United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA.
Microbiology (Reading). 2010 May;156(Pt 5):1459-1467. doi: 10.1099/mic.0.035857-0. Epub 2010 Jan 28.
Capsule depolymerase (CapD) is a gamma-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence.
荚膜分解酶 (CapD) 是一种γ-谷氨酰转肽酶，也是炭疽杆菌荚膜生物合成操纵子的产物。在本研究中，我们研究了调节 capD 表达对炭疽杆菌荚膜表型、与吞噬细胞相互作用和豚鼠毒力的影响。capD 的转录融合物被构建到编码热休克蛋白 60 (hsp60) 和延伸因子 Tu (EFTu) 的基因以及炭疽杆菌荚膜生物合成基因 capA 上。改变翻译信号以提高 capD 的表达，包括用共识序列替换推定的核糖体结合位点和用 ATG 替换 TTG 起始密码子。在野生型炭疽杆菌 Ames 的裂解物中，免疫印迹未检测到 CapD，但在工程菌株中检测到了 CapD 的共有核糖体结合位点。用免疫印迹检测到的 capD 过表达的菌株发现表面相关的荚膜较少，并且主要将较低分子量的荚膜释放到培养上清液中。capD 的过表达增加了对中性粒细胞吞噬杀伤的易感性，并增加了与巨噬细胞的粘附，导致在豚鼠感染模型中的适应性降低。这些数据表明，炭疽杆菌可能已经进化出较弱的 capD 表达，从而优化了荚膜介导的毒力。
