JAC Advance Access originally published online on August 5, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):752-759; doi:10.1093/jac/dkl319
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
*Corresponding author. Tel: +1-605-677-6681; Fax: +1-605-677-6381; E-mail: mchausse{at}usd.edu
Received 5 May 2006; returned 8 June 2006; revised 11 July 2006; accepted 12 July 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
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
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Pharyngeal infection with Streptococcus pyogenes can result in acute rheumatic fever, which causes
500 000 deaths each year worldwide.1 Prompt antibiotic treatment of streptococcal pharyngitis has decreased the mortality of this post-infection immune sequela in the United States from 15 000 annual deaths in the 1950s to the current level of 3500.1
Penicillin and erythromycin are typically used to treat pharyngeal infections; however, the incidence of erythromycin-resistant isolates of S. pyogenes is increasing due to the dissemination of mef and erm genes.2 Surprisingly, the pathogen remains sensitive to ß-lactam antibiotics, despite decades of prophylactic use and the acquisition of resistance among related species, such as Staphylococcus aureus and Streptococcus pneumoniae, and oral flora.3 To date, ß-lactamase production has not been reported in S. pyogenes. It is unclear whether physiological or genetic barriers prevent the acquisition or maintenance of ß-lactam resistance in S. pyogenes or whether resistance is imminent.4 Although all isolates of S. pyogenes are sensitive to penicillin in vitro, the clinical failure rate associated with eradicating the pathogen from the pharynx is 35%.5 Possible explanations for this include re-infection from exogenous sources, an intracellular reservoir of bacteria sequestered from the drug, ß-lactamase-producing normal flora and phenotypic tolerance.
Penicillin inhibits the activities of enzymes involved in cell wall metabolism, which makes certain bacteria susceptible to osmotic lysis; however, some species, including S. pyogenes, are killed in the absence of detectable lysis.6 One possible mechanism of penicillin-induced killing is the induction of a programmed cell death (PCD) response in bacteria, similar to apoptosis in eukaryotes.7,8 PCD is likely to be mediated by changes in gene expression and may be important for the survival of species exposed to adverse conditions, in developmental processes such as sporulation and in biofilm formation.7,8 Consistent with this theory, several regulatory loci influence penicillin-induced killing. These include both two-component regulatory systems, which alter gene transcription in response to changes in the environment, and global regulatory proteins. For example, LytRS of S. aureus is a two-component regulatory system7–9 that controls the expression of lrgAB, which inhibit penicillin-induced death.10–12 In S. pneumoniae, inactivation of VncS, which is the histidine kinase/phosphatase of the VncRS two-component system, confers tolerance to vancomycin.13 In addition, the CiaRH two-component regulatory system is involved in resistance to ß-lactam antibiotics and viability during the stationary phase of growth.14,15 Finally, SigB-dependent regulation is necessary for tolerance to ß-lactam antibiotics in Listeria monocytogenes.16 These studies, and others, indicate that complex regulatory responses to changes in cell wall integrity are important in the response to ß-lactam antibiotics and may result in either tolerance or cell death.
The rgg (regulator of glycosyltranferase G) gene encodes a transcriptional regulator that influences the expression of genes associated with virulence, catabolism and stress responses in S. pyogenes.17–21 The amino terminus of Rgg encodes a putative helix-turn-helix motif and Rgg binds to the promoters of at least some genes to control gene transcription.22–24 Inactivation of rgg in S. pyogenes strain NZ131 relieves exponential-phase repression of ArcABC25 and decreases the expression of at least one putative murein hydrolase, designated Mur1.2.19 Previously, penicillin tolerance in Streptococcus gordonii was associated with dysregulation of ArcABC26 and altered expression of murein hydrolases is known to influence penicillin-mediated killing of a variety of related bacteria. The observations suggested that Rgg-mediated gene regulation may contribute to penicillin-induced killing of S. pyogenes. The objectives of this study were (i) to determine whether rgg inactivation affects penicillin-induced killing and (ii) to determine whether killing is associated with changes in gene expression in response to penicillin exposure. The results indicated that rgg inactivation confers low-level tolerance to penicillin-mediated killing of S. pyogenes. In addition, proteomic results support a model in which tolerance is conferred by changes in the abundance of Rgg-regulated proteins independently of penicillin exposure and is not due to disruption of a regulatory response to penicillin.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bacterial strains and culture conditions
S. pyogenes strain NZ131 (serotype M49) and an rgg isogenic mutant strain, designated NZ131 rgg, were previously described.18,27 Briefly, the mutant strain was created by cloning an internal region of the rgg gene into the suicide vector pVA981-2, which confers resistance to erythromycin. Following electrotransformation, mutants were selected on agar plates containing erythromycin and confirmed by Southern blotting and nucleotide sequencing. Bacteria were cultured in Todd–Hewitt broth containing 0.2% (w/v) yeast extract (THY; BD Biosciences, Rockville, MD, USA) without agitation at 37°C in 5% CO2, unless otherwise stated.
