Li Ying, Cao Sanjie, Zhang Luhua, Yuan Jianlin, Lau Gee W, Wen Yiping, Wu Rui, Zhao Qin, Huang Xiaobo, Yan Qigui, Huang Yong, Wen Xintian
Research Center of Swine Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China.
Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
PLoS One. 2016 Sep 28;11(9):e0163364. doi: 10.1371/journal.pone.0163364. eCollection 2016.
Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a major cause of economic loss in swine industry worldwide. TolC, the key component of multidrug efflux pumps and type I secretion systems, has been well-studied as an exit duct for numerous substances in many Gram-negative bacteria. By contrast, little is known on the role of TolC in biofilm formation. In this study, a ΔtolC mutant was used to examine the importance of TolC in biofilm formation of A. pleuropneumoniae. Surface attachment assays demonstrated the essential role of TolC in initial attachment of biofilm cells. The loss of TolC function altered surface hydrophobicity, and resulted in greatly reduced autoaggregation in ΔtolC. Using both enzymatic treatments and confocal microscopy, biofilm composition and architecture were characterized. When compared against the wild-type strain, the poly-β-1, 6-N-acetyl-D-glucosamine (PGA), an important biofilm matrix component of A. pleuropneumoniae, was significantly reduced at the initial attachment stage in ΔtolC. These results were confirmed by mRNA level using quantitative RT-PCR. Additionally, defective secretion systems in ΔtolC may also contribute to the deficiency in biofilm formation. Taken together, the current study demonstrated the importance of TolC in the initial biofilm formation stage in A. pleuropneumoniae. These findings could have important clinical implications in developing new treatments against biofilm-related infections by A. pleuropneumoniae.
胸膜肺炎放线杆菌是猪传染性胸膜肺炎的病原体，是全球养猪业经济损失的主要原因。TolC是多药外排泵和I型分泌系统的关键组成部分，在许多革兰氏阴性细菌中作为多种物质的输出通道已得到充分研究。相比之下，关于TolC在生物膜形成中的作用知之甚少。在本研究中，使用ΔtolC突变体来研究TolC在胸膜肺炎放线杆菌生物膜形成中的重要性。表面附着试验证明了TolC在生物膜细胞初始附着中的重要作用。TolC功能的丧失改变了表面疏水性，并导致ΔtolC中的自聚集大大减少。使用酶处理和共聚焦显微镜对生物膜的组成和结构进行了表征。与野生型菌株相比，胸膜肺炎放线杆菌重要的生物膜基质成分多聚-β-1,6-N-乙酰-D-葡萄糖胺（PGA）在ΔtolC的初始附着阶段显著减少。这些结果通过定量RT-PCR的mRNA水平得到证实。此外，ΔtolC中分泌系统的缺陷也可能导致生物膜形成的不足。综上所述，本研究证明了TolC在胸膜肺炎放线杆菌生物膜形成初始阶段的重要性。这些发现可能对开发针对胸膜肺炎放线杆菌生物膜相关感染的新治疗方法具有重要的临床意义。
