Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis

Apoorva Bhatt, Laurent Kremer, Annie Z. Dai, James C. Sacchettini, William R. Jacobs

Research output: Contribution to journalArticle

83 Scopus citations

Abstract

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 β-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 α and epoxy mycolate biosynthesis and to accumulation of α′-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.

Original languageEnglish (US)
Pages (from-to)7596-7606
Number of pages11
JournalJournal of Bacteriology
Volume187
Issue number22
DOIs
StatePublished - Nov 2005

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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