Antibiotic treatment and killing assays
Penicillin G, penicillinase and cerulenin were obtained from Sigma (St Louis, MO, USA). The microdilution broth method was used to determine the MIC of penicillin. Penicillin concentrations ranging from 0.01 to 0.05 mg/L were made in 1 mL of THY and bacterial growth in 50 µL was detected by an increase in turbidity using a 1 cm path length cell in a Spectronic 20D+ spectrophotometer. Penicillin-induced killing was assessed using both broth media and agar plates. For broth assays, S. pyogenes was cultured in 10 mL of THY broth in 12 mL Corning tubes until the mid-exponential phase of growth (OD600 
0.3) and penicillin or diluent (water) was added at specified concentrations. After 1, 4 and 24 h incubation, viable cfu were enumerated by dilution plating on THY plates containing agar and 3 U of penicillinase to inactivate any residual penicillin. For plate assays, 100 mid-exponential phase cfu were spread on THY agar plates containing 0.024 mg/L penicillin (twice the MIC). The plates were incubated for 18 h at 37°C and 5% CO2 and then flooded with 100 µL (40 U) of penicillinase and incubated for an additional 18 h to allow tolerant bacteria to grow. To inhibit fatty acid biosynthesis (FAB), cerulenin was added at concentrations between 2 and 50 mg/L, as described previously.28
Protein preparations
The wild-type and mutant strains were grown at 37°C in 50 mL of THY. At the mid-exponential phase of growth (OD600 0.3), either 0.024 mg/L penicillin or an equal volume of diluent (water) was added to test and control cultures, respectively, and the cultures incubated for an additional 1 or 4 h at 37°C. The cultures were then centrifuged for 15 min at 13 000 g at 4°C to collect the bacteria. The bacterial pellets were suspended with 500 µL of lysis buffer {7 M urea, 2 M thiourea, 4% (w/v) 3-[3-(cholamidopropyl)-dimethyl-ammonio]-1-propane sulphonate (CHAPS), 1% (v/v) immobilized pH gradient buffer (IPG; GE Healthcare, Piscataway, NY, USA) and 75 mM dithiothreitol (DTT)}, frozen and stored at –80°C. Protein was isolated and quantified by using a FastPrep protein isolation kit, a PlusOne 2-D Clean-up kit and a PlusOne 2-D Quant kit, as described by the manufacturer (GE Healthcare, Piscataway, NY, USA).
Two-dimensional gel electrophoresis
An IPGphor isoelectric focusing system using immobiline dry strips (24 cm) with a linear pH range of 4–7 was used, as described by the manufacturer (GE Healthcare, Piscataway, NY, USA). IPG strips were hydrated with 0.2 mg of protein in 450 µL of sample buffer for 12 h at 20°C. Isoelectric focusing was performed with 500 V for 500 volt-hours (Vh), 1000 V for 1000 Vh and 8000 V for 96 000 Vh at 20°C. The strips were incubated in SDS equilibration buffer [50 mM Tris–HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% SDS and Bromophenol Blue] for 10 min. SDS–PAGE separation was performed with a DALT II six electrophoresis apparatus (GE Healthcare, Piscataway, NY, USA) and 10% acrylamide resolving gels (0.1 by 23.4 by 19.5 cm) containing 1% (w/v) SDS. Rhinohide (Molecular Probes, Eugene, OR, USA) was added to the acrylamide as a strengthening agent, as instructed by the manufacturer. The running buffer consisted of 0.25 M Tris, 1.92 M glycine and 1% (w/v) SDS and electrophoresis was performed for 18 h at 75 V. Proteins were stained with Sypro Ruby (Molecular Probes, Eugene, OR, USA), and digital images were acquired with a Typhoon imager (GE Healthcare, Piscataway, NY, USA). Analysis of the gels, including protein spot detection and quantification, was performed with PDQuest software (Bio-Rad, Hercules, CA, USA). Protein spots were quantified by summing the values of pixels comprising each protein spot. Gels were normalized based on the sum of all protein spots detected in each sample.
