Giorno Rebecca, Mallozzi Michael, Bozue Joel, Moody Krishna-Sulayman, Slack Alex, Qiu Dengli, Wang Rong, Friedlander Arthur, Welkos Susan, Driks Adam
Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, IL 60153, USA.
Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA.
Microbiology (Reading). 2009 Apr;155(Pt 4):1133-1145. doi: 10.1099/mic.0.023333-0.
Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and IunH (similar to an inosine-uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB-GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB-GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB-GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH-GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB-GFP and IunH-GFP resides in the region between the coat and the exosporium, called the interspace.
细菌芽孢具有一系列同心排列的保护结构，这些结构有助于休眠、存活并最终实现萌发。这些结构之一是芽孢衣，存在于所有芽孢中。然而，在炭疽芽孢杆菌中，芽孢被另一种形态复杂、了解较少的结构所包围，称为芽孢外壁。在此，我们对三种先前发现的芽孢外壁蛋白进行了表征，它们分别是ExsFA（也称为BxpB）、ExsFB（ExsFA / BxpB的高度相关旁系同源物）和IunH（类似于优先作用于肌苷 - 尿苷的核苷水解酶）。我们发现，在缺乏ExsFA / BxpB的情况下，芽孢外壁蛋白BclA不对称地聚集在最靠近芽孢囊中点的前芽孢极（即前芽孢靠近母细胞的极），而不是均匀地环绕芽孢外壁。ExsFA / BxpB可能在芽孢衣组装中也发挥作用，因为突变芽孢的表面缺乏野生型芽孢中可见的脊，且外观凹凸不平。ExsFA / BxpB对萌发也有适度但易于检测到的影响。尽管如此，在豚鼠的肌肉注射和气溶胶攻击模型中，exsFA / bxpB突变株仍具有完全的毒性。我们发现ExsFA / BxpB - GFP的定位模式是一个环，这与该蛋白位于芽孢外壁基底层的位置一致。相比之下，ExsFB - GFP荧光是一个实心椭圆形，表明ExsFB - GFP有独特的亚细胞定位。我们还使用这些融合蛋白来监测这些蛋白在芽孢形成过程中亚细胞定位的变化。在芽孢形成早期，两种融合蛋白都存在于整个母细胞细胞质中。随着芽孢形成的进展，GFP荧光从母细胞细胞质转移到前芽孢表面，并形成荧光环（对于ExsFA / BxpB而言）或实心椭圆形荧光（对于ExsFB而言）。IunH - GFP也产生了实心椭圆形荧光。我们认为至少一些ExsFB - GFP和IunH - GFP位于芽孢衣和芽孢外壁之间的区域，即间隙。
