C3-P-06Structome analysis of freeze-substituted virulentMycobacterium tuberculosisbased on direct enumeration of the serial ultrathin sections with TEM

Abstract

Serial ultrathin sections of virulent Mycobacterium tuberculosis H37Rv (ATCC 27294) were examined by transmission electron microscope and “structome” analysis (i.e., the quantitative three-dimensional structural analysis of a whole cell through direct enumeration and measurement at the electron microscopic level) was performed [ 1 ]. Five M. tuberculosis cells were cut into 24, 36, 69, 55, and 63 serial ultrathin cross sections, respectively. On average, the cells were 2.71 ± 1.05 μm in length, and the average diameter of the cell was 0.345 ± 0.029 μm. The outer membrane and plasma membrane surface areas were 3.04 ± 1.33 μm 2 and 2.67 ± 1.19 μm 2 , respectively. The cell and cytoplasm volumes were 0.293 ± 0.113 fl (= μm 3 ) and 0.210 ± 0.091 fl, respectively. The average total ribosome number per cell was 1,672 ± 568, and the ribosome density was 716.5 ± 171.4/0.1 fl. This is the first report of a structome analysis of M. tuberculosis cells prepared as serial ultrathin sections following cryofixation and rapid freeze substitution. These data are based on the direct measurement and enumeration of exquisitely preserved single-cell structures in transmission electron microscopy images rather than calculations or assumptions from indirect biochemical or molecular biological data, and may explain the slow growth of M. tuberculosis and enhance understanding of the structural properties related to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance, particularly in regard to the ratio of target to drug concentrations.