JAC Advance Access originally published online on August 5, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):752-759; doi:10.1093/jac/dkl319
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Proteomic analysis of a penicillin-tolerant rgg mutant strain of Streptococcus pyogenes
Division of Basic Biomedical Sciences, The Stanford School of Medicine of the University of South Dakota 414 East Clark Street, Vermillion, SD 57069, USA
Received 5 May 2006; returned 8 June 2006; revised 11 July 2006; accepted 12 July 2006
*Corresponding author. Tel: +1-605-677-6681; Fax: +1-605-677-6381; E-mail: mchausse{at}usd.edu
Objectives: To determine whether the transcriptional regulator Rgg contributes to penicillin-induced killing of Streptococcus pyogenes by altering a regulatory response to penicillin.
Methods: Penicillin-induced killing of a wild-type and isogenic rgg mutant strain was assessed in broth and solid media and in the presence of cerulenin, which inhibits fatty acid biosynthesis (FAB). Proteins from wild-type and rgg mutant cultures, either exposed to penicillin or not, were characterized by two-dimensional gel electrophoresis. Proteins of interest were identified with tandem mass spectrometry.
Results: The MIC of penicillin was 0.012 mg/L for both the wild-type strain NZ131 and an isogenic rgg mutant strain. The wild-type strain lost 1.9 log10 cfu/mL (
80-fold) after 24 h of exposure to 0.024 mg/L penicillin compared with controls; however, the mutant strain lost 0.3 log10 cfu/mL (2-fold) compared with controls. Changes in the proteome of wild-type and mutant cultures were assessed 1 and 4 h after exposure to penicillin. One hour exposure was associated with increased abundance (P < 0.05) of 12 proteins associated with FAB, the pentose phosphate pathway, glycolysis and stress responses in the wild-type strain. The abundance of 8 of 12 of these proteins was greater in samples obtained from the mutant strain, even prior to penicillin exposure. After 4 h of exposure, the abundance of 16 proteins was altered in one or both strains; however, a clear functional relationship was not evident. The addition of cerulenin slightly enhanced penicillin-induced killing of wild-type strain, which supported the proteomic results.
Conclusions: The results suggest that penicillin-independent changes in the cytoplasmic proteome of an rgg mutant strain of NZ131 confer tolerance to penicillin-mediated killing.
Keywords: microbial pathogenicity , antibiotics , group A streptococci , infectious diseases , mechanisms of action