Santiago-Tirado Felipe H, Onken Michael D, Cooper John A, Klein Robyn S, Doering Tamara L
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.
mBio. 2017 Jan 31;8(1):e02183-16. doi: 10.1128/mBio.02183-16.
The blood-brain barrier (BBB) protects the central nervous system (CNS) by restricting the passage of molecules and microorganisms. Despite this barrier, however, the fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that is estimated to kill over 600,000 people annually. Cryptococcal infection begins in the lung, and experimental evidence suggests that host phagocytes play a role in subsequent dissemination, although this role remains ill defined. Additionally, the disparate experimental approaches that have been used to probe various potential routes of BBB transit make it impossible to assess their relative contributions, confounding any integrated understanding of cryptococcal brain entry. Here we used an in vitro model BBB to show that a "Trojan horse" mechanism contributes significantly to fungal barrier crossing and that host factors regulate this process independently of free fungal transit. We also, for the first time, directly imaged C. neoformans-containing phagocytes crossing the BBB, showing that they do so via transendothelial pores. Finally, we found that Trojan horse crossing enables CNS entry of fungal mutants that cannot otherwise traverse the BBB, and we demonstrate additional intercellular interactions that may contribute to brain entry. Our work elucidates the mechanism of cryptococcal brain invasion and offers approaches to study other neuropathogens.
The fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that kills hundreds of thousands of people each year. One route that has been proposed for this brain entry is a Trojan horse mechanism, whereby the fungus crosses the blood-brain barrier (BBB) as a passenger inside host phagocytes. Although indirect experimental evidence supports this intriguing mechanism, it has never been directly visualized. Here we directly image Trojan horse transit and show that it is regulated independently of free fungal entry, contributes to cryptococcal BBB crossing, and allows mutant fungi that cannot enter alone to invade the brain.
血脑屏障（BBB）通过限制分子和微生物的通过来保护中枢神经系统（CNS）。然而，尽管存在这种屏障，真菌病原体新型隐球菌仍会侵入大脑，引发一种脑膜脑炎，据估计每年导致超过60万人死亡。隐球菌感染始于肺部，实验证据表明宿主吞噬细胞在随后的传播中起作用，尽管这一作用仍不明确。此外，用于探究血脑屏障转运各种潜在途径的不同实验方法使得无法评估它们的相对贡献，这混淆了对隐球菌进入大脑的任何综合理解。在这里，我们使用体外血脑屏障模型表明，“特洛伊木马”机制对真菌穿越屏障有显著贡献，并且宿主因子独立于游离真菌转运来调节这一过程。我们还首次直接成像了含有新型隐球菌的吞噬细胞穿越血脑屏障，表明它们是通过跨内皮孔道穿越的。最后，我们发现特洛伊木马式穿越使无法以其他方式穿越血脑屏障的真菌突变体能够进入中枢神经系统，并且我们证明了可能有助于进入大脑的其他细胞间相互作用。我们的工作阐明了隐球菌侵入大脑的机制，并提供了研究其他神经病原体的方法。
真菌病原体新型隐球菌侵入大脑，引发一种每年导致数十万人死亡的脑膜脑炎。针对这种进入大脑的途径提出的一种机制是特洛伊木马机制，即真菌作为宿主吞噬细胞内的乘客穿越血脑屏障（BBB）。尽管间接实验证据支持这一有趣的机制，但从未直接观察到。在这里，我们直接成像特洛伊木马式转运，并表明它独立于游离真菌进入受到调节，有助于隐球菌穿越血脑屏障，并使无法单独进入的突变真菌能够侵入大脑。
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