Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host.宿主纤溶酶（plasmin）是一种兼具纤维蛋白溶解活性、同时可作用于细胞外基质成分的纤溶酶，激活或招募宿主纤溶酶是细菌病原体常见的侵袭策略。鼠疫耶尔森氏菌（Yersinia pestis，腺鼠疫的致病菌）表达多功能表面蛋白酶Pla，该酶可激活纤溶酶并灭活纤溶抑制因子。Pla由pPla质粒编码。经皮内接种后，鼠疫耶尔森氏菌可在接种部位引流的淋巴节点（lymph node, LN）中增殖并造成该淋巴结的破坏。而亲缘关系密切、不含pPla质粒的假结核耶尔森氏菌（Y. pseudotuberculosis）则不具备这一能力。我们提出如下假说：腺鼠疫发生过程中，淋巴结内的组织损伤与细菌增殖存在关联，且二者均由pPla质粒驱动。我们利用一系列携带或不携带pPla质粒的鼠疫耶尔森氏菌与假结核耶尔森氏菌菌株，构建皮内注射小鼠感染模型开展实验。结果发现，pPla质粒并非细菌移位至淋巴结所必需的元件。我们还观察到，经pPla质粒消除处理的鼠疫耶尔森氏菌，其引发的组织病理损伤与野生型菌株并无二致。此外，假结核耶尔森氏菌的组织病理特征表现为：感染灶由炎性细胞浸润包裹，组织密度与滤泡结构均保持正常；引入pPla质粒后，该病理特征未发生改变。然而，pPla质粒的存在可使假结核耶尔森氏菌的载菌量提升至与鼠疫耶尔森氏菌相当的水平。与之类似，缺失pPla质粒会显著降低感染小鼠淋巴结内的鼠疫耶尔森氏菌载量。这种由pPla介导的载菌量增强效应，直接依赖于Pla的蛋白水解活性。对感染Pla缺陷型鼠疫耶尔森氏菌的淋巴结进行免疫组化（immunohistochemistry）检测，可见大量细菌裂解现象；而在野生型鼠疫耶尔森氏菌感染的样本中，则可见大量形态完整的微生物。综上，本研究表明，腺肿（bubo）中的组织损伤与细菌存活/增殖过程相互分离；Pla的核心作用并非改变宿主组织环境以促进鼠疫耶尔森氏菌的体内传播，而是保护细菌免受宿主的裂解破坏。