JAC Advance Access originally published online on October 31, 2006
Journal of Antimicrobial Chemotherapy 2007 59(1):35-42; doi:10.1093/jac/dkl442
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Proinflammatory activation of Toll-like receptor-2 during exposure of penicillin-resistant Streptococcus pneumoniae to ß-lactam antibiotics
1 Division of Genomic Medicine, University of Sheffield Medical School Beech Hill Road, Sheffield S10 2RX, UK 2 Department of Biological Sciences, University of Warwick Coventry, UK
*Corresponding author. Tel: +44-114-271-2027; Fax: +44-114-271-3892; E-mail: r.c.read{at}shef.ac.uk
Received 3 July 2006; returned 31 July 2006; revised 25 September 2006; accepted 4 October 2006
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Abstract |
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Objectives: Disease caused by penicillin-resistant Streptococcus pneumoniae (PRSP) is associated with more suppurative complications than disease caused by penicillin-susceptible S. pneumoniae (PSSP). Exposure of S. pneumoniae to ß-lactam antibiotics enhances the proinflammatory activation of human cells by pneumococci via Toll-like receptor-2 (TLR2). To test the hypothesis that penicillin resistance influences cellular TLR2 activation by ß-lactam-exposed pneumococci, we compared TLR2 induction by PSSP (MIC 0.06 mg/L) and a high-level PRSP clinical isolate (159; MIC 16 mg/L) following exposure to penicillin and cefotaxime.
Methods: Both organisms were treated with penicillin or cefotaxime at and around the MIC. TLR2 signalling was measured as relative IL-8 promoter activation in transfected HeLa cells.
Results: On exposure to penicillin, log-phase PSSP and PRSP induced TLR2-proinflammatory activation at levels significantly higher than unexposed bacteria, and maximal in each case at the MIC. Transformants containing low-affinity penicillin-binding proteins (PBP) 2x, 1a and 2b exhibited stepwise resistance to cefotaxime and penicillin. TLR2 activation following penicillin treatment was dependent on an abnormal cell wall (PBP1a and 2x) and autolysis (PBP2b). High affinity PBP2x was required for this effect to be observed in log-phase pneumococci exposed to cefotaxime at the MIC. Cefotaxime-mediated TLR2 activation was not observed in lag-phase transformants exposed to sub-lethal concentrations.
Conclusions: These data show that PRSP have similar TLR2-proinflammatory effects to PSSP when exposed to ß-lactam antibiotics but the antibiotic concentration relative to the MIC is critical. This has implications for treatment of pneumococcal disease when tissue concentrations of antibiotic are close to the MIC.
Keywords: S. pneumoniae , cefotaxime , penicillin-binding proteins , PBPs
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Introduction |
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Streptococcus pneumoniae is a major cause of community-acquired pneumonia and invasive disease including meningitis.1 ß-Lactam antibiotics such as penicillin or cefotaxime are agents used commonly for the treatment of pneumococcal disease. Resistance to ß-lactam antibiotics occurs because of alterations in the penicillin-binding proteins (PBPs) that cause decreased affinity for the antibiotic.2–4 S. pneumoniae has six PBPs; 1a, 1b, 2a, 2x, 2b and 3.2 Penicillin has the highest affinity for PBP2x, 2b and 1a, whereas cephalosporins target PBP1a, 2x and 2a.2,5–8 Antibiotic inhibition of PBP2b is associated with autolysis at the MIC.5–8 Inhibition of PBP2x prevents septal cross-links and daughter cell separation, producing bacteria with abnormal morphologies.5 PBP1a is also associated with septal cross-wall formation and may contribute to abnormal morphology following inhibition by antibiotic.9 Treatment of pneumococcal infections in experimental models and during clinical infection can result in paradoxical enhancement of inflammation as a result of the release of proinflammatory cell wall products such as lipoteichoic acid and peptidoglycan.10 Toll-like receptor-2 (TLR2) is a major pattern recognition receptor for the proinflammatory components of the cell wall of S. pneumoniae,11 and we have shown previously that penicillin enhances the TLR2-mediated proinflammatory activity of this organism.12 These enhancements are dependent on the MIC ratio, bacterial growth stage, duration of antibiotic exposure and pneumococcal autolysin.12
Cohort studies of individuals with penicillin-resistant S. pneumoniae (PRSP) infection have revealed an excess of suppurative complications of pneumococcal disease such as empyema but no increase in mortality.13 Treatment schedules for ß-lactam antibiotics are based on time–dose intervals that may promote troughs of sub-MIC or near-MIC concentrations of antibiotic within infected tissues. The treatment of pneumococcus-infected individuals with doses of antibiotics that achieve tissue concentrations that are sub-MIC will likely promote resistance development and local bacterial proliferation.14–17 It is possible that sub-MIC concentrations of antibiotics may also be associated with altered release of proinflammatory components from the pneumococcal cell wall—we have shown previously that treatment of lag-phase S. pneumoniae with sub-MIC concentrations of penicillin and cefotaxime enhances TLR2-mediated proinflammatory activity in transfected HeLa cells.12,18
As alteration in the PBPs can influence the nature of the antimicrobial activity of ß-lactam antibiotics, we postulated that penicillin-resistant pneumococci induce altered cellular proinflammatory characteristics during exposure to penicillin and cefotaxime. Therefore, we compared the relative penicillin and cefotaxime-induced TLR2 activation of penicillin-susceptible (PSSP) and penicillin-resistant (PRSP) clinical isolates of S. pneumoniae, and also the effect of stepwise mutations of PBP2x, 1a and 2b on this activity.
