Inhibition of AcpA phosphatase activity with ascorbate attenuates Francisella tularensis intramacrophage survival.
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 microM) and 2-phosphoascorbate (K(i) = 3.2 +/- 0.85 microM) 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.
McRae
Steven
S
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32610-3610, USA.
Pagliai
Fernando A
FA
Mohapatra
Nrusingh P
NP
Gener
Alejandro
A
Mahmou
Asma Sayed Abdelgeliel
AS
Gunn
John S
JS
Lorca
Graciela L
GL
Gonzalez
Claudio F
CF
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000502:physiology; D005604:Francisella tularensis / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D050296:Microbial Viability / Q000187:drug effects / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D000975:Antioxidants; D001426:Bacterial Proteins; D010744:Phosphoric Monoester Hydrolases; D001205:Ascorbic Acid