Running title: Alkaline phosphatase inhibition and Francisella survival 1 Francisella DnaK Inhibits Tissue Nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram negative pneumonic organism (Klebsiella pneumonia) suggesting the inhibition may be Francisella specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue nonspecific isozyme) is inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N-terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g., alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK coimmunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.