Protein identification
Proteins of interest were excised from the SDS–PAGE gels with a robotic spot cutter (Bio-Rad, Hercules, CA, USA). Protease digestion and subsequent peptide purification was performed with a robotic liquid handling system (Waters, Milford, MA, USA), as described previously.25 Briefly, peptides (5–10 µL of the tryptic digest solution) were separated with a C18 reverse-phase column (13 by 25 micron; LC Packings, Sunnyvale, CA, USA) by using a capillary liquid chromatography system and eluted directly into a Micromass electrospray ionization quadrupole/orthogonal time-of-flight mass (Q-ToF Micro; Waters) hybrid spectrometer with a 3–40% gradient of ACN-0.1% formic acid over 40 min and a flow rate of 20 nL/min. Spectra were obtained in positive ion mode, deconvoluted and analysed with MassLynx 4.0 software (Waters). Protein Lynx Global Server v. 2.1 (Waters) was used to query the non-redundant NCBI database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA). Proteins were identified by matching MS/MS spectra from at least two tryptic peptides or by de novo peptide sequence determination, when only one MS/MS match was identified. SPy designations and additional annotations are based on the SF370 genome sequence.29
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Inactivation of rgg alters penicillin-mediated killing
The MIC of penicillin was 0.012 mg/L for both wild-type and rgg mutant strains of NZ131. In children, the concentrations of penicillin 1 and 21 days after intramuscular injection are 0.08 and 0.01 mg/L, respectively.30 Therefore, to assess pathogen killing, isogenic strains grown with either liquid or solid media were exposed to a clinically relevant concentration (0.024 mg/L; twice the MIC) of penicillin. In broth cultures (Figure 1), the wild-type strain lost 1.9 log10 cfu/mL (80-fold) after 24 h of exposure to penicillin compared with controls; in contrast, the rgg mutant culture lost only 0.3 log10 cfu/mL (2-fold). Similar results were obtained with penicillin concentrations of 0.1 and 1 mg/L (data not shown). The generation times of the wild-type and mutant strains under these conditions were 100 and 90 min, respectively. Penicillin-induced killing was also assessed on agar plates. Only 0.5% (±0.7) of the wild-type cfu survived penicillin exposure. In contrast, 21% (±0.5) of rgg mutant cfu were tolerant to penicillin-mediated killing (P < 0.01). Together, the results indicated that rgg inactivation affects penicillin-induced killing. Although the magnitude of tolerance was low, we reasoned that the mutant strain could be useful in detecting potential penicillin-induced changes in gene expression associated with cell death or tolerance.
