B-260. Characterization of a Serine/Threonine Kinase in Bacillus anthracis

K. Bryant, J. Ballard;
Oklahoma Univ. Hlth. Sci. Ctr., Oklahoma City, OK.

Bacterial homologues of the eukaryote type serine/threonine kinases (STK) have been found to be important for a wide-variety of cellular events ranging from physiology to pathogenesis. Genome analysis of the pathogen Bacillus anthracis revealed four putative STKs of unknown function. In the current study, we characterized a STK in B. anthracis (termed STK-1) and examined its role in bacterial growth and virulence. First, to verify that STK-1 was a functional kinase, the catalytic domain of STK-1 (STKcat) was expressed and purified in Escherichia coli. An in vitro ATP-linked luminescence assay was used to confirm STKcat kinase activity. Next, to determine the cellular localization of STK-1, Western blots of subcellular fractions of B. anthracis were performed. Immunoanalysis revealed that STK-1 localized to the membrane. To gain insight into the function of STK-1, a genetic knockout of stk-1 (B. anthracis Δstk-1) was generated via homologous recombination. Interestingly, the resulting mutant strain showed no deficiencies in in vitro growth, germination, and sporulation when compared to the parent strain; however, when intracellular survival in RAW 264.7 macrophages and virulence in DBA/2 mice was examined, B. anthracis Δstk-1 exhibited clear defects. Within cultured macrophages, B. anthracis Δstk-1 exhibited a 50% decrease in survival, as compared to the parent strain. In DBA/2 mice, B. anthracis Δstk-1 spores exhibited an LD50 of 3.4x105 whereas the parent strain exhibited an LD50 of 4.5x104, indicating that STK-1 is required for full virulence. Moreover, comparison of survival curves of DBA/2 mice treated with B. anthracis Δstk-1 or the parent strain spores revealed significant differences. Collectively, these data suggest STK-1 may function as an infection-specific kinase important for survival within the host.