Choi Alexis H K, Slamti Leyla, Avci Fikri Y, Pier Gerald B, Maira-Litrán Tomás
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
J Bacteriol. 2009 Oct;191(19):5953-63. doi: 10.1128/JB.00647-09. Epub 2009 Jul 24.
We found that Acinetobacter baumannii contains a pgaABCD locus that encodes proteins that synthesize cell-associated poly-beta-(1-6)-N-acetylglucosamine (PNAG). Both a mutant with an in-frame deletion of the pga locus (S1Deltapga) and a transcomplemented strain (S1Deltapga-c) of A. baumannii were constructed, and the PNAG production by these strains was compared using an immunoblot assay. Deleting the pga locus resulted in an A. baumannii strain without PNAG, and transcomplementation of the S1Deltapga strain with the pgaABCD genes fully restored the wild-type PNAG phenotype. Heterologous expression of the A. baumannii pga locus in Escherichia coli led to synthesis of significant amounts of PNAG, while no polysaccharide was detected in E. coli cells harboring an empty vector. Nuclear magnetic resonance analysis of the extracellular polysaccharide material isolated from A. baumannii confirmed that it was PNAG, but notably only 60% of the glucosamine amino groups were acetylated. PCR analysis indicated that all 30 clinical A. baumannii isolates examined had the pga genes, and immunoblot assays indicated that 14 of the 30 strains strongly produced PNAG, 14 of the strains moderately to weakly produced PNAG, and 2 strains appeared to not produce PNAG. Deletion of the pga locus led to loss of the strong biofilm phenotype, which was restored by complementation. Confocal laser scanning microscopy studies combined with COMSTAT analysis demonstrated that the biovolume, mean thickness, and maximum thickness of 16-h and 48-h-old biofilms formed by wild-type and pga-complemented A. baumannii strains were significantly greater than the biovolume, mean thickness, and maximum thickness of 16-h and 48-h-old biofilms formed by the S1Deltapga mutant strain. Biofilm-dependent production of PNAG could be an important virulence factor for this emerging pathogen that has few known virulence factors.
我们发现鲍曼不动杆菌含有一个pgaABCD基因座，该基因座编码合成细胞相关的聚-β-(1-6)-N-乙酰葡糖胺（PNAG）的蛋白质。构建了鲍曼不动杆菌的pga基因座框内缺失突变体（S1Δpga）和转互补菌株（S1Δpga-c），并使用免疫印迹法比较了这些菌株的PNAG产生情况。缺失pga基因座导致鲍曼不动杆菌菌株不产生PNAG，用pgaABCD基因对S1Δpga菌株进行转互补完全恢复了野生型PNAG表型。鲍曼不动杆菌pga基因座在大肠杆菌中的异源表达导致合成了大量PNAG，而在携带空载体的大肠杆菌细胞中未检测到多糖。对从鲍曼不动杆菌分离的细胞外多糖物质进行核磁共振分析证实其为PNAG，但值得注意的是，只有60%的葡糖胺氨基被乙酰化。PCR分析表明，所检测的30株临床鲍曼不动杆菌分离株均有pga基因，免疫印迹分析表明，30株菌株中有14株强烈产生PNAG，14株菌株中等至弱产生PNAG，2株菌株似乎不产生PNAG。pga基因座的缺失导致强生物膜表型丧失，通过互补得以恢复。共聚焦激光扫描显微镜研究结合COMSTAT分析表明，野生型和pga互补的鲍曼不动杆菌菌株形成的16小时和48小时龄生物膜的生物体积、平均厚度和最大厚度显著大于S1Δpga突变菌株形成的16小时和- 48小时龄生物膜的生物体积、平均厚度和最大厚度。依赖生物膜产生PNAG可能是这种已知毒力因子较少的新出现病原体的一个重要毒力因子。