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Bacteria
Strains and their characteristics are listed in Table 1. All bacteria were grown in brain heart infusion broth supplemented with 10% heat inactivated fetal calf serum at 37°C, 5% CO2. PBP 1a, 2b and 2x genes were amplified from chromosomal DNA from 159, a high-level penicillin-resistant clinical isolate of S. pneumoniae19 by PCR using primers described previously.20 DNA fragments were cloned into a TATM vector plasmid (Invitrogen, UK) and plasmid extracted from transformed Escherichia coli using WizardTM miniprep kit (Promega).19
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Transformation of competent S. pneumoniae
D39 S. pneumoniae was transformed using a method adapted from Pozzi et al.21 Briefly, bacteria were cultured in C-medium containing 5 µL of 100 mM calcium chloride, 8 µL of 10% BSA, 10 µL of 10 ng/µL competence stimulating peptide-1 (CSP-1) (kindly donated by D. A. Morrison, University of Illinois, Chicago, USA) and 500 ng of plasmid DNA. Cells were incubated for 3 h at 37°C to permit expression of resistance. Finally, resistant cells were selected by culture of S. pneumoniae on blood agar plates containing increasing concentrations of cefotaxime and penicillin (mg/L).22 Strain D392x was generated by transforming the D39 isolate with a cloned PBP2x gene from isolate 159. Strain D392x1a was created by transforming strain D392x with the cloned PBP1a gene from isolate 159. Strain D392x1a2b was created by transforming the D392x1a strain with the cloned PBP2b gene from the 159 isolate. Antibiotics used for stepwise resistance selection were cefotaxime (strain D392x, strain strain D392x1a) and penicillin (strain D392x1a2b) respectively.
Antibiotics and MIC determination
Benzyl penicillin (penicillin) and cefotaxime were purchased from Sigma-Aldrich, UK. The MIC for bacteria to use in transfected HeLa cell experiments was determined using the macrobroth dilution method according to CLSI (formerly NCCLS) guidelines.23
Treatment of S. pneumoniae with antibiotic for TLR2 induction studies
D39 transformants of S. pneumoniae were exposed to 1/8 MIC or MIC for 6 h as described previously.12,18 To compare TLR2 induction by PRSP and PSSP, bacteria were treated with penicillin at a final concentration of 0, 0.0075, 0.015, 0.03, 0.06, 0.6, 2, 4, 8, 16 or 160 mg/L. Lag-phase bacteria (5 x 106 cfu/mL, optical density at 620 nm, 0.044), or early log growth (log growth) (OD620 0.099, adjusted to 5 x 106 cfu/mL) were treated with antibiotic for 6 h before inoculation onto transfected HeLa cells. Further antibiotic activity was stopped by use of 10 mg/L penicillinase (Bacillus cereus, Sigma-Aldrich, UK). Bacterial counts were checked spectrophotometrically and by viable count analysis. Controls included untreated bacteria, and broth plus antibiotic.
