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JAC Advance Access published online on March 5, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl560
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Gene expression profiling of the response of Streptococcus pneumoniae to penicillin

P. David Rogers1–5,*, Teresa T. Liu1, Katherine S. Barker1,4, George M. Hilliard5, B. Keith English3,4, Justin Thornton6, Edwin Swiatlo6,7 and Larry S. McDaniel6–8,

1 Department of Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA 2 Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA 3 Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA 4 Children's Foundation Research Center, Le Bonheur Children's Medical Center, Memphis, TN 38103, USA 5 Department of Molecular Sciences, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA 6 Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA 7 Division of Infectious Diseases, Department of Medicine, University of Mississippi Medical Center, Jackson, MS 39216, USA 8 Department of Surgery, University of Mississippi Medical Center, Jackson, MS 39216, USA

* Correspondence address. Children's Foundation Research Center of Memphis, Le Bonheur Children's Medical Center, 50 N. Dunlap Street, Room 304, West Patient Tower, Memphis, TN 38103, USA. Tel: +1-901-572-5387; Fax: +1-901-448-1741; E-mail: drogers{at}utmem.edu

Received 19 October 2006; returned 8 November 2006; revised 2 January 2007; accepted 3 January 2007


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Objectives: The aim of this study was to identify changes in the gene expression profile of Streptococcus pneumoniae in response to a subinhibitory concentration of penicillin in an effort to better understand mechanisms by which this organism copes with this stress.

Methods: S. pneumoniae serotype 2 strain D39 was grown for 1 h in the presence or absence of penicillin at a concentration equivalent to half the MIC (0.03 mg/L). RNA was isolated and gene expression profiles were compared using DNA microarrays. Differential expression of select genes was confirmed by real-time RT–PCR.

Results: A total of 386 genes were found to be responsive to penicillin. Up-regulated genes included those of the ciaR–ciaH operon, luxS, genes encoding cell envelope proteins and genes of the pst locus. Down-regulated genes included genes involved in competence, genes encoding capsular polysaccharide biosynthesis proteins, genes involved in fatty acid chain elongation and genes of the polyamine transporter operon.

Conclusions: Altered expression of these genes reflects a protective response to perturbation of the bacterial cell wall by penicillin. Such genes may represent potential therapeutic targets for enhancing the activity of penicillin against this organism and provide insight into novel mechanisms of penicillin resistance.

Key Words: antibiotics , genomics , microarrays


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Streptococcus pneumoniae is a major cause of pneumonia, meningitis, bacteraemia, otitis media and sinusitis in humans. The pneumococcus is a significant health threat, especially in the elderly and in children under the age of 5 years. Although penicillin exhibits activity against many isolates of S. pneumoniae, increased resistance to this antibiotic over the last three decades has significantly hindered its clinical utility.1

Penicillins exhibit bactericidal activity against S. pneumoniae; however, their precise mechanism of action is unclear. They inhibit bacterial growth by interfering with the synthesis of the cell wall. Specifically, they bind to the transpeptidase enzyme, thus inhibiting the cross-linking of strands of peptidoglycan.2,3 Penicillin-binding proteins (PBPs), found in the cell membrane, represent additional targets of penicillins.4 S. pneumoniae has developed mechanisms of resistance to penicillins, such as alterations in PBP encoding genes as well as in non-PBP-encoding genes that may be involved in other steps of biosynthesis of cell wall components.5

Microorganisms respond to environmental changes by altering the expression of genes and gene products critical to their continued survival. Gene and protein expression profiling have proven useful in understanding the defence mechanisms employed by microorganisms to cope with specific external toxic stresses such as the presence of an antibiotic in its immediate environment.6–11 Recently, microarray analysis has been successfully used to examine the effects of translation inhibitors on global transcription patterns of S. pneumoniae R6.12

The present study was undertaken to identify the global stress response of S. pneumoniae to the effects of penicillin. Using a functional genomic approach, we identified genes that are differentially expressed in S. pneumoniae in response to exposure to a subinhibitory concentration of penicillin. Such changes in the S. pneumoniae gene expression profile give insight into the mechanism of action of (and resistance to) this agent, as well as the acute stress response to this antibiotic.


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Bacteria and growth conditions

S. pneumoniae serotype 2 strain D39 was grown in 100–500 mL of Todd-Hewitt broth containing 0.5% yeast extract to an optical density at 600 nm (OD600) of 0.1. Cells were then grown in the presence or absence of penicillin at a concentration of 0.5x MIC (0.03 mg/L) for approximately two doubling times (~1 h). Cells were then collected by centrifugation and stored at –80°C.

RNA isolation

S. pneumoniae cell pellets were thawed on ice before resuspending in 2 mL of pre-heated (95°C) Trizol Max reagent (Invitrogen, Carlsbad, CA, USA). The cell suspension was then heated for 4 min at 95°C. Aliquots (250 µL) of the cell lysate were added to new microcentrifuge tubes containing 1 mL of Trizol reagent (Invitrogen) per tube, mixed gently and then stored on ice for 5 min. Chloroform (200 µL) was added to each tube, the tubes shaken for 15 s and then incubated on ice for 3 min. Separation of the aqueous phase from the organic phase was accomplished by centrifugation (~18 000 g) for 15 min at 4°C at which time the aqueous phase was transferred to new tubes containing 0.5 mL of isopropanol. The solution was mixed by inversion, and tubes were incubated on ice for 10 min. Total RNA was collected by centrifugation (~18 000 g) for 10 min at 4°C after which RNA pellets were washed in 70% ethanol (in DEPC-treated water) followed by 2 min centrifugation. The supernatants were removed carefully with a micropipette, and RNA pellets were then air-dried on ice for ~5 min. Each pellet was resuspended in 50 µL of DEPC-treated water, and the RNA solutions for each sample (within a single experiment) pooled into a single tube. To remove the DNA that contaminated the RNA samples, five 2 µL aliquots of each RNA sample were incubated in a 100 µL reaction mixture containing 1x DNase I reaction buffer and ~50 U of DNase I (Invitrogen). The reactions were incubated for 10 min at 37°C, 1 µL of 0.5 M EDTA, pH 8.0 was added to stop the reaction, an equal volume of phenol/chloroform/isoamyl alcohol (25 : 24 : 1; Sigma) was then added, and the samples shaken and incubated on ice for 10 min. Aqueous phase separation was accomplished by centrifugation (~18 000 g) for 5 min at 4°C at which time the aqueous phase was transferred to new tubes containing 100 µL of isopropanol. The solution was mixed by inversion, and tubes were incubated on ice for 30 min. Total RNA was collected by centrifugation (~18 000 g) for 10 min at 4°C after which RNA pellets were washed in 70% ethanol (in DEPC-treated water) followed by 2 min centrifugation. The supernatants were removed carefully with a micropipette, and RNA pellets were then air-dried on ice for ~5 min. The pellets from the five original aliquots were resuspended in a total of 30 µL of DEPC-treated water. RNA integrity was visualized by electrophoresis, and RNA concentrations were approximated by spectrophotometry at 260 and 280 nm.

Probe preparation and microarray hybridization

The S. pneumoniae microarray was manufactured by Eurogentec (Seraing, Belgium) in collaboration with the Molecular Genetics group at the University of Groningen, The Netherlands and Laboratory of Pediatrics, Erasmus Medical Center, Rotterdam, The Netherlands. Primers for each of the 2088 putative ORFs in the S. pneumoniae genome were designed to amplify a specific region of each ORF.

Total RNA sample (25 µg) and 5 µg of LuxA RNA control (Promega, Madison, WI, USA) were added to a mixture of specific primer mix, dNTPs (including Cy3- or Cy5-dCTP) (NEN Life Sciences, Boston, MA, USA), DTT in 5x first-strand buffer. The reaction mixture was denatured at 65°C for 5 min and incubated at 42°C for 5 min, after which RNasin (Promega) and Superscript II RT (LifeTechnologies/Invitrogen) were added to the mixture. The reaction proceeded at 42°C for 1 h, after which additional Superscript II RT was added, and the reaction mixture incubated at 42°C for an additional hour. To stop the reaction, EDTA and NaOH were added; the mixture was incubated at 65°C for 20 min and acetic acid was added. Five microlitres each of the Cy3- and Cy5-labelled probes were mixed with calf thymus DNA and EGT hybridization buffer (Eurogentec), incubated at 65°C for 2 min and snap-cooled. The mixture was applied to the array slides under glass cover slips. Hybridization was performed at 37°C overnight in a humidified chamber. To wash the slides, the cover slip was removed and the slide incubated at room temperature in 0.2x SSC (20x SSC stock consists of 3 M sodium chloride and 0.3 M sodium citrate) + 0.1% SDS for 5 min, rinsed at room temperature with 0.2x SSC for 5 min and spin-dried for 5 min. Slides were scanned using a ChipReader microarray scanner (Virtek Vision Intl, Waterloo, Ontario, Canada).

Data analysis

GenePix 1.0 software (Molecular Devices, Sunnyvale, CA, USA) was used for image analysis. The local background values were calculated from the area surrounding each feature and subtracted from the total feature signal values. These adjusted values were used to determine the differential gene expression for each feature. A normalization factor was applied to account for systematic differences in the probe labels by using the differential gene expression ratio to balance the Cy5 signals. Only features with a mean balanced differential expression ratio ≥2.0 or ≤–2.0 for both features representing a given cDNA on the array in two biologically and technically independent experiments were considered to be differentially expressed. DNA sequences were annotated on the basis of BLASTn searches using the StreptoPneumoList database (http://genolist.pasteur.fr/streptopneumolist/index.html).

Real-time, RT–PCR

An aliquot of the RNA preparations used in the microarray experiments was saved for quantitative real-time RT–PCR follow-up studies. First-strand cDNAs were synthesized from 2 µg of total RNA in a 21 µL reaction volume using the SuperScript First-Strand Synthesis System for RT–PCR (Invitrogen) as per the manufacturer's instructions. Quantitative real-time PCRs were performed in triplicate using the 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Independent PCRs were performed using the same cDNA for both the gene of interest and 16S rRNA, using the SYBR® Green PCR Master Mix (Applied Biosystems). Gene-specific primers were designed for the gene of interest and 16S rRNA using Primer Express® software (Applied Biosystems) and the Oligo Analysis and Plotting Tool (Qiagen, Valencia, CA, USA). Primer sequences are listed in Table 1. The PCR conditions consisted of AmpliTaq Gold activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified using the software provided with the 7000 Sequence Detection System. The change in fluorescence of SYBR Green I dye in every cycle was monitored by the system software, and the threshold cycle (CT) above background for each reaction was calculated. The CT value of 16S rRNA was subtracted from that of the gene of interest to obtain a CT value. The CT value of an arbitrary calibrator (e.g. untreated sample) was subtracted from the CT value of each sample to obtain a dCT value. The gene expression level relative to the calibrator was expressed as 2–CT. The mean expression levels between two groups were compared using Student's t-test.


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  		Table 1.. Sequences of primers used for real-time RT–PCR assays

 

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Whole genome microarray comparisons of mRNA levels were performed for strain D39 treated with and without exposure to a subinhibitory concentration of penicillin. Two separate and independent cultures were used for biological replicates. A total of 386 genes were found to be differentially expressed upon exposure to penicillin. Of these, 128 showed an increase in expression and 258 showed a decrease in expression. Penicillin-responsive genes are shown in Tables 2 and 3. The category of genes with the largest number of responses (beyond those encoding hypothetical proteins and proteins of unknown function) was those involved in transport and binding (10%), energy metabolism (7%), regulatory functions (7%), signal transduction (5%), cellular processes (4%), protein fate (4%), cell envelope (3%) and DNA metabolism (3%). To validate differential expression of genes identified by microarray analysis, we performed real-time RT–PCR for five genes of interest. All five genes were up-regulated in response to penicillin by at least 4-fold and were directionally consistent with changes in expression observed by microarray analysis (Figure 1).


Figure 1
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  		Figure 1.. Quantitative real-time RT–PCR analysis of selected genes identified as up-regulated by microarray experiments. Data are shown as means ± SD. The mean expression levels between two groups were compared using Student's t-test.

