Proteomic analysis of a penicillin-tolerant rgg mutant strain of Streptococcus pyogenes.
OBJECTIVE
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 log(10) cfu/mL ( approximately 80-fold) after 24 h of exposure to 0.024 mg/L penicillin compared with controls; however, the mutant strain lost 0.3 log(10) cfu/mL ( approximately 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.
Chaussee
Michelle A
MA
Division of Basic Biomedical Sciences, The Stanford School of Medicine of the University of South Dakota 414 East Clark Street, Vermillion, SD 57069, USA.
McDowell
Emily J
EJ
Rieck
Lindsey D
LD
Callegari
Eduardo A
EA
Chaussee
Michael S
MS
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D004268:DNA-Binding Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010403:Penicillin Resistance; D010406:Penicillins / Q000494:pharmacology; D020543:Proteome; D013297:Streptococcus pyogenes / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*
D001426:Bacterial Proteins; D003470:Culture Media; D004268:DNA-Binding Proteins; D010406:Penicillins; D020543:Proteome; D015534:Trans-Activators; C074779:rgg protein, Streptococcus