Abstract
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 pneumoniae) 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) was 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 co-immunoprecipitated 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.
Highlights
Pulmonary Francisella infection resulted in reduction of plasma alkaline phosphatase activity
Despite the high mortality rate in untreated individuals, little is understood regarding F. tularensis virulence factors or the innate and adaptive immune responses operating at the sites of primary infection
Protein bands of interest (Figs. 4B and 5A) were digested with trypsin, and the resulting peptides analyzed by capillary HPLC-electrospray ionization tandem mass spectrometry as described under “Experimental Procedures.”
Summary
Pulmonary Francisella infection resulted in reduction of plasma alkaline phosphatase activity. Results: Francisella heat shock protein DnaK binds to alkaline phosphatase reducing enzymatic activity. Liver damage resulting from pulmonary F. novicida infection was assessed by analyzing liver function enzymes in plasma and a marked decrease in total alkaline phosphatase (AP) activity as early as 48 h after pulmonary challenge was observed. This observation of decreased AP was unexpected because most reported pathogen infections give rise to increased AP activity. We provide evidence that heat shock protein DnaK of F. novicida binds to AP-reducing enzymatic activity This is the first report of such a novel mechanism used by a pathogen to evade the host’s defense
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