Fig.1 Schematic illustration of intracellular persister-mediated immune suppression and TESAN-mediated persister clearance in macrophages. (a) The key components of TESAN. (b) Schematic illustration of orthogonal Screening. (i) Orthogonal array. (ii) Multiparametric evaluation including cell viability, antimicrobial activity, Zeta potential, critical micelle concentration (CMC), and morphological transformation. (c) Stimuli-responsive assembly of TESAN. (d) Intracellular persisters continuously secrete effector proteins that block the fusion of autophagosomes with lysosomes, thereby suppressing host immune function in macrophages (left). TESAN remains biologically inert during intracellular trafficking via charge shielding, undergoes gradual protonation in acidic lysosomes to enable mild lysosomal escape, and is subsequently activated by LasB to release antimicrobial components. This activation further induces in situ self-assembly into a nanofiber network on the bacterial surface, which restricts effector secretion and diffusion, thereby restoring host immune function (right).#

Fig.2 Molecular design, screening, and characterization of TESAN. (a) Chemical structures of Peptide 1 and Peptide 2 with orthogonal screening matrix (C1–C9) varying F (_F_1–_F_3), E (_E_2, _E_4, _E_6), and R (_R_0–_R_2). (b) Cell viability of C1–C9 in RAW264.7 at 64 μM and 256 μM. (c) MIC values of C1–C9 against P. aeruginosa 27853 and PAO1. (d) Zeta potential of C1–C9 under pH 7.4, pH 5.0, and pH 7.4 + LasB conditions. (e) CMC of C1–C9 before and after LasB treatment. (f) Representative TEM images of C1–C9 before and after LasB treatment. Scale bar: 200 nm. (g) Radar plots evaluating cell compatibility (b), antibacterial activity (c), charge modulation (d), enzyme-activated self-assembly (e), and morphological transformation (f). (h) CMC value of TSAN ± LasB, ESAN ± LasB, and TESAN ± LasB. (i) Secondary structure distribution of TSAN ± LasB, ESAN ± LasB, and TESAN ± LasB by CD spectroscopy.#

在体外抗菌活性测试中，如图3所示，TESAN能够通过OprF结合基团快速识别铜绿假单胞菌。在细菌分泌的LasB酶作用下，TESAN在细菌表面原位组装，迅速诱导细菌细胞膜发生生理性刚化、去极化以及通透性改变。透射电镜与扫描电镜观察证实，成型的纳米纤维网络严密包裹细菌，直接导致细菌外膜破裂和胞浆渗漏，展现出强效的直接捕获与杀灭能力。

Fig.3 Extracellular antibacterial activity of TESAN. (a) Time-dependent OD600 of P. aeruginosa treated with TESAN at 8–64 μM. (b) Laurdan GP of P. aeruginosa treated with TESAN at 8–64 μM for 2 h. (c) Time-dependent DiSC3(5) fluorescence of P. aeruginosa treated with TESAN at 8–64 μM. (d) Representative confocal fluorescence microscopy images of AO/PI-stained P. aeruginosa treated with TESAN at 8–64 μM for 2 h. Green, AO; red, PI. Scale bar: 20 μm. (e) Time-dependent OD600 of P. aeruginosa treated with PBS, TSAN, ESAN, or TESAN (32 μM). (f) Laurdan GP of P. aeruginosa treated with PBS, TSAN, ESAN, or TESAN (32 μM) for 2 h. (g) Time-dependent DiSC3(5) fluorescence of P. aeruginosa treated with PBS, TSAN, ESAN, or TESAN (32 μM). (h) Representative confocal fluorescence microscopy images of AO/PI-stained P. aeruginosa treated with PBS, TSAN, ESAN, or TESAN (32 μM) for 2 h. Scale bar: 20 μm. (i) Representative TEM images of P. aeruginosa treated with PBS, TSAN, ESAN, or TESAN (32 μM) for 2 h. Scale bar: 200 nm. (j) Representative SEM images of P. aeruginosa treated with PBS, TSAN, ESAN, or TESAN (32 μM) for 2 h. Scale bar: 1 μm (upper row); 200 nm (lower row). n = 3 independent experiments. Data are presented as mean ± s.d. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.#

细胞水平的研究揭示了TESAN克服胞内感染与修复免疫的双重机制，如图4所示。TESAN通过甘露糖受体介导的内吞作用进入感染巨噬细胞，并迅速完成溶酶体逃逸进入细胞质。在细胞质中，TESAN精准定位胞内残留菌并被LasB酶激活，成功实现了对巨噬细胞内残留菌99.98%的清除率。更为关键的是，原位生成的纳米纤维网络在细菌表面构建了物理屏障，使细菌效应蛋白ExoS的表达和分泌降至基线水平。这一空间阻断效应成功解除对宿主自噬系统的抑制，显著促进了LC3阳性自噬体与LAMP1阳性溶酶体的融合，彻底恢复了巨噬细胞内源性的自噬清除功能。

