Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
Biotechnology Research Center, Southwest University, Beibei, Chongqing, PR China.
Microbiology (Reading). 2012 Jul;158(Pt 7):1843-1851. doi: 10.1099/mic.0.058867-0. Epub 2012 Apr 13.
Neuronal calcium sensor proteins and their homologues participate in transducing extracellular signals that affect intracellular Ca(2+) levels, which in turn regulate enzyme activities, secretion, gene expression and other biological processes. The filamentous fungus Beauveria bassiana is a broad-host-range pathogen of insects that acidifies the extracellular milieu during growth and pathogenesis towards target hosts. A collection of B. bassiana random insertion mutants were screened on pH indicator plates and one mutant was isolated that displayed reduced acidification. The random insertion site was mapped to a gene that displayed homology to the neuronal calcium sensor/frequenin protein family and was designated Bbcsa1. To validate the role of Bbcsa1 in B. bassiana, a targeted gene-knockout was constructed. Data confirmed that Bbcsa1 was not an essential gene and the ΔBbcsa1 strain displayed delayed acidification of the medium when grown in Czapek-Dox medium, as compared with the wild-type parent. HPLC profiling of secreted metabolites did not detect any major changes in the production of organic acids, although downregulation of the membrane H(+) pump/ATPase was noted in the mutant. A slight growth-deficient phenotype was observed for the ΔBbcsa1 strain on Czapek-Dox and potato dextrose media, which was accentuated at high calcium concentrations (500 mM) and 1.5 M sorbitol, but was unaffected by EDTA or SDS. Perturbations in vacuole morphology were also noted for the mutant. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the ΔBbcsa1 mutant when applied topically, representing the natural route of infection, but no significant effect was observed when fungal cells were directly injected into target hosts. These results suggest that Bbcsa1 participates in pre-penetration or early penetration events, but is dispensable once the insect cuticle has been breached.
神经元钙传感器蛋白及其同源物参与转导影响细胞内 Ca(2+)水平的细胞外信号，进而调节酶活性、分泌、基因表达和其他生物过程。白僵菌是一种广泛宿主范围的昆虫病原体，在生长和向目标宿主发病过程中会使细胞外环境酸化。对一组白僵菌随机插入突变体进行了 pH 指示剂平板筛选，分离出一个显示酸化能力降低的突变体。随机插入位点被定位到一个与神经元钙传感器/频率蛋白家族同源的基因，被命名为 Bbcsa1。为了验证 Bbcsa1 在白僵菌中的作用，构建了一个靶向基因敲除突变体。数据证实 Bbcsa1 不是必需基因，与野生型亲本相比，ΔBbcsa1 菌株在 Czapek-Dox 培养基中生长时培养基酸化延迟。HPLC 分析分泌代谢物没有检测到有机酸产生的任何重大变化，尽管突变体中膜 H(+)泵/ATP 酶的下调。与野生型亲本相比，ΔBbcsa1 菌株在 Czapek-Dox 和马铃薯葡萄糖培养基上的生长缺陷表型略有增加，在高钙浓度（500 mM）和 1.5 M 山梨醇下更为明显，但不受 EDTA 或 SDS 影响。突变体的液泡形态也受到干扰。使用作为靶宿主的家蚕进行昆虫生物测定表明，ΔBbcsa1 突变体在经皮给药时的毒力降低，代表了自然感染途径，但当真菌细胞直接注射到靶宿主中时，没有观察到显著影响。这些结果表明 Bbcsa1 参与了预穿透或早期穿透事件，但一旦昆虫表皮被穿透，它就是可有可无的。