 


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  		Table 2.. Genes up-regulated in S. pneumoniae serotype 2 strain D39 in response to exposure to penicillin at 0.5x MIC for 1 h

 


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  		Table 3.. Genes down-regulated in S. pneumoniae serotype 2 strain D39 in response to exposure to penicillin at 0.5x MIC for 1 h

 

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In the present study, we have examined the effects of a subinhibitory concentration of penicillin on the gene expression profile of S. pneumoniae. ß-Lactam antibiotics are known to interact with PBPs at subinhibitory concentrations, and cell lysis and shedding of membrane components have been observed at such concentrations.13–15 We postulated that acute changes in gene expression would reflect stress responses that might afford protection from this class of antibiotic. We therefore examined changes in the gene expression profile of S. pneumoniae in response to penicillin using DNA microarray analysis.

Penicillin induced the expression of the CiaR–CiaH operon and down-regulated competence genes including the operon encoding the ComDE two-component signal transduction system (TCSTS). The CiaRH system was the first TCSTS identified in S. pneumoniae and appears to be a negative regulator of competence.16 This TCSTS was identified during studies of laboratory-derived stepwise acquisition of ß-lactam resistance in S. pneumoniae strain R6. In addition to mutations in PBPs, mutations associated with resistance in this strain were also present in the histidine protein kinase gene ciaH.16–18 Indeed, specific gain-of-function mutations in ciaH, such as R6ciaHC306, have been identified, which confer ß-lactam resistance, suggesting that this TCSTS may control genes critical to the biosynthesis and maintenance of the cell wall.16,18–20 Interestingly, mutant R6ciaHC306 has also been shown to exhibit a deficiency in transformation.16 Microarray analysis was previously used to show that the entire competence regulon was shut down in this mutant. Recently, the CiaRH system was shown to be critical for survival in the presence of cell wall inhibitors, suggesting a central role for this system in cell integrity.21

The ComDE TCSTS is required for response to the comC-encoded competence stimulating peptide (CSP), which regulates competence for genetic transformation.22 In addition to the CiaRH system, evidence was recently provided suggesting that LuxS may also contribute to the down-regulation of competence in S. pneumoniae. Indeed, we observed LuxS to be up-regulated in response to penicillin.23 Our findings are consistent with these studies, suggesting that induction of the CiaR–CiaH system and down-regulation of comD–comE (and other genes involved in competence) may represent a mechanism by which S. pneumoniae defends itself against cell wall damage secondary to penicillin. Recently, Prudhomme et al.24 demonstrated that certain antibiotics induce competence in S. pneumoniae. This was observed in response to aminoglycosides, fluoroquinolones and mitomycin C. No induction of competence was observed in response to the ß-lactam ampicillin or the third-generation cephalosporin cefotaxime. These findings are consistent with our observation of down-regulation of comD–comE in response to penicillin.

Several genes encoding cell envelope proteins were responsive to penicillin. Up-regulated genes included two LysM domain proteins (SP0107 and SP2063), a cell wall surface anchor family protein (SP1833) and a membrane protein (SP1972). The exact function of these proteins is poorly understood. Down-regulated genes included those encoding capsular polysaccharide biosynthesis proteins Cps4A, Cps4B and Cps4C, a putative capsular polysaccharide biosynthesis protein (SP1837), a cell wall surface anchor family protein (SP1992), the glycosyl transferase CpoA and the rod shape-determining proteins MreC and MreD. Like ciaH, mutations in the gene encoding CpoA have been shown to confer ß-lactam resistance and a defect in competence development. It has been suggested that competence is dependent upon proper cell wall maintenance.25 MreC and MreD are suspected to function as part of a complex required for some aspect of peptidoglycan biosynthesis.26 Down-regulation of these genes in response to penicillin may elicit changes in the cell wall that provide protection against the ß-lactam antibiotics.

Penicillin substantially induced the expression of the Pst locus (PstSACB and PhoU) in S. pneumoniae. This system has been shown to participate in the uptake of inorganic phosphate and appears to play a role in transformation and penicillin tolerance as deficiencies in both phenotypes were observed upon insertion duplication mutagenesis of pstB.27 Recently, pstS was identified through proteomic analysis as being up-regulated in association with experimentally induced penicillin resistance.28 Further examination revealed that the pstS, pstC and phoU transcripts were also up-regulated, suggesting a role for phosphate transport in this process. Additionally, pstS was also found to be up-regulated in penicillin-resistant clinical isolates. Disruption of pstS significantly reduced penicillin resistance, demonstrating direct involvement of this system in penicillin resistance. Although the mechanism by which the Pst system contributes to this phenotype is unclear, its activation in response to penicillin exposure likely represents a defensive stress response to this antibiotic.

