title: Non-acid-fastness in Mycobacterium tuberculosis ΔkasB mutant correlates with the cell envelope electron density.
abstract: The acid-fastness is the most important and the most specific characteristics in mycobacteria, the mechanism of which is not clear but may be attributed to the lipid rich cell wall of this bacterium. While the exact component(s) responsible for this staining method remained unidentified, a Mycobacterium tuberculosis mutant, attenuated strain that produced shorter mycolic acids with defects in trans-cyclopropanation was shown to be acid fast negative. In this study, we examined the ultrastructure of the cell envelope (CE) of the mutant strain ΔkasB (missing a beta-ketoacyl-ACP synthase involved in mycolic acid biosynthesis), the parental CDC1551 (wild type strain) and kasB complemented strain, and compared ultrastructural differences among them with conventional transmission electron microscopy (TEM) and cryo-transmission electron microscopy (CEM). Conventional TEM revealed that there were no detectable differences in the thickness of the cell envelope among three strains (wild-type: 43.35 ± 6.13 nm; ΔkasB: 45.98 ± 11.32 nm; complement: 40.71 ± 6.3 nm). However, CEM data demonstrated that the region between the inner and outer membranes of the mutant strain, which is composed mainly of cell wall anchored mycolic acids (MA), showed a significant decrease in electron density as compared to the wild type and kasB complement strain (567.1 ± 372.7 vs. 301.4 ± 262.1, or vs. 235.2 ± 174.9, p < 0.02 or p < 0.001, respectively). These results suggested that altered MA patterns in the kasB mutant may have affected the packing of the lipid rich layer of the M. tuberculosis cell envelope, resulting in a reduced electron density of this layer as seen by CEM and loss of acid-fastness in light microscopical observation, and we propose a novel model of the cell envelope structure in tubercle bacilli.
authors.affiliation: Department of Mycobacterium Reference and Research, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24, Matsuyama, Kiyose, Tokyo 204-8533, Japan. hyamada@jata.or.jp
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamada
authors.forename: Apoorva
authors.initials: A
authors.lastname: Bhatt
authors.forename: Radostin
authors.initials: R
authors.lastname: Danev
authors.forename: Nagatoshi
authors.initials: N
authors.lastname: Fujiwara
authors.forename: Shinji
authors.initials: S
authors.lastname: Maeda
authors.forename: Satoshi
authors.initials: S
authors.lastname: Mitarai
authors.forename: Kinuyo
authors.initials: K
authors.lastname: Chikamatsu
authors.forename: Akio
authors.initials: A
authors.lastname: Aono
authors.forename: Koji
authors.initials: K
authors.lastname: Nitta
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Nagayama
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics*; D000143:Acids; D001426:Bacterial Proteins / Q000235:genetics*; D002473:Cell Wall / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D004583:Electrons; D017353:Gene Deletion; D005798:Genes, Bacterial; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000472:pathogenicity / Q000648:ultrastructure; D013194:Staining and Labeling; D014774:Virulence / Q000235:genetics
chemical_list: D000143:Acids; D001426:Bacterial Proteins; C444656:KasB protein, Mycobacterium tuberculosis; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase