蹭行运动和胞外藻酸盐在铜绿假单胞菌生物被膜形成中的作用

The Role of Twitching Motility Mediated by Pili and Extracellular Alginate in Pseudomonas aeruginosa Biofilms Development

目的比较3株铜绿假单胞菌即含新的mucA基因突变的黏液型PA17、mucA基因经典突变的黏液型PDO300、含野生型mucA基因的非黏液型PAO1生物被膜早期形成和成熟形态,探讨铜绿假单胞菌纤毛介导的蹭行运动和胞外基质藻酸盐在黏液型及非黏液型铜绿假单胞菌生物被膜形成中的作用。方法用PA17、PAO1、PDO300的单菌落穿刺接种1%琼脂糖平板,肉眼观察细菌纤毛介导的蹭行运动,测定细菌运动环评估3株细菌的蹭行运动能力;在生物被膜形成早期采用结晶紫对膜内黏附细菌染色,测定其吸光度(A)值,建立24 h内3株细菌的黏附曲线;建立未饱和铜绿假单胞菌生物被膜模型,检测早期和成熟期的胞外藻酸盐分泌;将含绿色荧光蛋白的pUCP/UV质粒转化3株铜绿假单胞菌,在激光共聚焦显微镜下观察静态生物被膜不同时期的形态和结构。结果黏液型PA17、非黏液型PAO1菌株蹭行运动的直径小于黏液型PDO300(P<0.05),PA17、PAO1的运动能力弱于PDO300菌株;黏液型PDO300在早期生物被膜形成中黏附能力最强,非黏液型PAO1次之,黏液型PA17最弱;在未饱和生物被膜早期和成熟期黏液型菌株PA17、PDO300藻酸盐合成均明显高于非黏液型菌株PAO1(P<0.05),而PA17和PDO300在生物被膜相同时期的藻酸盐合成无明显差异(P>0.05);激光共聚焦显微镜下PA17菌株在生物被膜形成中,不可逆黏附形成明显迟于非黏液型PAO1和黏液型PDO300,PA17成熟生物被膜形态呈薄膜状,与非黏液型PAO1成熟生物被膜类似;而PDO300不可逆黏附形成早,成熟生物被膜形态呈蘑菇状。结论纤毛介导的蹭行运动是早期生物被膜形成中黏附的重要方式,可能影响成熟生物被膜的形态;而藻酸盐不是早期黏附的主要决定因素,与成熟生物被膜形态无关。

Objective To compare the development and feature of three strains of Pseudomonas aeruginosa containing mucus-type PA17 with the new mucA gene mutation,mucinous PDO300 with the classic mucA gene mutation and non-mucoid PAO1 with wild-type mucA gene,and explore the role of twitching motility mediated by pili and extracellular alginate in Pseudomonas aeruginosa biofilms development.Methods Single colony of PA17,PAO1,PDO300 was inoculated separately in 1% agarose plates,twitch motility mediated by bacterial pili was observed under the naked eye,and the zone of twitch motility was measured to assess the movement of bacteria.The adhesion of bacterium in initial biofilms was detected by crystal violet staining and expressed as absorbance（A）value,and the adhesion curve within 24 h was set up.The alginate was determined in unsaturated biofilms model.pUCP/UV plasmid containing green fluorescent protein was transfected into Pseudomonas aeruginosa,and at the same time confocal laser scanning micro多scopy was used to observe biofilms formation in static state.The results were statistically analyzed by SPSS12.0.Results The diameters of the zone in mucin-type PA17 and non-mucoid strain PAO1 were less than those of mucinous type PDO300（P〈0.05）,suggesting the ability to twitch motility by strains PA17 and PAO1 was less than PDO300.PDO300 irreversibly adhered to the abotic surface at first,PAO1 secondly,and PA17 at last.The alginate product of mucoid PA17,PDO300 in unsaturated biofilms was significantly increased as compared with that of nonmucoid PAO1（P〈0.05）,but there was no significant difference between mucoid PA17 and PDO300（P〈0.05）.Under the confocal laser scanning microscopy,the formation of irreversible adhesion by mucus-type PA17 was significantly later than non-mucinous-type PAO1 and mucoid PDO300.PA17 slowly shaped into the flat-film features in the mature biofilms,which was similar to PAO1,while mucus-type PDO300 shaped irreversible adhesion at first,and the features of PDO300 biofilms were like as the mushroom.Conclusion Twitching motility plays an important role in early adhesion of biofilms formation and possibly impacts on mature biofilms architecture.The alginate is not a key factor in early adhesion and not related with biofilms feature.