Exposure to penicillin resulted in the down-regulation of genes encoding enzymes involved in fatty acid metabolism, particularly those associated with fatty acid chain elongation. These included fabK, fabD, fabG, fabF, fabZ, accC, accD and accA. Alterations in other cell membrane components such as phospholipids have been shown to have an impact on susceptibility to other antibiotic classes such as the macrolides.29 It is therefore possible that alterations in fatty acid biosynthesis gene expression may lead to changes in the cell membrane that would favour cell survival in the presence of penicillin.

Putrescine, spermidine and cadaverine are all polyamines that are present in all cell types and are required for a number of physiological processes, particularly through interactions with ATP, DNA and RNA.30–34 Interestingly, penicillin exposure resulted in the down-regulation of the polyamine transporter (Pot) operon.35 Genes of this operon have recently been implicated in pneumococcal pathogenesis.35–37 Although it is unclear why S. pneumoniae might suppress expression of this operon in response to penicillin, it is tempting to speculate that this antibiotic might elicit secondary effects that alter the virulence factors of this organism in favour of the host. Polyamines are required in high concentrations in rapidly dividing cells. It is possible that down-regulation of the Pot operon may reflect slowed growth in response to penicillin.

Recently, Haas et al.38 examined changes in the gene expression profiles of S. pneumoniae strains T4 (TIGR4) and Tupelo (vancomycin tolerant). Interestingly, many genes that responded to penicillin in the present study responded similarly to vancomycin in the previous study. These included the up-regulation of the stress response genes htpX and lemX, the genes encoding {alpha}-amylase (amy), 4-{alpha}-glucotransferase (malQ), a glycogen phosphorylase protein (SP2106), a prolyl oligopeptidase family protein (SP1343) and neuraminidase B (nanB) and the TCSTS genes ciaR and ciaH. Down-regulated genes common to both penicillin and vancomycin exposure included the genes encoding the rod shape-determining proteins MreC and MreD, adenylate kinase (adk), lysine decarboxylase (cad), spermidine synthase (speE), carboxynorspermidine decarboxylase (nspC), a carbon–nitrogen hydrolase family protein (SP0922), the ß subunit of ribonucleoside-diphosphate reductase 2 (nrdF), the capsular polysaccharide biosynthesis proteins Cps4A-C and adhesion lipoprotein (lmb), the CiaRH regulon PTS system genes manN, manM and manL, the iron compound ABC transporter genes piuD and piuA and genes encoding an ATP-dependent RNA helicase (SP1586), transcription-repair coupling factor (mfd) and lactoylglutathione lyase (gloA). The CiaRH system has been shown to respond to cell wall inhibitors and plays a major role in ensuring cell wall integrity.23 Genes that respond similarly to both penicillin and vancomycin may represent a common stress response to cell wall inhibitors, some of which may be critical in protecting the cell from the effects of these agents.

There was no apparent change in the expression of genes encoding choline binding proteins, which are surface proteins that play a role in pneumococcal virulence.39 This included the well-characterized proteins PspA and PspC.40,41 The data suggest that these proteins do not respond to the stress of penicillin exposure and support the idea that these proteins are more responsive to host factors that affect expression during the natural history of pneumococcal infection such as carriage in the nasopharynx and persistence during bacteraemia.42

In the present study, we have identified S. pneumoniae genes that are differentially expressed in response to exposure to a subinhibitory concentration of penicillin. Many of these genes have previously been implicated in resistance or tolerance to penicillin, suggesting their altered expression here is part of a protective response to perturbation of the bacterial cell wall. Such genes may represent potential therapeutic targets for enhancing the activity of penicillin against this organism. Likewise, other changes in gene expression identified here may suggest novel mechanisms of penicillin resistance. Further study of these genes in this context is warranted.


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B. K. E. receives grant support from Cubist Pharmaceuticals.


   Acknowledgements
 
We thank Elizabeth Meals for technical assistance associated with this work. This work was supported by a grant from Abbott Laboratories.


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