Effects of benzothiazinone and ethambutol on the integrity of the corynebacterial cell envelope

Abstract

The mycomembrane (MM) is a hydrophobic layer formed by mycolic acids covering the surface of Mycobacteria and related species. This group includes important pathogens such as Mycobacterium tuberculosis, Corynebacterium diphtheriae, but also the biotechnologically important strain Corynebacterium glutamicum. The MM contributes to the impermeability of the cell envelope and thereby also protects bacteria from antibiotics. This makes biosynthesis of the MM an attractive target for antibiotic intervention. The first line anti-tuberculosis drug ethambutol (EMB) interferes with the synthesis of the arabinogalactan (AG), which is a structural scaffold for covalently attached mycolic acids that form the inner leaflet of the MM. Similarly, the new drug candidate, benzothiazinone 043 (BTZ) affects the synthesis of the AG component of the cell wall. We previously showed that C. glutamicum cells treated with a sublethal concentration of EMB lose the integrity of the MM. In this study we examined the effects of a sublethal concentration of BTZ. Our work shows that 1 {micro}g ml-1 BTZ efficiently blocks the apical growth machinery and reduces cell proliferation, however the integrity of the MM is largely preserved and the effects of {beta}-lactam antibiotics are only additive, not synergistic. Transmission electron microscopy (TEM) analysis revealed a distinct middle layer in the septum of control cells considered to be the inner leaflet of the MM covalently attached to the AG. It functions as a greasy slide for the lateral flow of mycolic acids in the outer leaflet. This layer was not detectable in the septa of BTZ or EMB treated cells, which suggests that the greasy slide is impaired and the confluency of the MM is thereby reduced. In addition, we observed that EMB treated cells have a thicker and less electron dense peptidoglycan (PG) layer consistent with the report that EMB also inhibits the glutamate racemase MurI. We conclude that EMB and BTZ have distinct differences in their modes of action. While EMB and BTZ both effectively block elongation growth, BTZ also strongly reduces septal cell wall synthesis. This renders BTZ treated cells likely more tolerant to antibiotics that act on growing bacteria.