|
Penicillin alters the S. pyogenes proteome
One possible explanation of the tolerance of the mutant strain was that inactivation of rgg disrupted an Rgg-dependent regulatory response to penicillin that normally results in cell death. Alternatively, differences in gene expression in the mutant strain, regardless of penicillin exposure, could confer tolerance. To test these models and to determine whether penicillin exposure influences the wild-type proteome, we compared soluble cellular proteins isolated from the mutant and wild-type strains after exposure to penicillin with controls. The strains were grown to the mid-exponential phase of growth, the cultures were divided and either penicillin (0.024 mg/L) or an identical volume of diluent (water) was added. After 1 and 4 h of incubation, cellular proteins were isolated and separated with two-dimensional gel electrophoresis. Samples obtained from the wild-type strain after 1 h of exposure to either penicillin or water contained an average of 385 and 447 protein spots, respectively. After 4 h, 516 and 518 protein spots were detected in treated and control samples, respectively. In samples isolated from the rgg mutant strain, 475 and 656 proteins were detected after 1 h of exposure to penicillin or diluent, respectively; after 4 h, 547 and 464 protein spots were detected in treated and control samples, respectively. Several proteins were excised from the gels and identified with tandem mass spectrometry, which assisted in spot matching and gel to gel comparisons. Statistically significant differences in protein abundance following penicillin exposure (P < 0.05) were identified in each strain and the results are reported in Tables 1 and 2. For comparison purposes, the results obtained from both strains are provided, even if a significant difference in protein abundance was detected in only one of the strains. The results confirmed a previous report that Rgg influences the abundance of proteins involved in metabolism and stress responses, which further validated the data.25
|
|
After 1 h of penicillin exposure, the abundance of 12 proteins, corresponding to 11 loci, was altered in at least one of the strains compared with controls (Table 1). Proteins involved in the metabolism of murein (MurD, UDP-N-acetylmuramoylalanine-d-glutamate ligase), pyruvate (AcoB, pyruvate dehydrogenase), fatty acid biosynthesis [FabF, 3-oxoacyl-(acyl-carrier-protein) synthase] and purine biosynthesis (PurA, adenylosuccinate synthetase) and glycolysis (Pgi, glucose-6-phosphate isomerase; Plr, glyceraldehyde-3-phosphate dehydrogenase; PyK, pyruvate kinase) were more abundant in wild-type and rgg mutant cultures exposed to penicillin compared with controls (Table 1). ClpX (ATP-dependent Clp protease subunit X) was detected in samples obtained from the wild-type strain only after the addition of penicillin (Table 1). In samples obtained from the rgg mutant strain, ClpX was detected in both the control and penicillin-exposed samples but was more abundant after penicillin exposure (Table 1). The abundance of the heat shock protein GroEL also increased after penicillin exposure in both strains (Table 1). Although statistically significant (P < 0.05), the magnitude of changes in response to penicillin was generally low (Figure 2), which may reflect the finding that only a fraction of the rgg mutant culture (
20%, based on results obtained using solid media) was tolerant to killing. In addition, we note that proteins not detected in the gels may be present below the limits of detection (1 ng) or may not be soluble under the conditions used.
|
After 4 h of penicillin exposure, the abundance of 16 proteins was altered in one strain or the other compared with controls (Table 2). Several were functionally related to each other including proteins involved in the pentose phosphate pathway and purine metabolism (PrsA, ribose-phosphate pyrophosphokinase; GuaB, inosine monophosphate dehydrogenase), lipid metabolism (AdhA, alcohol dehydrogenase I; GlmM, phospho-sugar mutase) oxidoreductases (GapN, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; SodA, superoxide dismutase), protein synthesis [RplA, 50S ribosomal protein L1; Tuf, elongation factor EF-Tu; TrmU, tRNA-(5-methylaminomethyl-2-thiouridylate)] and chaperones (DnaK, GroEL). Of note, the abundance of AdhA, which is probably involved in glycerolipid metabolism, decreased in the wild-type strain in response to penicillin but increased in the mutant strain (Table 2). In addition, GlmM, which is involved in lipid and cell wall biosynthesis was 10-fold more abundant in samples obtained from the mutant strain compared with the wild-type strain (Table 2). In contrast to changes identified after 1 h of penicillin exposure, a functionally related pattern of change was not apparent after 4 h of penicillin exposure.
Penicillin-induced killing is more effective in the exponential phase of growth. Although this is generally attributed to cell division, other growth phase-associated phenotypes may play a role. Some of the changes in protein expression associated with the mutant strain prior to penicillin exposure suggested that the proteome of the rgg mutant strain in the exponential phase of growth was similar to that of the wild-type strain in the stationary phase of growth. To test this idea further, the quantities of all the proteins detected in stationary-phase cultures of the wild-type strain were compared with the amount of each matched protein isolated from the rgg mutant strain during the exponential phase of growth. The results showed that the exponential-phase proteome of the mutant strain is similar (P < 0.001) to that of the wild-type strain in the stationary phase of growth (R2 = 0.82). In contrast, similar analysis of the exponential and stationary phase proteomes of the wild-type strain showed little correlation (R2 = 0.46).