HeLa cell transfection, stimulation and the dual luciferase reporter assay
HeLa cells do not express TLR2 or CD14.24 Expression plasmids for human TLR2 and CD14 together with luciferase-tagged reporter vectors were transiently transfected into HeLa cells (seeded at 1.5 x 105 cells/well) using Superfect transfection reagent (UK-Qiagen) as described elsewhere.24,25 Empty vector (pcDNA3, Invitrogen) in place of TLR2 was used as a transfection control. Reporter and expression plasmids were purified from recombinant E. coli using the endotoxin-free maxi purification kit (UK-Qiagen) according to the manufacturer's guidelines. Reporter vectors were ptk-rluc (Promega) that contains the Renilla luciferase gene tagged to a constitutive reporter and pIL-8 pluc, which contains sequences of the human IL-8 gene (GenBank M2813C) cloned into pGL3-basic vector (Promega) to initiate synthesis of firefly luciferase as described elsewhere.25 Twenty-four hours after transfection, 10 µL volumes of test and control were applied to the transfected cells and incubated for 6 h as described previously.25 HeLa cells were passively lysed and firefly and Renilla luciferase levels determined using the dual luciferase reporter assay (Promega, UK) according to the manufacturer's instructions. Luminescence was measured as relative light units using an ML3000 luminometer. The number of relative light units is directly proportional to the level of IL-8 promoter activity in normalized data. Data were further corrected by normalization with empty-vector control.12,18
Statistical analysis
All data were analysed for normal distribution using the Kolmogrov–Smirnov test.26 Comparison of D39 penicillin-susceptible strain with PBP transformant or comparison of strain D39 with 159 PRSP clinical isolate were analysed using the unpaired Student's t-test. Antibiotic-treated versus-untreated strains were analysed using the ANOVA with Bonferroni correction.27 For the Bonferroni significance (P) was set at 0.025 for two, 0.016 for three, or 0.0045 for eleven separate comparisons.
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Penicillin-induced enhancement of TLR2 activation by PSSP and PRSP is maximal at the MIC for each strain
Table 1 lists details and characteristics of S. pneumoniae strains used. HeLa cells, transfected with cDNA encoding TLR2, were inoculated with clinical isolates of PSSP or PRSP exposed to penicillin concentrations ranging from 0 to 160 mg/L. Subsequent activation of the reporting IL-8 promoter by log-phase bacteria was enhanced maximally by exposure to a concentration of penicillin equivalent to the MIC for each of the isolates (Table 1). The effect of penicillin treatment on TLR2 stimulation by PRSP and PSSP is shown in Figure 1. When compared with untreated bacteria, exposure of both strains to penicillin at the MIC (PSSP, 0.06 mg/L; PRSP, 16 mg/L) significantly enhanced IL-8 promoter activation via TLR2. There was no enhanced proinflammatory activity of PRSP at concentrations less than the MIC; in particular, at the concentration of penicillin that enhanced PSSP activation of HeLa cells (MIC 0.06 mg/L). Notably, enhanced TLR2 activation was not observed when PSSP was treated at concentrations higher than its MIC, probably because of relatively low cumulative numbers of bacteria over the 6 h incubation with higher doses of the antibiotic.
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P < 0.0045, D39 treated versus untreated (ANOVA with Bonferroni).Additionally, as reported previously,28 treatment of lag-phase PSSP at 1/8 MIC (but not the MIC) enhanced IL-8 activation. However, in contrast the same concentration of penicillin (0.0075 mg/L) did not enhance proinflammatory activation by PRSP (data not shown).
Low-affinity PBP2x, PBP1a and PBP2b each increase the MIC for D39 S. pneumoniae of penicillin and cefotaxime
Table 1 shows the MIC antibiogram for D39 and D39 PBP transformants with respect to penicillin and cefotaxime. Transformation of isolate D39 with low-affinity PBP2x increased the MIC of penicillin by 2-fold to 0.12 mg/L and cefotaxime by 8-fold to 1 mg/L rendering the organism moderately resistant to these antibiotics (CLSI).29 Further transformation of strain D392x with low-affinity PBP1a increased the penicillin MIC more than 15-fold. Cefotaxime resistance of strain D392x1a also increased 20-fold to 2.5 mg/L producing an intermediately cefotaxime-resistant organism. Further transformation with low-affinity PBP2b to produce strain D392x1a2b increased the penicillin MIC to 16 mg/L producing a high-level penicillin-resistant transformant (MIC > 2 mg/L),29 which is the same level of MIC as the resistant clinical isolate (159) used in this study.
