Background.

Mycobacterium paratuberculosis (Mptb) persists in host macrophages and in the environment. Protein expression patterns may vary depending upon its growth phase.

Purpose.

To contrast protein expression of Mptb ATCC 19698 in persistent, stationary and reactivation growth phases.

Methods and materials.

Mptb was cultured in Middlebrook 7H9 broth with OADC, Tween 80 and mycobactin J at 37°C. After 5 years in undisturbed upright tissue culture flasks with tightly closed caps the cells were viable, nonreplicating and thus considered persistent. Cells were harvested from the persistent phase cultures, from reactivated cultures (cells from the persistent phase cultures put to new media) and from stationary growth cultures (i.e., 4 week old cultures from frozen seed lot). Proteins from the three growth phases were evaluated by SDS-PAGE.

Results.

Persistent Mptb cells were reactivated when inoculated into 7H9 medium with OADC, but not in modified Watson-Reid or 7H9 medium enriched with glycerol and dextrose. Soluble proteins extracted from the persistent Mptb yielded 8 protein bands on SDS-PAGE gels. However, about 25 protein bands were seen from the stationary growth phase cells and the reactivated Mptb cells. Expression of a 40 kDa protein was higher and expression of two proteins (27 and 23 kDa) was lower in reactivated Mptb than in cells from the stationary culture.

Conclusions.

Mptb persists in a viable state for 5 years or longer. Fewer soluble proteins were found in persistent phase cells. Persistent Mptb resumes growth in 7H9 medium enriched with OADC, but not in 7H9 with glycerol and dextrose or in modified Watson-Reid medium. This finding implies that fatty acids may be needed to support reactivation of persistent Mptb.