铜绿假单胞菌分泌蛋白Pec1抑制巨噬细胞自噬及影响铜绿假单胞菌清除效应初步观察

doi:10.3877/cma.j.issn.1674-1358.2022.06.003

目的

观察铜绿假单胞菌（PA）分泌蛋白Pec1通过抑制小鼠肺泡巨噬细胞（MH-S）自噬而影响其对铜绿假单胞菌清除效应。

方法

体外予Pec1蛋白、PAO1灭活菌、PAO1灭活菌+ Pec1蛋白、PAO1活菌、Pec1蛋白敲除活菌（PAO1Δpec1）与小鼠肺泡巨噬细胞（MH-S）共培养，实时定量逆转录聚合酶链反应（qRT-PCR）检测自噬标志物微管相关蛋白轻链3（LC3）的mRNA表达水平；Western blot检测自噬标志物LC3蛋白表达水平；透射电镜下观察MH-S细胞自噬小体的形成；通过细菌探针荧光原位杂交技术（FISH）联合激光扫描共聚焦显微镜检测MH-S细胞胞内活菌、MH-S细胞破壁后菌落计数，计算MH-S细胞胞内杀菌效率。LC3的mRNA及蛋白表达水平、透射电镜下MH-S细胞自噬小体的数量、MH-S细胞胞内杀菌效率呈正态分布，组间比较采用两独立样本t检验。

结果

Pec1蛋白组、PAO1灭活菌+ Pec1蛋白组和PAO1活菌组自噬标志物LC3的mRNA表达水平分别为（0.624 ± 0.071）、（0.614 ± 0.069）和（0.752 ± 0.098），分别低于对照组（1.071 ± 0.147）（t = 2.723、P = 0.044）、PAO1灭活菌组（1.098 ± 0.144）（t = 2.950、P = 0.028）和Pec1蛋白敲除活菌组（1.214 ± 0.229）（t = 2.816、P = 0.037），差异均有统计学意义；Pec1蛋白组、PAO1灭活菌+ Pec1蛋白组和PAO1活菌组蛋白LC3-Ⅱ/ LC3-Ⅰ比值分别为（0.396 ± 0.063）、（0.384 ± 0.070）和（0.771 ± 0.080），分别低于对照组（1.000 ± 0.043）（t = 8.130、P < 0.001）、PAO1灭活菌组（0.947 ± 0.055）（t = 7.573、P < 0.001）和Pec1蛋白敲除活菌组（1.080 ± 0.185）（t = 4.166、P = 0.002），差异均有统计学意义；Pec1蛋白组、PAO1灭活菌+ Pec1蛋白组和PAO1活菌组自噬小体数量分别为（1.330 ± 0.577）、（1.670 ± 0.577）和（1.330 ± 0.577），分别低于对照组（4.330 ± 0.577）（t = 5.692、P < 0.001）、PAO1灭活菌组（4.670 ± 0.577）（t = 5.692、P < 0.001）和Pec1蛋白敲除活菌组（4.000 ± 1.000）（t = 5.060、P < 0.001），差异均有统计学意义。通过细菌探针FISH联合激光扫描共聚焦显微镜检测MH-S胞内活菌，计算出MH-S细胞对PAO1及PAO1Δpec1的胞内杀菌效率分别为（39.3 ± 3.4）%和（82.2 ± 1.3）%，两组差异具有统计学意义（t = 4.908、P = 0.005）；通过菌落计数计算出MH-S细胞对PAO1及PAO1Δpec1的胞内杀菌效率分别为（18.4 ± 4.1）%和（42.2 ± 1.4）%，差异具有统计学意义（t = 8.576、P < 0.001）。

结论

Pec1可能通过抑制MH-S细胞自噬削弱对铜绿假单胞菌清除。

关键词: 铜绿假单胞菌, Pec1, 肺泡巨噬细胞, 自噬

Objective

To investigate the effect of secreted protein Pec1 of Pseudomonas aeruginosa on the clearance of Pseudomonas aeruginosa by inhibiting the autophagy of mouse alveolar macrophages (MH-S).

Methods

Pec1, inactivated bacteria of PAO1, inactivated bacteria of PAO1 + Pec1, viable bacteria of PAO1, Pec1 protein knockout viable bacteria of PA (PAO1ΔPec1) and mouse alveolar macrophages (MH-S) were co-cultured in vitro. The mRNA expression level of autophagy marker microtubule-associated protein light chain 3 (LC3) was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The levels of autophagy marker LC3 protein was detected by Western blot. The formation of autophagosomes in MH-S cells was observed by transmission electron microscopy. The count of MH-S intracellular viable bacteria was detected by microbial fluorescence in situ hybridization (FISH) combined with confocal laser scanning microscope and the intracellular bacterial load was evaluated by colony-forming units (CFU) assay, and the intracellular bactericidal efficiency of MH-S cells was calculated. The mRNA and protein expression levels of LC3, the number of autophagosomes in MH-S cells under transmission electron microscope, and the intracellular bactericidal efficiency of MH-S cells were normally distributed, and the comparison between groups was performed by two independent samples t-test.

