Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis
Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis
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DOI：
10.1128/jb.187.22.7596-7606.2005
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发表时间：
2005-11-01
影响因子：
3.2
通讯作者：
Jacobs, WR
中科院分区：
文献类型：
--
作者：
Bhatt, A;Kremer, L;Jacobs, WR
抑制或失活InhA，脂肪酸合酶II （FASII）酶，导致分枝杆菌细胞裂解。为了确定合成霉菌酸的FASII复合体的其他酶的失活是否也会导致裂解，我们表征了耻毛分枝杆菌中两种β -酮酰基-酰基载体蛋白合成酶KasA和KasB的重要性。利用特殊的转导进行等位基因交换，耻垢分枝杆菌可以产生零kasB突变体，而不是kasA突变体，这表明与kasB不同，kasA是必不可少的。为了确认kasA的重要性，并详细说明kasA缺失后发生的分子事件，我们开发了CESTET（条件表达专门化转导必要性测试），这是一种结合条件基因表达和专门化转导的遗传工具。使用CESTET，我们能够在ha和kasA突变体中生成条件null。以耻垢分枝杆菌为对照，研究了KasA耗竭对耻垢分枝杆菌的影响。无论是InhA还是KasA的耗竭都会导致细胞裂解，但在裂解之前有不同的生化和形态学事件。虽然InhA缺失诱导了一个80 kda的复合物，其中包含了分枝杆菌酰基载体蛋白KasA和AcpM，但KasA缺失并没有诱导出相同的复合物。InhA或KasA的缺失导致α和环氧真菌酸生物合成的抑制以及α '-真菌酸的积累。此外，扫描电镜显示，与InhA耗尽时观察到的气泡相反，KasA耗尽导致细胞表面出现“皱褶”外观。因此，我们的研究支持进一步探索KasA作为分枝杆菌药物开发的靶点。
Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null inhA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development.