Protein Export and Surface Anchoring in Gram+ Bacteria

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DESCRIPTION (provided by applicant): Among the low G+C Gram+ bacteria are many important human pathogens, including some classified as potential bioterrorism agents (i.e. Bacillus anthracis, category A and Listeria monocytogenes, category B). Many of these organisms have proteins on their surface that lack typical Sec-dependent N-terminal secretion signals and thus use unknown mechanisms for export through the cell membrane (cm). Some of these proteins are apparently involved in pathogenesis since they are effective for passive immunization of mice. Aims 1 and 2 of this work are directed at identification and characterization of new Sec-independent protein secretion systems in these organisms. Once secreted through the cm in Gram+ organisms, proteins are either released into the extracellular milieu or anchored to the cell surface. The general mechanism for anchoring proteins to the cell wall (cw) in Gram+ bacteria requires a transpeptidase, called sortase, that recognizes and cleaves a short amino acid motif preceding a C-terminal hydrophobic region and charged tail. We recently characterized two sortases in the group A streptococcus (GAS) that anchor distinct subsets of proteins with the LPXTG motif. At a similar location in the genome of other GAS strains, we have identified genes encoding additional proteins with homology to sortase. In Aim 3 we will characterize the function in the GAS of these proposed new sortases and test their ability to anchor their predicted substrate proteins, which we find encoded nearby. We will also characterize a predicted protein with homology to signal peptidase I (encoded in the same region) that we propose will be needed for secretion of these proteins. The greater understanding of secretion and cell wall anchoring in Gram+ bacteria that results from this work should provide new targets for broad spectrum antibacterial therapy and potential new vaccine vectors. In addition, it should provide commercially useful new methods for large-scale production of proteins.