Inhibition of fatty acid biosynthesis enhances penicillin-induced killing
Penicillin exposure was associated with an increase in the abundance of several FAB enzymes. To determine whether FAB is involved in cell death, penicillin-induced killing of the wild-type and rgg mutant strains was assessed in the presence of cerulenin, which inhibits FAB by inactivating ß-ketoacyl-synthases.31 Cerulenin slightly enhanced killing of the wild-type strain. Although the synergic effect of adding cerulenin was small, there was an apparent dose-dependent effect on bacterial cell killing (Figure 3). In contrast, 2 and 5 mg/L cerulenin did not enhance penicillin-induced killing of an rgg mutant strain and in some cases the exposed cultures had slightly more cfu/mL compared with the control cultures, which resulted in a negative value. However, the addition of 50 mg/L cerulenin enhanced penicillin-induced killing of an rgg mutant strain.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The results of the current study indicate that inactivation of rgg in S. pyogenes strain NZ131 is associated with decreased susceptibility of the pathogen to penicillin-induced killing. Although the magnitude of tolerance was small, this is the first locus of S. pyogenes to be identified that alters penicillin-mediated killing. Thus, it was of interest to use the mutant strain in an attempt to identify changes in the proteome specifically associated with penicillin-induced cell death. Using a proteomic approach, changes were identified among proteins involved in FAB, the pentose phosphate pathway, glycolysis and stress responses after penicillin exposure. Most of these penicillin-responsive proteins were more abundant in the rgg mutant strain, even in the absence of penicillin. Thus the tolerance associated with rgg inactivation is most likely due to penicillin-independent changes in the expression of one or more Rgg-regulated genes. Therefore, the results do not support a model whereby penicillin exposure specifically induces an Rgg-dependent regulatory response that results in bacterial cell death.
Stationary-phase bacteria, or bacteria otherwise starved for essential nutrients, are tolerant to penicillin-induced killing.32 Such phenotypic tolerance has been attributed to changes in protein composition.33 In several ways, the phenotype of the NZ131 rgg mutant strain during the exponential phase of growth resembles that of the wild-type strain in the stationary phase of growth. For example, the NZ131 rgg mutant strain expresses ArcABC, which facilitates the fermentation of arginine, during the exponential phase of growth; in contrast, the wild-type strain expresses the operon during the stationary phase of growth.25 Similar changes in the expression of the arcABC operon were associated with penicillin-tolerant mutants of S. gordonii.26 In S. gordonii, the mutations mapped outside the arc loci and were hypothesized to be in a gene encoding a global regulatory protein; however, it remains unclear whether any of the mutations were in orthologues of rgg. Moreover, the rgg mutant strain ferments arginine and serine during the exponential phase of growth even in the presence of glucose, similar to the catabolic activity of the wild-type strain during the stationary phase of growth.21 Finally, the exponential-phase proteome of the rgg mutant strain correlated directly with that of the wild-type strain in the stationary phase of growth, supporting the idea that the loss of growth phase-dependent regulation in the mutant strain contributes to tolerance.
Changes in the expression of stress-responsive proteins alter penicillin-associated killing of a variety of bacteria. For example, in S. aureus the abundance of GroES and transcription of groEL increases after exposure to oxacillin.34 Additional oxacillin-responsive genes include clpP and clpB.35 ClpX was more abundant among samples from both the wild-type and rgg mutant strains of S. pyogenes after 1 h of exposure to penicillin and was more abundant in the rgg mutant strain compared with the wild-type strain. Interestingly, in Bacillus subtilis, ClpX inhibits the assembly of FtsZ in a ClpP-independent manner.36 FtsZ is a tubulin-like protein essential for cell division.37 Thus penicillin-induced changes in the abundance of ClpX may affect FtsZ and disrupt proper assembly of cell wall precursors at the division septum, which may directly result in bacterial cell death.38 Finally, HtrA, which was more abundant in the rgg mutant strain, also contributes to penicillin tolerance in L. monocytogenes.39 Clearly the pleiotropic nature of the rgg mutant strain complicates the identification of changes in gene expression which are directly responsible for tolerance to penicillin.