Results

The mRNA expression levels of autophagy marker LC3 in Pec1 protein group, inactivated bacteria of PAO1 + Pec1 protein group and viable bacteria of PAO1 group were (0.624 ± 0.071), (0.614 ± 0.069) and (0.752 ± 0.098), respectively, which were significantly lower than those of control group (1.071 ± 0.147) (t = 2.723, P = 0.044), inactivated bacteria of PAO1 group (1.098 ± 0.144) (t = 2.950, P = 0.028) and Pec1 protein knockout viable bacteria group (1.214 ± 0.229) (t = 2.816, P = 0.037), with significant differences. The LC3-Ⅱ/LC3-Ⅰratios of Pec1 protein group, inactivated bacteria of PAO1 + Pec1 protein group and live bacteria of PAO1 group were (0.396 ± 0.063), (0.384 ± 0.070) and (0.771 ± 0.080), respectively, which were significantly lower than those of the control group (1.000 ± 0.043) (t = 8.130, P < 0.001), inactivated bacteria of PAO1 group (0.947 ± 0.055) (t = 7.573, P < 0.001) and Pec1 protein knockout viable bacteria group (1.080 ± 0.185) (t = 4.166, P = 0.002), with significant differences. The number of autophagosomes in Pec1 protein group, inactivated bacteria of PAO1 +Pec1 protein group and live bacteria of PAO1 group were (1.330 ± 0.577), (1.670 ± 0.577) and (1.330 ± 0.577), respectively, which were significantly lower than those of control group (4.330 ± 0.577) (t = 5.692, P < 0.001), inactivated bacteria of PAO1 group (4.670 ± 0.577) (t = 5.692, P < 0.001) and Pec1 protein knockout viable group (4.000 ± 1.000) (t = 5.060, P < 0.001), with significant differences. The intracellular viability of MH-S cells was detected by bacterial probe FISH combined with confocal laser scanning microscope. The intracellular bactericidal efficiency of MH-S cells against PAO1 and PAO1ΔPec1 were calculated (39.3 ± 3.4)% and (82.2 ± 1.3)%, with significant difference (t = 4.908, P = 0.005). By the colony-forming units (CFU) assay, the intracellular bactericidal efficiency of MH-S cells against PAO1 and PAO1ΔPec1 was calculated as (18.4 ± 4.1)% and (42.2 ± 1.4)%, respectively, with significant difference (t = 8.576, P < 0.001).

Conclusion

Pec1 may inhibit the clearance of Pseudomonas aeruginosa by inhibiting autophagy in MH-S cells.

Key words: Pseudomonas aeruginosa, Pec1, Alveolar macrophage, Autophagy

图1 Pec1蛋白刺激对MH-S细胞LC3蛋白及mRNA表达水平的影响注：A、B分别为Western blot及其半定量比较；C：各组LC3 mRNA相对表达水平

表1 TEM单个视野下（× 15 000）各组MH-S细胞自噬小体的数量

研究组	对照组	t值	P值
1.330 ± 0.577（Pec1组）	4.330 ± 0.577 （空白组）	5.692	< 0.001
1.670 ± 0.577（PAO1灭活菌+ Pec1组）	4.670 ± 0.577（PAO1灭活菌组）	5.692	< 0.001
1.330 ± 0.577（PAO1活菌组）	4.000 ± 1.000（PAO1Δpec1组）	5.060	< 0.001

表1 TEM单个视野下（× 15 000）各组MH-S细胞自噬小体的数量

研究组	对照组	t值	P值
1.330 ± 0.577（Pec1组）	4.330 ± 0.577 （空白组）	5.692	< 0.001
1.670 ± 0.577（PAO1灭活菌+ Pec1组）	4.670 ± 0.577（PAO1灭活菌组）	5.692	< 0.001
1.330 ± 0.577（PAO1活菌组）	4.000 ± 1.000（PAO1Δpec1组）	5.060	< 0.001

图2 TEM下观察各组MH-S细胞自噬小体形成注：白色箭头示MH-S细胞内自噬小体（× 15 000）

图3 Pec1蛋白对MH-S细胞清除PA的影响注：A、B分别为PAO1活菌及PAO1Δpec1感染MH-S细胞1 h及2 h破壁裂解后的菌落计数、相应胞内杀菌效率；C、D为CLSM下MH-S细胞吞入胞内的PAO1活菌及PAO1Δpec1情况（× 600），DAPI（蓝）染细胞核，CY3（红）染PAO1及PAO1Δpec1；E为根据CLSM观察结果计算的胞内杀菌效率

图4 自噬激动剂Rap促进MH-S细胞对PA的清除注：A、B为两种不同处理方式后的菌落计数及胞内杀菌效率

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Inhibition of autophagy of macrophages by secreted protein Pec1 of Pseudomonas aeruginosa and its effect on clearance of Pseudomonas aeruginosa

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Inhibition of autophagy of macrophages by secreted protein Pec1 of Pseudomonas aeruginosa and its effect on clearance of Pseudomonas aeruginosa