New Morphologies of Hib Adhesion Pili.
New Morphologies of Hib Adhesion Pili.
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Hib 粘附菌毛的新形态。
DOI：
10.1093/micmic/ozad067.466
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Bullitt,Esther
中科院分区：
文献类型：
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作者：
Thairatana,Siriratt;Doran,Matthew;Sonani,RaviR;Egelman,EdwardH;Bullitt,Esther
流感嗜血杆菌是一种侵入性物种，已知可在幼儿和免疫抑制个体中引起脑膜炎、肺炎和其他严重感染。最流行的类型是B型流感嗜血杆菌（Hib），一种革兰氏阴性细菌，定植在人类鼻咽部。Hib细菌表面含有丝状菌毛，这是细菌结合和粘膜细胞定植的关键（如[7]）。虽然对Hib的致病性至关重要，但Hib菌毛的分子结构尚未得到高分辨率的解析。为了高分辨率地解决Hib菌毛结构，我们发现最有效的表达菌毛的方法是在新鲜制备的脑心输液液培养基中培养细菌，并补充两种新鲜制备的红细胞因子，β-NAD和血红素组氨酸[修改自6]。采用热萃取法对纯化进行优化，与传统的细菌毛剪法相比，该方法获得了更纯净的样品和更短的流程。我们的冷冻电镜结构揭示了Hib菌毛的锯齿形1-start螺旋组织，这与之前基于极低分辨率负染色EM[4]提出的三重对称相矛盾。这种形态使人联想到古菌毛，如Csu[5]和CupE[1]。一个AlphaFold[2]预测的Hib毛蛋白亚基HifA的结构被拟合到低温电镜密度图中，以检查毛蛋白亚基之间的相互作用。从AlphaFold模型中，我们看到HifA的前16个残基形成n端延伸（NTE），插入相邻亚基的凹槽中。我们观察到另一种相互作用，其中氨基酸130-149与邻近亚基接触，从而加强了毛细丝结构。结合，NTE和这种额外的相互作用，预计将有助于防止毛断裂。我们的数据显示，当悬浮在不同的缓冲中时，毛与毛之间的相互作用是不同的。在Tris缓冲液中悬浮的菌毛产生了单独的菌毛，但毛与毛之间没有明显的相互作用。然而，悬浮在MOPS缓冲液中，产生了二维和三维的毛束。这些对Hib菌毛形成的新四级结构的观察扩大了我们对细菌定植和感染的认识；我们现在期望这些菌毛能够形成一种生物膜，促进流感嗜血杆菌细菌的持续结合。我们在这里提出了一种新的Hib菌毛的三维结构，并讨论了其对治疗学发展的影响。(7、8)
Haemophilus influenza is an invasive species known to cause meningitis, pneumonia, and other serious infections in young children and immunosuppressed individuals. The most prevalent type is H. influenza type B (Hib), a gram-negative bacterium that colonizes the human nasopharynx [3]. The surface of Hib bacteria contains filamentous pili that are key to bacterial binding and colonization of mucosal cells (eg [7]). Although vital for the pathogenicity of Hib, the molecular structure of Hib pili has not been resolved to high resolution. With the aim of solving the Hib pili structure to high resolution, we discovered that the most efficient way to express the pili was to grow the bacteria in freshly made brain heart infusion liquid media, supplemented with two freshly made erythrocyte factors, β-NAD, and heme-histidine [modified from 6]. Heat extraction was performed to optimize purification, which resulted in a purer sample and shorter protocol than the traditional method of shearing pili from the bacteria. Our cryo-EM structure of Hib pili revealed the zigzag shaped 1-start helical organization, which contradicted the previously proposed three-fold symmetry based upon very low resolution negative stain EM [4]. This morphology is reminiscent of archaic pili such as Csu [5] and CupE [1]. An AlphaFold [2] predicted structure for the Hib pilin subunit, HifA, was fitted into the cryo-EM density map to examine the interactions between pilin subunits. From the AlphaFold model, we see that the first 16 residues of HifA form the N-terminal extension (NTE) that is inserted into a groove in the adjacent subunit. We observed an additional interaction in which amino acids 130-149 are in contact with the adjacent subunit thereby strengthening the pilus filament structure. In combination, the NTE and this additional interaction are expected to aid in preventing pilus breakage. Our data show diverse pili-pili interactions when suspended in different buffers. Pili suspended in Tris buffer resulted in individual pili without significant pili-pili interactions. Suspension in MOPS buffer, however, resulted in two-and three-dimensional bundles of pili. These observations of new quaternary structures formed by Hib pili are expanding our knowledge of bacterial colonization and infection; we now expect these pili to be capable of forming a biofilm that facilitates sustained binding of H. influenzae bacteria. We present here a new 3-D structure of Hib pili and discuss its implications for the development of therapeutics.[7, 8]