Low-affinity PBP mutants of S. pneumoniae retain penicillin-enhanced activation of TLR2 that is conditional on the MIC ratio
We postulated that cumulative mutation of the PBPs might alter the observed enhancement of TLR2 activation during penicillin exposure of S. pneumoniae. None of the mutants exhibited altered TLR2 activation in the absence of penicillin (Figure 2). However, treatment of strain D392x containing low-affinity PBP2x in 1/8 MIC penicillin during lag growth or the MIC during log-phase resulted in similar TLR2 activation when compared with penicillin-treated D39 at the same MIC ratio (P > 0.05 both comparisons, Student's t-test, Figure 2a,b,e and f). Compared with untreated bacteria there were significant increases in TLR2 induction by D392x during exposure to 1/8 MIC penicillin during lag phase and exposure to MIC penicillin during early log phase (P < 0.016, both comparisons, ANOVA with Bonferroni, Figure 2b and f).
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The D39 transformant strain D392x1a containing low-affinity PBP2x and PBP1a and the multiple PBP strain D392x1a2b containing low-affinity PBP2x, PBP1a and PBP2b exhibited significantly greater TLR2 activation than untreated bacteria when treated with 1/8 MIC during lag phase (P < 0.016, for both comparisons, ANOVA with Bonferroni, Figure 2c and d) and MIC treatment during log phase (P < 0.016, for both comparisons, ANOVA with Bonferroni, Figure 2g and h).
A significant change in proinflammatory activity was also observed upon comparison of wild-type D39 and strain D392x1a when exposed to 1/8 MIC penicillin (lag-phase organisms, Figure 2a and c) and to MIC penicillin (log-phase organisms, Figure 2e and g). Both transformants induced significantly less IL-8 promoter activation than wild-type (P < 0.05, both comparisons, Student's t-test), though the size of the effect was small. As expected, the absolute concentrations of penicillin capable of enhancing proinflammatory activity were apparently influenced by PBP mutation. For example a concentration of 0.12 mg/L penicillin enhanced the proinflammatory activity of log-phase strain D392x, but not strain D391a2x (Figure 2f and g).
Mutation of PBPs alters TLR2 activation in the presence of cefotaxime
Exposure of the D392x strain to cefotaxime at 1/8 MIC during lag growth resulted in a significant decrease in TLR2 signalling when compared with D39 at the same MIC ratio (P < 0.005, Student's t-test, Figure 3a and b) and did not enhance TLR2 activation when compared with untreated strain D392x (P > 0.025, ANOVA with Bonferroni, Figure 3b). Treatment of lag-phase strain D392x1a in 1/8 MIC cefotaxime exhibited a similar reduction in TLR2 activity when compared with wild-type isolate D39 (P < 0.005, Student's t-test, Figure 3a and c) but again did not enhance TLR2 activation when compared with untreated D392x1a (P > 0.025, ANOVA with Bonferroni, Figure 3c). Culture of the D392x transformant in MIC cefotaxime during log growth produced a significant increase in TLR2 signalling when compared with both wild-type D39 (P < 0.005, Student's t-test, Figure 3d and e) and untreated D392x (P < 0.025, ANOVA with Bonferroni, Figure 3e) at the same MIC ratios. Log D392x1a treated with MIC cefotaxime exhibited TLR2 signalling that did not differ from untreated bacteria (P < 0.025, ANOVA with Bonferroni, Figure 3f) or wild-type isolate D39.
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Work presented here shows that proinflammatory activation of cellular TLR2 by penicillin- or cefotaxime-exposed S. pneumoniae is dependent on the ratio of the local antibiotic concentration to the MIC for the target strain. The clinical relevance of this work is that ß-lactam-mediated inflammation within human tissue during treatment of pneumococcal disease may be closely related to the concentration of drug achieved relative to the bacterial MIC, which will be higher for penicillin-resistant pneumococci. We have shown previously that on exposure to penicillin, the activation by pneumococci of human cells via TLR2 is enhanced, which may be attributed to the release of cell wall components as a consequence of autolysis.12 This activity was shown to be maximal at sub-MIC concentrations in the case of lag-phase bacteria, and maximal at the MIC in the case of log-phase bacteria. Here we not only show that enhanced TLR2 induction also occurs in PRSP but that this is also critically dependent on the MIC ratio. Treatment of both PRSP and PSSP with MIC penicillin resulted in significant enhancement of IL-8 promoter activation but PRSP did not exhibit this activity at 1/8 MIC. The strain 159 PRSP used in this study has an altered cross-linked peptidoglycan (data not shown) consistent with the observations of Garcia-Bustos and Tomasz30 and Severin et al.31 of altered peptidoglycan of resistant organisms compared with susceptible S. pneumoniae. Antibiotic susceptible D39 has a normal cell wall (data not shown) and this may account, in part, for the differences between strains at this concentration. Stepwise transformation of PBPs increased the MIC, and these transformants retained penicillin-enhanced TLR2 activation that was seen at the same MIC ratio as the wild-type strain.
