Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity.
FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H2O2. Streptococcus pneumoniae produces exceptionally high levels of H2O2, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H2O2 inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H2O2 production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H2O2. In vitro exposure of the spxB mutant to various H2O2 concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H2O2 or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H2O2 produced by S. pneumoniae.
Benisty
Rachel
R
Pediatric Infectious Disease Unit, Soroka University Medical Center, Faculty of Health Sciences, Beer Sheva, Israel.
Cohen
Aharon Yehonatan
AY
Feldman
Alexandra
A
Cohen
Zvi
Z
Porat
Nurith
N
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain; D002569:Cerulenin / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007114:Immunization; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D047468:Immunoprecipitation; D009154:Mutation / Q000235:genetics; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011008:Pneumococcal Infections / Q000235:genetics / Q000378:metabolism*; D011771:Pyruvate Oxidase / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D001426:Bacterial Proteins; D005227:Fatty Acids; D007074:Immunoglobulin G; D016877:Oxidants; D012333:RNA, Messenger; D002569:Cerulenin; D006861:Hydrogen Peroxide; D011771:Pyruvate Oxidase; D003545:Cysteine