FAB and penicillin exposure
Enzymes involved in FAB including AcoB, AcoL, AccB, FabG and FabF were more abundant in both wild-type and rgg mutant samples after 1 h of exposure to penicillin compared with controls (Table 1). As noted previously, the enzymes were also more abundant in the mutant strain compared with the wild-type strain, even in the absence of penicillin. In Streptococcus mutans, the synthesis of fatty acids increases nearly 70-fold after penicillin exposure.40 Although the regulation of FAB is not well characterized in S. pyogenes, it is typically coordinated with the synthesis of macromolecules such as phospholipids and peptidoglycan. Thus, the increase in FAB and coincident inhibition of peptidoglycan synthesis by penicillin would seem to be detrimental to the bacterium; however, cerulenin increased penicillin-induced killing of S. pyogenes suggesting that FAB protects against bacterial cell death. Although the effect of cerulenin on killing was <1 log unit, the results were consistent with previous reports using related bacteria. For example, the addition of cerulenin also increases penicillin-induced killing of Enterococcus faecalis and Escherichia coli.41–43 Penicillin also causes lipoteichoic acid to be released from the S. pyogenes cell wall44 and the excretion of lipids into the medium.45 Because lipids and lipoteichoic acids inhibit the activity of murein hydrolases,46–49 one possibility is that increased FAB in the absence of cell division promotes lipid excretion, which protects the pathogen from cell death by inhibiting murein hydrolase activity. We did not identify murein hydrolases in this study of predominately cytoplasmic proteins, presumably because they are secreted and associated with the cell wall. Additional information regarding streptococcal murein hydrolases and their role in penicillin-induced killing of S. pyogenes is necessary to test this possibility.
Many features of penicillin-induced killing of S. pyogenes are poorly understood at the molecular level including the fortuitous lack of penicillin resistance among clinical isolates, which seems unusual after several decades of prophylactic use to prevent acute rheumatic fever and the emergence of resistance in related bacteria. In this work, we identified and characterized a mutant strain of S. pyogenes that confers low-level tolerance to penicillin-mediated killing. In addition, we characterized the proteomes of the mutant and parental strain following penicillin exposure. Although clearly limited by the inability to detect all proteins (including membrane proteins), the results suggest that tolerance is due to changes in expression associated with rgg inactivation and not due to an altered regulatory response to penicillin exposure. Thus the stationary-phase pattern of gene expression in S. pyogenes, which is similar to that of the rgg mutant strain in the exponential phase of growth, could confer tolerance and thereby contribute to the clinical failure rate associated with penicillin treatment. Clearly, a better understanding of the mechanism of ß-lactam-induced bacterial cell death is important in identifying the cause of clinical treatment failures, in assessing the risk of penicillin resistance emerging in S. pyogenes, and in the development of new antimicrobial compounds.
Transparency declarations
None to declare.
|
Acknowledgements |
|---|
We thank I. Biswas, A. Dmitriev and K. Weaver for reviewing the manuscript. The project was supported by grant 0360026Z from the Greater Midwest affiliate of the American Heart Association and grants RO1401507 and RR16479-02 from the National Institutes of Health.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 Thom T, Haase N, Rosamond W, et al. (2006) Heart disease and stroke statistics—2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 113:e85–151.
2
Desjardins M, Delgaty KL, Ramotar K, et al. (2004) Prevalence and mechanisms of erythromycin resistance in group A and group B Streptococcus: implications for reporting susceptibility results. J Clin Microbiol 42:5620–3.
3
Sprunt K, Redman W, Leidy G. (1968) Penicillin resistant alpha streptococci in pharynx of patients given oral penicillin. Pediatrics 42:957–68.
4 Horn DL, Zabriskie JB, Austrian R, et al. (1998) Why have group A streptococci remained susceptible to penicillin? Report on a symposium. Clin Infect Dis 26:1341–5.[Web of Science][Medline]
5
Kaplan EL and Johnson DR. (2001) Unexplained reduced microbiological efficacy of intramuscular benzathine penicillin G and of oral penicillin V in eradication of group A streptococci from children with acute pharyngitis. Pediatrics 108:1180–6.
6
Horne D and Tomasz A. (1977) Tolerant response of Streptococcus sanguis to ß-lactams and other cell wall inhibitors. Antimicrob Agents Chemother 11:888–96.
7
Lewis K. (2000) Programmed death in bacteria. Microbiol Mol Biol Rev 64:503–14.