Alterations in the affinity of the PBPs for ß-lactam antibiotics are major factors in the formation of PRSP.2–4,32 Changes in the genes encoding PBP2x, 2b and 1a are prerequisites for penicillin resistance19 whereas changes in PBP2x and 1a are required for cephalosporin resistance.33
We have shown that increased TLR2 activity following penicillin treatment is at least independent of a low-affinity PBP2x. However, in D392x1a transformants that contain low-affinity PBP1a as well as PBP2x, TLR2 activation was significantly reduced when compared with wild-type. Both PBP2x and PBP1a are required for septal cross-wall synthesis and inhibition of these proteins produces bacteria with abnormally large morphologies.5,9,34 In the absence of high affinity PBP2x, penicillin will interact first with PBP2b and 1a at the lowest concentration producing a cell with some altered morphology and lysis at the MIC. However, these differences in the affinity profile of the PBPs have not affected TLR2 inducibility following penicillin treatment of the PBP2x transformant. Altered PBP2x and PBP1a in the D392x1a transformant has caused a significant reduction in TLR2 induction when compared with the wild-type parent. In the absence of high affinity PBP1a and 2x, penicillin will first saturate PBP2b at the lowest concentration, an event that permits lysis of morphologically normal cells at the MIC. PBP2b is the ‘lysis-associated’ PBP of S. pneumoniae. Inhibition of PBP2b at sub-MIC concentrations causes release of large cell wall fragments and cell lysis at the MIC. PBP2b inactivity is linked with N-acetyl-L-alanine amidase (LytA) activation. LytA is the major autolysin of S. pneumoniae and the presence of an osmotically vulnerable cell wall in PBP2b-inhibited cells stimulates LytA activation producing ‘holes’ in the cell wall resulting in secretion of large cell wall structure at sub-MIC and lysis at the MIC.5,35 LytA activity has been shown to induce release of branched stem peptides that are proinflammatory.28,36–38 Indeed we have shown previously that the absence of a functional LytA enzyme in a lytA negative transformant of D39 failed to produce increased TLR2-mediated IL-8 promoter activity in 1/8 MIC penicillin-treated S. pneumoniae when compared with wild-type.12 Moreover we have shown that penicillin at MIC and sub-MIC induces enhanced TNF-
and IL-1ß gene expression and protein release by primary human macrophages compared with bacteria not treated with penicillin.35 The results described here suggest that the proinflammatory consequences of penicillin treatment may be attributable to saturation of PBP1a and PBP2b although the magnitude of this effect is not great. Abnormal morphologies caused by PBP2x and PBP1a inhibition together with cell lysis are prerequisites for proinflammatory activation of TLR2. Abnormal morphology may optimize interaction of the cell wall with TLR2; this together with the presence of large quantities of lysed or partially lysed cell wall structures prime maximal TLR2 interaction.
D392x1a2b possesses low-affinity PBP2x, 1a and 2b. Consequently these proteins have similar affinities for penicillin resulting in an inflammatory profile that is similar to penicillin-susceptible bacteria although this effect requires a much higher dose of penicillin for full saturation. The degree of TLR2 activation following penicillin treatment of these cells was no different from D39 at respective MIC ratios and growth phases.
