Spore-forming bacteria as pharmaceutical factories

Abstract

Many Bacilli form dormant structures, spores, which routinely survive physical, chemical and biological challenge. How do spores encapsulate their contents, and could it be possible to mimic or adapt them to make better drug products? Key spore component 2,6-dipicolinic acid (DPA) is necessary for protein stability, but investigation of its role is confounded with that of water content. Tests replacing DPA with chemical analogues were not successful, but led to the novel approach of doping spores with extra DPA. Various Bacillus species, in the main B. subtilis and B. thuringiensis, have been engineered to produce, for the first time, fragments of a humanized monoclonal antibody: kindly provided by Medimmune. It is possible to produce antibodies inside the spore core. This work proposes for the first time that such therapeutic spores might lack an outer coat, and demonstrates that antibody production continues. It is understood they continue to resist most challenges including wet heat, which damages proteins, yet they become susceptible to lysozyme. Such spores, further developed to lyse in presence of intestinal, though not gastric fluid, may have use in oral delivery. Cry1Ac of B. thuringiensis offers an alternative; large protein crystals are produced during sporulation. Here, various fluorescent fusions that maintain fluorescence over months were developed and investigated in detail. For the first time, fluorescent antibody crystals were produced. In addition to drug delivery, these fusions offer a novel approach to crystallization of intransigent proteins, for structural studies. This work shows that it might be feasible to use spores as pharmaceutical formulations. A number of challenges remain: from fabrication, through limits of current good manufacturing practice, to engineering biology to protect the environment and commercial interests. Bacterial spores have for the past century and a half been known for their dormancy, and it’s about time engineers took advantage.