing. In summary, our data highlight the complex infection strategies developed by pathogenic bacteria to achieve chronic infection.IMPORTANCEDuring the transition from acute to chronic Pseudomonas aeruginosa infection, bacteria modulate the host's immune microenvironment to evade immune responses, ensuring long-term survival. Clinical studies have confirmed that the effector protein TesG (secreted by a type I secretion system) can serve as a potential clinical biomarker for chronic P. aeruginosa lung infections. Our findings indicate that TesG promotes the alternative activation of macrophages through the regulation of NLRC5, thereby suppressing inflammatory responses and facilitating the progression of chronic pulmonary infections. These discoveries enhance our understanding of the complex interplay between P. aeruginosa and the host, laying the groundwork for developing precise diagnostic and therapeutic strategies targeting chronic pulmonary infections.

Keywords: NLRC5; TesG; infection immunity; macrophage polarization.

Fig 1

TesG induces the polarization of macrophages in vitro. iBMDMs were incubated overnight with purified TesG protein (0–15 μg/mL) or controls. (A) Representative phase-contrast images. Arrows indicate cells with M2-like morphology. M2 positive control: IL-4/IL-13 (20 ng/mL each); heat-inactivated TesG control: 10 µg/mL. Scale bar, 100 µm. (B) Flow cytometric assessment of M2 polarization. Overlay histogram shows CD206 expression profiles in iBMDMs, and the bar chart quantifies the proportion of CD206⁺ cells (n = 4–6). (C) The mRNA expression of macrophage polarization-related genes in iBMDMs treated overnight with TesG (10 µg/mL) or PBS. Data points represent biological replicates within individual experiments; bar graphs show mean ± SEM. Similar results were observed in three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; and ns, not significant (B: one-way ANOVA with Tukey’s multiple comparison test; C: two-tailed unpaired Student’s t test).

Fig 2

TesG induces the polarization of macrophages in vivo. (A) Constructed mouse model of chronic lung infection. Chronic lung infections were modeled by tracheal injection of 50 µL of agarose bead-encapsulated wild-type PAO1 (1 × 10⁶ CFU) or ∆tesG mutant strains. Mouse lung tissues were collected at 7 and 14 days post-treatment, and single-cell suspensions were prepared for macrophage detection by flow cytometry. (B and C) Polarization of AM and IM at 7 days post-infection. (D and E) Macrophage polarization profiling at 14 days post-infection. Data are from one representative experiment with five to seven mice per group; points represent individual mice, bars show mean ± SEM. Similar results were observed in two independent experiments. Unpaired t-test: *P < 0.05; **P < 0.01.

Fig 3

TesG induces the polarization of macrophages via NLRC5. (A) The mRNA expression level of Nlrc5 was upregulated in iBMDMs following overnight treatment with 10 µg/mL TesG protein. (B) Western blot analysis demonstrates that TesG progressively induced NLRC5 protein expression. Protein band intensities and mRNA relative expression levels were quantified as the ratio of the indicated protein or mRNA to GAPDH. These values were then normalized to the average expression levels in the PBS-treated or TesG 0 h-treated group, which were set to 1.00. (C) Immunofluorescence staining shows TesG-induced NLRC5 (red) expression in iBMDMs. Membranes were stained with DiO (green) and nuclei with DAPI (blue). Scale bar: 10 µm. (D) Interference efficiency of Nlrc5 siRNA in iBMDMs. (E) Flow cytometry analysis of macrophage polarization following TesG treatment in Nlrc5-knockdown iBMDMs. All experiments included three to five biological replicates; all data are presented as the mean ± SEM. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; and ns, not significant (A and D: two-tailed unpaired Student’s t test; E: two-way ANOVA with Tukey’s multiple comparison test).

Fig 4

TesG regulates the polarization of macrophages through known functions of NLRC5. (A) iBMDMs were treated overnight with either TesG protein (10 µg/mL) alone or in combination with NLRC5 pathway inhibitors (5 µM Ac-YVAD-cmk, caspase-1 inhibitor; 1 µM peficitinib, JAK inhibitor; and 0.1 µM IKK16, IKK-2 inhibitor), and macrophage polarization was analyzed by flow cytometry. Data are presented as mean ± SEM of three to four biological replicates, with statistical significance determined by one-way ANOVA with Tukey’s multiple comparison test: *P < 0.05; **P < 0.01; ***P < 0.001; and ns, not significant.

Fig 5

TesG/NLRC5 signaling regulates the polarization of macrophages in vivo. Construction of chronic infection models with PAO1 and ∆tesG strains in both C57BL/6 WT and Nlrc5 knockout mice. (A) Alveolar macrophage polarization at 7 days post-infection. (B) Interstitial macrophage polarization at 7 days post-infection. Data represent mean ± SEM from one representative experiment with four to seven mice per group; points show individual mice. Similar results were observed in two independent experiments. Two-way ANOVA with Tukey’s multiple comparison test: *P < 0.05; **P < 0.01; ***P < 0.001; and ns, not significant.