Hammerschmidt Sven, Wolff Sonja, Hocke Andreas, Rosseau Simone, Müller Ellruth, Rohde Manfred
Research Center for Infectious Diseases, University of Würzburg, Röntgenring 11, 97070 Würzburg, Germany.
Infect Immun. 2005 Aug;73(8):4653-67. doi: 10.1128/IAI.73.8.4653-4667.2005.
The capsular polysaccharide of Streptococcus pneumoniae represents an important virulence factor and protects against phagocytosis. In this study the amount of capsular polysaccharide present on the bacterial surface during the infection process was illustrated by electron microscopic studies. After infection of A549 cells (type II pneumocytes) and HEp-2 epithelial cells a modified fixation method was used that allowed visualization of the state of capsule expression. This modified fixation procedure did not require the use of capsule-specific antibodies. Visualization of pneumococci in intimate contact and invading cells demonstrated that pneumococci were devoid of capsular polysaccharide. Pneumococci not in contact with the cells did not show alterations in capsular polysaccharide. After infection of the cells, invasive pneumococci of different strains and serotypes were recovered. Single colonies of these recovered pneumococci exhibited an up-to-10(5)-fold-enhanced capacity to adhere and an up-to-10(4)-fold-enhanced capacity to invade epithelial cells. Electron microscopic studies using a lysine-ruthenium red (LRR) fixation procedure or cryo-field emission scanning electron microscopy revealed a reduction in capsular material, as determined in detail for a serotype 3 pneumococcal strain. The amount of polysaccharide in the serotype 3 capsule was also determined after intranasal infection of mice. This study illustrates for the first time the phenotypic variation of the polysaccharide capsule in the initial phase of pneumococcal infections. The modified LRR fixation allowed monitoring of the state of capsule expression of pathogens during the infectious process.
肺炎链球菌的荚膜多糖是一种重要的毒力因子，可抵御吞噬作用。在本研究中，通过电子显微镜研究阐明了感染过程中细菌表面存在的荚膜多糖的量。在感染A549细胞（II型肺细胞）和HEp-2上皮细胞后，使用了一种改良的固定方法，该方法能够观察到荚膜表达状态。这种改良的固定程序不需要使用荚膜特异性抗体。对紧密接触和侵入细胞的肺炎球菌的观察表明，肺炎球菌没有荚膜多糖。未与细胞接触的肺炎球菌在荚膜多糖方面没有显示出变化。细胞感染后，回收了不同菌株和血清型的侵袭性肺炎球菌。这些回收的肺炎球菌的单个菌落表现出高达10⁵倍的粘附能力增强和高达10⁴倍的侵袭上皮细胞能力增强。使用赖氨酸-钌红（LRR）固定程序或低温场发射扫描电子显微镜进行的电子显微镜研究显示，荚膜物质减少，这在对3型肺炎球菌菌株的详细测定中得到证实。在小鼠鼻内感染后也测定了3型荚膜中的多糖量。本研究首次阐明了肺炎球菌感染初始阶段多糖荚膜的表型变异。改良的LRR固定方法能够在感染过程中监测病原体的荚膜表达状态。
