Department of Molecular Genetics and Infection Biology, Interfaculty Institute of Genetics and Functional Genomics, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany.
Front Cell Infect Microbiol. 2021 Feb 15;10:613467. doi: 10.3389/fcimb.2020.613467. eCollection 2020.
has evolved versatile strategies to colonize the nasopharynx of humans. Colonization is facilitated by direct interactions with host cell receptors or binding to components of the extracellular matrix. In addition, pneumococci hijack host-derived extracellular proteases such as the serine protease plasmin(ogen) for ECM and mucus degradation as well as colonization. expresses strain-dependent up to four serine proteases. In this study, we assessed the role of secreted or cell-bound serine proteases HtrA, PrtA, SFP, and CbpG, in adherence assays and in a mouse colonization model. We hypothesized that the redundancy of serine proteases compensates for the deficiency of a single enzyme. Therefore, double and triple mutants were generated in serotype 19F strain EF3030 and serotype 4 strain TIGR4. Strain EF3030 produces only three serine proteases and lacks the SFP encoding gene. In adherence studies using Detroit-562 epithelial cells, we demonstrated that both TIGR4Δ and 19F mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. Consistent with these results, we show that the mutants of strain 19F, which preferentially colonizes mice, abrogate nasopharyngeal colonization in CD-1 mice after intranasal infection. The bacterial load in the nasopharynx was monitored for 14 days. Importantly, mutants showed significantly lower bacterial numbers in the nasopharynx two days after infection. Similarly, we detected a significantly reduced pneumococcal colonization on days 3, 7, and 14 post-inoculations. To assess the impact of pneumococcal serine proteases on acute infection, we infected mice intranasally with bioluminescent and invasive TIGR4 or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. We imaged the acute lung infection in real-time and determined the survival of the mice. The TIGR4 mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. In conclusion, our results showed that pneumococcal serine proteases contributed significantly to pneumococcal colonization but played only a minor role in pneumonia and invasive diseases. Because colonization is a prerequisite for invasive diseases and transmission, these enzymes could be promising candidates for the development of antimicrobials to reduce pneumococcal transmission.
已经进化出多种策略来定植人类的鼻咽部。定植是通过与宿主细胞受体的直接相互作用或与细胞外基质成分的结合来促进的。此外，肺炎链球菌劫持宿主来源的细胞外蛋白酶，如丝氨酸蛋白酶纤溶酶原（物），用于 ECM 和粘液降解以及定植。表达依赖于菌株的多达四种丝氨酸蛋白酶。在这项研究中，我们评估了分泌或细胞结合的丝氨酸蛋白酶 HtrA、PrtA、SFP 和 CbpG 在粘附测定和小鼠定植模型中的作用。我们假设丝氨酸蛋白酶的冗余性弥补了单一酶的缺乏。因此，在血清型 19F 菌株 EF3030 和血清型 4 菌株 TIGR4 中生成了双突变体和三突变体。菌株 EF3030 仅产生三种丝氨酸蛋白酶，并且缺乏 SFP 编码基因。在使用 Detroit-562 上皮细胞进行的粘附研究中，我们证明了缺乏丝氨酸蛋白酶或仅表达 CbpG、HtrA 或 PrtA 的 TIGR4Δ和 19F 突变体粘附到 Detroit-562 细胞的能力降低。与这些结果一致，我们表明，优先定植于小鼠的 19F 菌株的突变体在鼻腔感染后可消除 CD-1 小鼠的鼻咽定植。在 14 天内监测鼻咽中的细菌负荷。重要的是，突变体在感染后两天在鼻咽中的细菌数量明显减少。同样，我们在接种后第 3、7 和 14 天检测到肺炎球菌定植的显著减少。为了评估肺炎链球菌丝氨酸蛋白酶对急性感染的影响，我们通过鼻腔感染用生物发光和侵袭性 TIGR4 或表达仅 CbpG、HtrA、PrtA 或 SFP 的同工三突变体感染小鼠。我们实时成像急性肺感染并确定小鼠的存活率。仅表达 PrtA 的 TIGR4 突变体显示出明显的衰减，并且在急性肺炎模型中毒性降低。总之，我们的结果表明，肺炎链球菌丝氨酸蛋白酶对肺炎链球菌定植有重要贡献，但在肺炎和侵袭性疾病中只起次要作用。因为定植是侵袭性疾病和传播的前提，这些酶可能是开发减少肺炎球菌传播的抗菌药物的有前途的候选物。