The effect of cefotaxime treatment on TLR2-mediated proinflammatory activity by PBP transformants produced some interesting results; the presence of low-affinity PBP2x in the D392x transformant has resulted in major decreases in TLR2 signalling following 1/8 MIC treatment during lag growth when compared with D39. This suggests that in 1/8 MIC cefotaxime-treated D39, saturation of PBP2x is a major factor associated with enhanced TLR2 activation. Cefotaxime has high affinity for PBP2x and PBP1a inducing morphological changes in the organism preventing bacterial growth (PBP2x inhibition).4,5,34,39 Abnormal morphologies caused by cefotaxime activity may account for the greater TLR2 induction in these cells. In contrast at the MIC, early log treatment of the low-affinity PBP2x transformant, (D392x) exhibited greater TLR2 signalling induction than both untreated bacteria and cefotaxime susceptible D39. Treatment of wild-type D39 in cefotaxime has shown enhanced TLR2-mediated proinflammatory activity when compared with untreated bacteria (this work and Moore et al.,18) that is consistent with earlier work by Tuomanen et al.,37,38 that show heightened meningeal inflammation among animals exposed to ceftriaxone-treated S. pneumoniae. However, it is uncertain why wild-type bacteria should exhibit lower activation than the transformant but it is speculated that the results may be attributable to different susceptibilities of early log-phase bacteria to antibiotic when compared with lag-phase bacteria,12 and the presence of high affinity PBP2b in wild-type early log cells may interfere with optimal inflammatory activation of cefotaxime-treated cells.
In addition to the inflammatory role of LytA, we have shown that the PBPs are equally important in penicillin-mediated TLR2 activation by S. pneumoniae, following exposure to penicillin. Penicillin inhibition (by acylation) of PBP2b and PBP1a is proinflammatory via TLR2 and this has been shown by use of PBP transformants of S. pneumoniae. Penicillin treatment enhances TLR2 activation as a result of the inhibition of PBP1a and PBP2b and the consequential activation of LytA. Cefotaxime treatment enhances TLR2 activation attributable to the inhibition of PBP2x. The consequences of PBP inhibition in the bacterium are different and both are proinflammatory via TLR2.
Our observations are consistent with other studies of cell wall component release reported in the literature. Penicillin treatment of S. pneumoniae results in the release of non-linked LTA and PGN at the MIC.40 It is noteworthy that in PSSP we see a reduction in IL-8 activation at concentrations above the MIC (Figure 1). It has been shown previously (by radiolabelling of cell walls of pneumococci prior to penicillin exposure) that release of cell wall products is greater at the MIC than at sub-MIC or supra-MIC concentrations.41 The decline in activity at supra-MIC concentrations under our experimental conditions is likely attributable to the rate of killing of pneumococci at higher penicillin concentrations. We have shown previously12 that viable counts of D39 (albeit reduced) could still be recovered after 3 h incubation with penicillin at the MIC, but at 10x MIC, no bacteria are recovered at 3 h (data not shown). Therefore the lack of enhanced TLR2 activation by D39 exposed to supra-MIC concentrations is probably attributable to the relative paucity of cell wall material available for the interaction under the experimental conditions used.
The proinflammatory moiety causing the effects observed in our system is most likely to be peptidoglycan. The reason for this is that LTA can induce proinflammatory activation via TLR2/CD14 but only when in heterodimer associations with TLR1.24 In contrast PGN can induce TLR2/CD14 activation in the absence of a heterodimer,11 although co-expression with TLR6 enhances the response.24 HeLa cells do not inherently express TLR124 or TLR6 (data not shown). We have found in experiments using S. pyogenes LTA at biological concentrations that no signal in HeLa cells transfected with TLR2/CD14 occurs, but a significant response is induced when cells are cotransfected with TLR1 (data not shown).
We have shown that both PRSP and PSSP have almost equal TLR2-proinflammatory activity at their respective MICs, consistent with the concept that PRSP have equivalent virulence to PSSP.42 Both the high-level penicillin-resistant D392x1a2b transformant and susceptible S. pneumoniae exhibited similar proinflammatory activities at their respective MIC.43,44 Factors such as sub-MIC dosing and tolerance may promote ß-lactam failure in vivo as a result of failed bacterial clearance and development of penicillin resistance.15,45 We postulate that infections with PRSP may produce suppurative complications not only because of the failure of the antibiotic to inhibit bacterial growth,13,45 but also because the tissue concentration of drug during treatment is favourable to induction of inflammation.
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None to declare, other than the financial support identified in the Acknowledgements section.
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Present address. Micropathology Ltd, University of Warwick Science Park, Barclays Venture Centre, Sir William Lyons Road, Coventry CV4 7EZ, UK 
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Acknowledgements |
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We thank Professor Alexander Tomasz for the analysis of bacterial cell walls and for his advice and support. We thank Linda Goodwin for technical support. Financial support for this work was provided by the Bayer Corporation and the British Society for Antimicrobial Chemotherapy.
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