Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840, USA.
J Bacteriol. 2010 Dec;192(23):6154-9. doi: 10.1128/JB.00818-10. Epub 2010 Sep 24.
Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.
贝纳柯克斯体是人类 Q 热的病原体，它在宿主细胞内占据一个独特的位置，在那里它在经过修饰的酸性吞噬体或寄生性空泡（PV）中复制。PV 膜富含胆固醇，宿主胆固醇代谢的抑制会对 PV 的生物发生和病原体的复制产生负面影响。PV 膜胆固醇的确切来源尚不清楚，也不知道细菌是否主动转移和/或修饰宿主细胞胆固醇或甾醇前体。贝纳柯克斯体缺乏从头合成胆固醇的酶;然而，该生物体编码了一种真核生物样Δ24 甾醇还原酶同源物 CBU1206。该酶在其他原核生物中不存在，预测它在胆固醇或麦角固醇生物合成的最后一步中还原甾醇双键在碳 24 位。在本研究中，我们检查了 CBU1206 的功能活性。氨基酸比对显示与土壤变形虫 Naegleria gruberi 的 Δ24 甾醇还原酶具有最大的序列同一性（51.7%）。通过在酿酒酵母 erg4 突变体中表达该蛋白，在半乳糖诱导的启动子控制下，检查了 CBU1206 的活性。erg4 是一种酵母 Δ24 甾醇还原酶，负责麦角固醇合成的最后还原步骤。与 Erg4-绿色荧光蛋白（GFP）一样，CBU1206-GFP 融合蛋白定位于酵母内质网。CBU1206 的异源表达挽救了 S. cerevisiae erg4 在存在布雷菲德菌素 A 和环己酰亚胺时对生长的敏感性，并导致麦角固醇的新合成。这些数据表明 CBU1206 是一种活性甾醇还原酶，并表明该酶在贝纳柯克斯体细胞内生长过程中可能作用于宿主甾醇。
