Inhibition of AcpA Phosphatase Activity with Ascorbate Attenuates Francisella tularensis Intramacrophage Survival

Abstract

Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (K i ‫؍‬ 380 ؎ 160 M) and 2-phosphoascorbate (K i ‫؍‬ 3.2 ؎ 0.85 M) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia.Francisella tularensis, the causative agent of human tularemia, is a highly infectious bacterium that could represent a serious bio-threat if genetically modified to resist antibiotic treatment (1-3). Four acid phosphatases are encoded by F. tularensis: AcpA, AcpB, AcpC, and Hap. A recent work indicated that a quadruple knockout mutant strain showed attenuated virulence and diminished macrophage vacuolar escape (4), thus the pharmacological inhibition of acid phosphatase activity is an excellent target to prevent the proliferation of these bacteria inside infected cells.Acid phosphatases are produced by many intracellular pathogenic organisms. These enzymes are able to function inside host cell endosomes by hydrolyzing the phosphoester (P-O) bonds of phosphorylated molecules. Acid phosphatases have been identified in Coxiella (5), Bordetella (6), and Legionella (7, 8), and their activity has been directly linked with virulence in some of these organisms. Several of these enzymes have been purified, their biochemical characteristics described, and their structure solved. Despite evidence suggesting that these phosphatases suppress the respiratory burst of human neutrophils (7, 9, 10), their natural substrates, biological role, and molecular events involved during infection are still under study.A combination of several factors makes it difficult to identify the intracellular substrate that these enzymes utilize to aid the bacteria during the infection process. For example, phosphate is the most abundant functional group present in the intracellular pool of cellular metabolites (11). In addition, most of the phosphatases previously...

Identification of a Small Molecule That Modifies MglA/SspA Interaction and Impairs Intramacrophage Survival of Francisella tularensis

“…Currently, Tularemia can be treated with antibiotics such as streptomycin and gentamicin [22]. However, the identification of new therapeutics is very significant, since Francisella can easily be genetically modified and therefore its sensitivity to known antibiotics could be compromised [23]–[25].…”

Mechanisms of Francisella tularensis Intracellular Pathogenesis

“…The importance of acid phosphatases in resistance of F. novicida to ROS can also be seen following treatment of neutrophils and MDMs with reagents that block assembly and/or function of NADPH oxidase, or in macrophages from p47 phox2/2 mice, where the ability of F. novicida DacpA and/or DacpABCH mutants to survive intracellularly is improved (Mohapatra et al 2010). Additionally, ascorbate treatment of murine or human macrophages infected with the LVS blocks acid phosphatase production and inhibits intracellular growth of the bacterium, a phenotype that is dependent on production of AcpA (McRae et al 2009). Thus, AcpA and other predicted acid phosphatases appear to play an important role in NADPH oxidase inhibition and resistance to ROS following infection by F. novicida.…”

The Acid Phosphatase AcpA Is SecretedIn Vitroand in Macrophages by Francisella spp

“…The Schu S4 acpA mutant was constructed as described earlier for the LVS acpA mutant (35). Briefly, a 1-kb fragment upstream and a 1-kb fragment downstream of acpA were amplified with primers JG1795/ JG1796 and JG1797/JG1798, respectively, and cloned into the SalI and BamHI, and BamHI and SmaI sites of pJC84, respectively.…”