Colin Harwood

Optimisation of Bacillus subtilis for the secretion of heterologous proteins Therapeutic proteins (including those required for experimental purposes and clinical trials) are major products of biomanufacturing processes and considerable time and expense are expended to maximise the yield and quality of proteins produced in heterologous hosts. The production host of choice is the Gram-negative bacterium Escherichia coli for which many strains and expression systems have been developed. However, one size does not fit all: E. coli is not suitable for the production of many proteins, either because it is not able to carry out appropriate post-translational modifications (e.g. glycosylation) or because it does not facilitate their folding into a native (i.e. functional) configuration. The former can be overcome by use of more expensive eukaryotic host production systems, while the latter can often be overcome by secreting proteins from the cytoplasm. Secretion has three major potential advantages over intracellular accumulation: the secreted target protein is usually natively folded; yields can be as high or higher than that obtained from intracellular E. coli host/vector systems; there is a reduced requirement for expensive extraction and purification procedures, with reduced risks of contamination with host proteins and nucleic acids.

Although various microbial protein secretion systems have been developed, all currently have limitations that restrict their use and value as a tool for the Biomanufacturing industry. These systems include secretion into the periplasm of E. coli and subsequent selective release and purification; production in the yeast Pichia pastoris; production in Bacillus subtilis and close relatives. In all cases, the yields are relatively low compared with that of the intracellular E. coli systems (mg rather than g per litre).

Bacillus subtilis is widely used for the commercial production of industrial enzymes, particularly for the food and detergents’ industries. Its advantage is its capacity to secrete native Bacillus proteins into the culture medium at concentrations in excess of ten of grams per litre. However, experience with the use of this bacterium for the production of heterologous proteins has been mixed with, in most cases, generally low yields. The aim of this proposal is to exploit knowledge of the genomics and systems biology of B. subtilis to develop this bacterium as a host/vector system for the production of high quality heterologous proteins for clinical trials and for high-throughput X-ray crystallographic studies. The aim is not to replace E. coli as the primary host for heterologous protein production, but to expand the range of host/vector systems that are available for problematical proteins.

Response of Bacillus anthracis to stress in the environment and in host cells Infectious disease is the culmination of a battle between the pathogen and its host. Central to the host’s innate defence mechanism are phagocytic cells; macrophages and dendritic cells. Phagocytes express a series of antibacterial factors, including reactive oxygen and nitrogen species, antibacterial peptides and enzymes. They can also impose increased acidification. In spite of these defence mechanisms, several clinically important pathogens can proliferate inside macrophages. The interaction between B. anthracis and host macrophages is central to its pathogenesis. However, an understanding of the cellular and molecular interactions between B. anthracis and the immune system is far from complete. B. anthracis avoids and even breaches cells of the immune system: vegetative cells avoid macrophages by virtue of their possession of a poly-D-glutamic acid capsule, while engulfment of spores by alveolar macrophages and their subsequent efficient germination within the macrophage is an important component of its ability to cause infection. However, it is not clear if B. anthracis can multiply in macrophages. We are dissecting the B. anthracis-macrophage relationship through a combined cellular, genetic and molecular approach.