8 Bayles KW. (2003) Are the molecular strategies that control apoptosis conserved in bacteria? Trends Microbiol 11:306–11.[CrossRef][Web of Science][Medline]
9 Bayles KW. (2000) The bactericidal action of penicillin: new clues to an unsolved mystery. Trends Microbiol 8:274–8.[CrossRef][Web of Science][Medline]
10
Rice KC, Firek BA, Nelson JB, et al. (2003) The Staphylococcus aureus cidAB operon: evaluation of its role in regulation of murein hydrolase activity and penicillin tolerance. J Bacteriol 185:2635–43.
11
Groicher KH, Firek BA, Fujimoto DF, et al. (2000) The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance. J Bacteriol 182:1794–1801.
12
Brunskill EW and Bayles KW. (1996) Identification of LytSR-regulated genes from Staphylococcus aureus. J Bacteriol 178:5810–12.
13 Novak R, Henriques B, Charpentier E, et al. (1999) Emergence of vancomycin tolerance in Streptococcus pneumoniae. Nature 399:590–3.[CrossRef][Medline]
14 Zahner D, Kaminski K, van der Linden M, et al. (2002) The ciaR/ciaH regulatory network of Streptococcus pneumoniae. J Mol Microbiol Biotechnol 4:211–16.[Web of Science][Medline]
15
Mascher T, Heintz M, Zähner D, et al. (2006) The CiaRH system of Streptococcus pneumoniae prevents lysis during stress induced by treatment with cell wall inhibitors and by mutations in pbp2x involved in ß-lactam resistance. J Bacteriol 188:1959–68.
16
Begley M, Hill C, Ross RP. (2006) Tolerance of Listeria monocytogenes to cell envelope-acting antimicrobial agents is dependent on SigB. Appl Environ Microbiol 72:2231–4.
17 Lyon WR, Gibson CM, Caparon MG. (1998) A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes. EMBO J 17:6263–75.[CrossRef][Web of Science][Medline]
18
Chaussee MS, Ajdic D, Ferretti JJ. (1999) The rgg gene of Streptococcus pyogenes NZ131 positively influences extracellular SPE B production. Infect Immun 67:1715–22.
19
Chaussee MS, Watson RO, Smoot JC, et al. (2001) Identification of Rgg-regulated exoproteins of Streptococcus pyogenes. Infect Immun 69:822–31.
20
Chaussee MS, Sylva GL, Sturdevant DE, et al. (2002) Rgg influences the expression of multiple regulatory loci to coregulate virulence factor expression in Streptococcus pyogenes. Infect Immun 70:762–70.
21
Chaussee MS, Somerville GA, Reitzer L, et al. (2003) Rgg coordinates virulence factor synthesis and metabolism in Streptococcus pyogenes. J Bacteriol 185:6016–24.
22
Vickerman MM, Wang M, Baker LJ. (2003) An amino acid change near the carboxyl terminus of the Streptococcus gordonii regulatory protein Rgg affects its abilities to bind DNA and influence expression of the glucosyltransferase gene gtfG. Microbiology 149:399–406.
23
Rawlinson EL, Nes IF, Skaugen M. (2005) Identification of the DNA-binding site of the Rgg-like regulator LasX within the lactocin S promoter region. Microbiology 151:813–23.
24
Neely MN, Lyon WR, Runft DL, et al. (2003) Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes. J Bacteriol 185:5166–74.
25
Chaussee MA, Callegari EA, Chaussee MS. (2004) Rgg regulates growth phase-dependent expression of proteins associated with secondary metabolism and stress in Streptococcus pyogenes. J Bacteriol 186:7091–99.
26
Caldelari I, Loeliger B, Langen H, et al. (2000) Deregulation of the arginine deiminase (arc) operon in penicillin-tolerant mutants of Streptococcus gordonii. Antimicrob Agents Chemother 44:2802–10.
27
Chaussee MS, Gerlach D, Yu CE, et al. (1993) Inactivation of the streptococcal erythrogenic toxin B gene (speB) in Streptococcus pyogenes. Infect Immun 61:3719–23.
28
Daneo-Moore L, Bourbeau P, Weinstein R, et al. (1979) Effects of cerulenin on antibiotic-induced lysis of Streptococcus faecalis (S. faecium). Antimicrob Agents Chemother 16:858–61.
29
Ferretti JJ, McShan WM, Ajdic D, et al. (2001) Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 98:4658–63.
