Identification-Characterization of Factors from Francisella novicida that inhibit Host Tissue non-specific Alkaline Phosphatase

Date
2011
Authors
Lakshmana Chetty, Senthilnath
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Abstract

Francisella tularensis is an intracellular Gram negative bacterium that can causes the pneumonic tularemia in humans. Francisella tularensis subsp. tularensis is classified as one of the most infectious pathogenic bacteria because inhalation of only a few organisms will cause disease and significant mortality. Given the rapid spread of F. tularensis following infection and the high mortality rate, new methods for treating and rapidly identifying Francisella infections are in great need. We have observed significant inhibition of host plasma Alkaline Phosphatase (ALP) activity following intranasal challenge of mice with F. tularensis. This inhibition appears specific to Francisella. Thus, we have directed our efforts at identification/partial characterization of this inhibitory factor.

We report here that the F. novicida (U112) cell lysates exhibit significant inhibition (40%) of the tissue non-specific alkaline phosphatase isoenzyme (TNAP). The inhibitory activity appears to be proteinaceous in nature due to the loss of inhibition in ALP activity following incubation of bacterial lysate for 10 minutes at 75°C. Native PAGE gel analysis of lysate material revealed that the inhibitory factor migrates as 130 and 260 kDa molecular weight species. Following DEAE-anion exchange chromatography and subsequent electrophoretic separation, the 130 kDa band was identified by proteomic analysis to be molecular chaperone DnaK (Hsp70). Immunoprecipitation of DnaK from cell lysates with anti-DnaK antibody indicated the presence of two co-precipitating bands at ∼60 kDa and 72 kDa which were identified by proteomic analysis to be HtpG(Hsp90) and GroEL(Hsp60), respectively. Addition of anti-Dnak monoclonal antibody to cell lysate material resulted in loss of TNAP inhibition in a dose dependent manner. Mutation of related genes resulted in lesser (∼10%) inhibition when compared to the U112 wild type strain. These findings suggest that DnaK and two other heat shock proteins play a contributing role in the inhibition of host TNAP.

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Integrative Biology