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

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

Abstract

Abstract:

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

Changwen Zhang, Shaoqing Lin, Minjie Lv, Xiao Jin, Changjun Zhu, Ganzhu Feng. Inhibition of autophagy of macrophages by secreted protein Pec1 of Pseudomonas aeruginosa and its effect on clearance of Pseudomonas aeruginosa[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2022, 16(06): 370-376.

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References 25

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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

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

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