Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages.
Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages.
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DOI：
10.1371/journal.pone.0096015
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Hube B
中科院分区：
文献类型：
--
作者：
Kasper L;Seider K;Gerwien F;Allert S;Brunke S;Schwarzmüller T;Ames L;Zubiria-Barrera C;Mansour MK;Becken U;Barz D;Vyas JM;Reiling N;Haas A;Haynes K;Kuchler K;Hube B
光滑念珠菌目前是侵袭性念珠菌病的第二大常见病因。我们以前的工作表明C. glabrata适应于在巨噬细胞中的细胞内存活，并在非酸化的晚期内体阶段吞噬体内复制。相比之下，在酸化的成熟吞噬体中发现热杀死的酵母。在本研究中，我们的目的是阐明导致抑制吞噬体酸化和成熟的过程。我们发现，吞噬体含有活的C。glabrata细胞不与预先标记的溶酶体融合，并且具有低的吞噬体水解酶活性。酸化抑制的发生与巨噬细胞类型（人/鼠）、分化（M1-/M2-型）或活化状态（维生素D3刺激）无关。我们观察到，与热灭活酵母相比，在活酵母内化后，模式识别受体下游的巨噬细胞MAPK或NFκB信号级联没有差异活化，但Syk活化在含有活酵母的巨噬细胞中衰减得更快。因此，活酵母向未成熟吞噬体的递送可能不是由经由MAPK或NFκB信号传导的初始识别事件触发的，但可能涉及Syk活化。虽然C. glabrata吞噬体，该质子泵对细胞内存活的影响是低的，因为用巴弗洛霉素A1阻断V-ATP酶活性对真菌活力没有影响。主动pH调节是真菌改变吞噬体pH的一种可能策略。glabrata在体外以氨基酸作为唯一碳源生长时，能够碱化其细胞外环境。通过筛选C. glabrata突变体库中，我们确定了重要的基因环境碱化，进一步测试了它们对吞噬体pH值的影响。我们发现，缺乏真菌甘露糖基转移酶导致严重减少碱化在体外和C。glabrata酸化吞噬体。因此，蛋白质甘露糖基化可能在C.光滑的
Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.
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