30 Peloso UC, De Souza JC, Botino MA, et al. (2003) Penicillin concentrations in sera and tonsils after intramuscular administration of benzathine penicillin G to children. Pediatr Infect Dis J 22:1075–8.[Web of Science][Medline]
31 Heath RJ, White SW, Rock CO. (2001) Lipid biosynthesis as a target for antibacterial agents. Prog Lipid Res 40:467–97.[CrossRef][Web of Science][Medline]
32 Charpentier E and Tuomanen E. (2000) Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae. Microbes Infect 2:1855–64.[CrossRef][Web of Science][Medline]
33 Tuomanen E. (1986) Phenotypic tolerance: the search for ß-lactam antibiotics that kill nongrowing bacteria. Rev Infect Dis 8:Suppl 3, S279–91.[Web of Science][Medline]
34 Singh VK, Jayaswal RK, Wilkinson BJ. (2001) Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS Microbiol Lett 99:79–84.
35
Utaida S, Dunman PM, Macapagal D, et al. (2003) Genome-wide transcriptional profiling of the response of Staphylococcus aureus to cell-wall-active antibiotics reveals a cell-wall-stress stimulon. Microbiology 149:2719–32.
36 Weart RB, Nakano S, Lane BE, et al. (2005) The ClpX chaperone modulates assembly of the tubulin-like protein FtsZ. Mol Microbiol 57:238–49.[CrossRef][Web of Science][Medline]
37 Weiss DS. (2004) Bacterial cell division and the septal ring. Mol Microbiol 54:588–97.[CrossRef][Web of Science][Medline]
38
Giesbrecht P, Kersten T, Maidhof H, et al. (1998) Staphylococcal cell wall: morphogenesis and fatal variations in the presence of penicillin. Microbiol Mol Biol Rev 62:1371–414.
39
Stack HM, Sleator RD, Bowers M, et al. (2005) Role for HtrA in stress induction and virulence potential in Listeria monocytogenes. Appl Environ Microbiol 71:4241–7.
40 Brissette JL and Pieringer RA. (1985) The effect of penicillin on fatty acid synthesis and excretion in Streptococcus mutans BHT. Lipids 20:173–9.[Web of Science][Medline]
41
Carson DD, Pieringer RA, Daneo-Moore L. (1981) Effect of cerulenin on cellular autolytic activity and lipid metabolism during inhibition of protein synthesis in Streptococcus faecalis. J Bacteriol 146:590–604.
42
Shungu DL, Cornett JB, Shockman GD. (1980) Lipids and lipoteichoic acid of autolysis-defective Streptococcus faecium strains. J Bacteriol 142:741–6.
43
Rodionov DG and Ishiguro EE. (1996) Dependence of peptidoglycan metabolism on phospholipid synthesis during growth of Escherichia coli. Microbiology 142:2871–77.
44
Kessler RE and van de Rijn I. (1981) Effects of penicillin on group A streptococci: loss of viability appears to precede stimulation of release of lipoteichoic acid. Antimicrob Agents Chemother 19:39–43.
45
Horne D, Hakenbeck R, Tomasz A. (1977) Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J Bacteriol 132:704–17.
46 Cleveland RF, Holtje JV, Wicken AJ, et al. (1975) Inhibition of bacterial wall lysins by lipoteichoic acids and related compounds. Biochem Biophys Res Commun 67:1128–35.[CrossRef][Web of Science][Medline]
47
Cleveland RF, Wicken AJ, Daneo-Moore L, et al. (1976) Inhibition of wall autolysis in Streptococcus faecalis by lipoteichoic acid and lipids. J Bacteriol 126:192–7.
48
Holtje JV and Tomasz A. (1975) Biological effects of lipoteichoic acids. J Bacteriol 124:1023–27.
49
Tomasz A and Waks S. (1975) Mechanism of action of penicillin: triggering of the pneumococcal autolytic enzyme by inhibitors of cell wall synthesis. Proc Natl Acad Sci USA 72:4162–6.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  W. M. McShan, J. J. Ferretti, T. Karasawa, A. N. Suvorov, S. Lin, B. Qin, H. Jia, S. Kenton, F. Najar, H. Wu, et al. Genome Sequence of a Nephritogenic and Highly Transformable M49 Strain of Streptococcus pyogenes J. Bacteriol., December 1, 2008; 190(23): 7773 - 7785. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||