Mycobacterium tuberculosis Chaperonin 10 Stimulates Bone Resorption: A Potential Contributory Factor in Pott's Disease

Pott's disease (spinal tuberculosis), a condition characterized by massive resorption of the spinal vertebrae, is one of the most striking pathologies resulting from local infection with Mycobacterium tuberculosis (Mt; Boachie-Adjei, O., and R.G. Squillante. 1996. Orthop. Clin. North Am. 27:95–103). The pathogenesis of Pott's disease is not established. Here we report for the first time that a protein, identified by a monoclonal antibody to be the Mt heat shock protein (Baird, P.N., L.M. Hall, and A.R.M. Coates. 1989. J. Gen. Microbiol. 135:931–939) chaperonin (cpn) 10, is responsible for the osteolytic activity of this bacterium. Recombinant Mt cpn10 is a potent stimulator of bone resorption in bone explant cultures and induces osteoclast recruitment, while inhibiting the proliferation of an osteoblast bone–forming cell line. Furthermore, we have found that synthetic peptides corresponding to sequences within the flexible loop and sequence 65–70 of Mt cpn10 may comprise a single conformational unit which encompasses its potent bone-resorbing activity. Our findings suggest that Mt cpn10 may be a valuable pharmacological target for the clinical therapy of vertebral tuberculosis and possibly other bone diseases.

A chaperonin from a thermophilic bacterium, Thermus thermophilus

Unlike Escherichia coli chaperonins, a chaperonin (cpn) from a therm ophilic bacterium , Thermus thermophilus , consisting of homologues to GroEL (cpn 60) and GroES (cpn 10) is co-purified as a large complex. Thermus chaperonin shows a bullet-like shape in the side view seen by electron microscopy, and antibody against cpn 10 binds only to the round side of the bullet. We conclude that a single cpn 60-heptam er ring with two stripes stacks into two layers and a cpn 10 oligomer binds to one side of the layers. The purified Thermus chaperonin contains endogenously bound ADP, and incubation with ATP causes a partial dissociation of chaperonin into cpn 60 monomers and a cpn 10 heptam er. The effect of Thermus chaperonin on protein refolding upon dilution from guanidine HCl is different at three temperature ranges. At high temperatures above 55°C, where the native proteins are stable but their spontaneous foldings fail, the chaperonin induces productive folding in an ATP-dependent manner. At middle temperatures (25-55°C) where spontaneous foldings of the enzymes occur, the chaperonin slows down the rate of folding without changing the final yield of productive folding. At lower temperatures below 25°C where spontaneous foldings also occur, the chaperonin arrests the folding even in the presence of ATP. When a solution of relatively heat labile protein is incubated at high temperatures, and then residual activity of the protein is measured at its optimal temperature after incubation with ATP, the temperature that causes irreversible heat denaturation of the protein is elevated about 10°C by inclusion of Thermus chaperonin in the solution. Furthermore, once the folding intermediate of a protein is captured by Thermus chaperonin, it retains the ability to resume productive folding even after exposure to the otherwise denaturing high temperature. These results indicate that during heat denaturation proteins assume the common structure which is recognizable by the chaperonin. Finally, a ‘folding intermediate reservoir’ model to explain the effect of chaperonin is proposed, and is compared with a ‘marsupium ’ model.