Abstract
The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis.
Highlights
Interactions between microbes and immune cells play a critical role in microbial pathogenesis
J774A.1 macrophages pretreated with DMSO control or compounds (10 M) for 1 h were infected with B. anthracis Sterne spores (5 m.o.i.)
Further characterization studies showed that the two most potent compounds, NSC 95397 and NSC 270012 (Fig. 1B), did not inhibit 1) the growth of B. anthracis spores (Fig. 1C), 2) the enzymatic activity of anthrax LF [22], or 3) cellular proteasome activity [22], a host component exploited by anthrax lethal toxin (LT) to kill susceptible macrophages [29]
Summary
Interactions between microbes and immune cells play a critical role in microbial pathogenesis. Chemical Genetic Screening and Phosphatase Profiling—To assess whether inhibition of host phosphatase function would elicit protection against B. anthracis-induced cell death, a chemical genetic approach was used wherein a focused library of known Cdc25 phosphatase inhibitors [22] and their analogs (supplemental Table 1) were screened in infected macrophages.
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