Fig.4 Intracellular antibacterial activity and immune restoration of TESAN. (a) Schematic illustration of the experimental workflow for evaluating intracellular bacterial clearance in a RAW264.7 macrophage infection model. (b) Representative time-dependent confocal fluorescence microscopy images of Lyso-Tracker and intracellular Cy5.5-labeled TESAN in infected RAW264.7 macrophages after TESAN (32 μM) incubation for 0.5–5 h (left), with corresponding quantification of their colocalization (right). Green, Lyso-tracker; magenta, TESAN; blue, Hoechst. Scale bar: 10 μm. n = 6 independent experiments. (c) Representative super-resolution images of intracellular AO-labeled P. aeruginosa and Cy5.5-labeled TESAN (32 μM) in RAW264.7 macrophages. Green, P. aeruginosa; magenta, TESAN; blue, Hoechst. Scale bar: 2 μm. (d) Representative colony images and quantification of intracellular P. aeruginosa in infected RAW264.7 macrophages under different treatments (32 μM) for 6 h. (e) Representative bio-TEM images of intracellular P. aeruginosa in infected RAW264.7 macrophages after TESAN (32 μM) incubation for 0, 4, and 6 h. Scale bar: 500 nm. (f) Western blot analysis of ExoS protein expression in infected RAW264.7 macrophages under different treatments for 6 h. (g) Quantification of ExoS protein levels normalized to GAPDH in (f). (h) Quantitative real-time PCR (qRT-PCR) analysis of ExoS mRNA expression in infected RAW264.7 macrophages under different treatments. (i) Representative super-resolution images of intracellular AO-labeled P. aeruginosa with LC3 and LAMP1 in infected RAW264.7 macrophages under different treatments. Green, P. aeruginosa; red, LAMP1; magenta, LC3; blue, Hoechst. Scale bar: 2 μm. (j) Quantification of colocalization among AO-labeled P. aeruginosa, LC3, and LAMP1 in (i). (k) Fluorescence intensity line profiles of P. aeruginosa, LC3, and LAMP1 across the TESAN-treated cells shown in (i). n = 3 independent experiments. Data are presented as mean ± s.d. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. ns, not significant; **p < 0.01; ***p < 0.001; and ****p < 0.0001.#

在小鼠肺部急性感染模型中，TESAN展现出卓越的体内治疗效果和生物安全性，如图5所示。气管给药后，TESAN能够特异性聚集于感染肺部，并持续驻留超过72小时。治疗结果显示，TESAN使小鼠肺部的细菌载量降低了4.64个数量级（即减少超过99.99%），显著减轻了肺组织炎症损伤。同时，TESAN有效调控了肺部免疫微环境，促进促炎型M1巨噬细胞向抗炎型M2巨噬细胞转变，并恢复了免疫细胞平衡。最终，TESAN将感染小鼠的7天生存率从对照组的10%大幅提升至70%。

Fig.5 In vivo efficacy of TESAN in a pulmonary infection model. (a) Schematic illustration of P. aeruginosa-induced pulmonary infection model in female BALB/c mice. Intratracheal instillation of TESAN, TSAN, ESAN at a dose of 1 mg/kg (50 μL per mouse). (b) Representative near-infrared fluorescence (NIRF) images of mice at different time points (0–96 h) after intratracheal instillation of different treatments (Ex/Em = 675/693 nm). (c) Ex vivo NIRF images of major organs (H, heart; Li, liver; S, spleen; Lu, lung; K, kidney) collected at 48 h postadministration of different treatments. (d) Quantification of P. aeruginosa CFU in excised lung tissues on day 5 postinfection. (e) Representative H&E-stained lung sections from mice treated with different treatments on day 5 postinfection. Scale bar: 50 μm (upper row); 1 mm (lower row). (f) Representative immunofluorescence images (left) and flow cytometry plots (right) of lung tissues from mice with different treatments on day 5 postinfection. Green, CD206; red, CD86; blue, DAPI. Scale bar: 50 μm. (g–i) Levels of IL-6 (g), TNF-α (h), and IL-10 (i) in infected lung tissues on day 5 postinfection, measured by ELISA. (j) Representative flow cytometry plots showing CD8+ T cells (CD3+CD8+) and CD4+ T cells (CD3+CD4+) in lung tissues on day 5 postinfection. (k) Kaplan–Meier survival curves for mice from different treatment groups. n = 3 independent mice for (b–j) and n = 10 independent mice for (k). Data are presented as mean ± s.d. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.#

为评估临床转化潜力，研究团队进一步利用小鼠及肺炎患者肺泡灌洗液（BALF）提取的肺泡巨噬细胞进行了验证，如图6所示。超分辨率显微成像清晰显示，TESAN能够精准识别患者来源巨噬细胞内部的铜绿假单胞菌。在患者临床样本中，TESAN同样实现了高达99.91%的胞内细菌消除率，证实了该策略在真实临床复杂微环境中的有效性与通用性。

Fig.6 Identification and clearance of intracellular P. aeruginosa in BALF samples from mice and patients. (a) Schematic illustration of the experimental workflow for evaluating intracellular bacterial identification and clearance using alveolar macrophages isolated from P. aeruginosa-infected mice and patient-derived BALF samples. (b, c) Representative super-resolution images of intracellular P. aeruginosa and TESAN (32 μM) in alveolar macrophages isolated from BALF of P. aeruginosa-infected mice (b) and patients (c), with corresponding fluorescence intensity line profiles. Red: TESAN; Green: CD68; blue: Hoechst staining for cell nuclei and bacteria. Scale bar: 5 μm. (d, e) Quantification of intracellular P. aeruginosa CFU in alveolar macrophages isolated from BALF of infected mice (d) and patients (e) before and after TESAN treatment (32 μM). (f) Representative colony images corresponding to (d) and (e). n = 7 biologically independent samples, each measured in three independent experiments. Data are presented as mean ± s.d. Statistical significance was determined by two-tailed paired t test comparing control vs TESAN-treated groups. *p < 0.05; ****p < 0.0001.#