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The impact of pneumolysin on the macrophage response to Streptococcus pneumoniae is strain-dependent.

Harvey RM, Hughes CE, Paton AW, Trappetti C, Tweten RK, Paton JC - PLoS ONE (2014)

Bottom Line: In recent times a clinically significant and internationally successful serotype 1 ST306 clone has been found to express a non-cytolytic variant of Ply (Ply306).Strains that expressed cytolytic Ply were found to induce a significant increase in IL-1β release from macrophage-like cells compared to the non-cytolytic and Ply-deficient strains in a background-independent manner, confirming the requirement for pore formation in the Ply-dependent activation of the NLRP3 inflammasome.However, cytolytic activity in the D39 background was found to induce increased expression of the genes encoding GM-CSF (CSF2), p19 subunit of IL-23 (IL23A) and IFNβ (IFNB1) compared to non-cytolytic and Ply-deficient D39 mutants, but had no effect in the A0229467 background.

View Article: PubMed Central - PubMed

Affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia.

ABSTRACT
Streptococcus pneumoniae is the world's leading cause of pneumonia, bacteremia, meningitis and otitis media. A major pneumococcal virulence factor is the cholesterol-dependent cytolysin, which has the defining property of forming pores in cholesterol-containing membranes. In recent times a clinically significant and internationally successful serotype 1 ST306 clone has been found to express a non-cytolytic variant of Ply (Ply306). However, while the pneumococcus is a naturally transformable organism, strains of the ST306 clonal group have to date been virtually impossible to transform, severely restricting efforts to understand the role of non-cytolytic Ply in the success of this clone. In this study isogenic Ply mutants were constructed in the D39 background and for the first time in the ST306 background (A0229467) to enable direct comparisons between Ply variants for their impact on the immune response in a macrophage-like cell line. Strains that expressed cytolytic Ply were found to induce a significant increase in IL-1β release from macrophage-like cells compared to the non-cytolytic and Ply-deficient strains in a background-independent manner, confirming the requirement for pore formation in the Ply-dependent activation of the NLRP3 inflammasome. However, cytolytic activity in the D39 background was found to induce increased expression of the genes encoding GM-CSF (CSF2), p19 subunit of IL-23 (IL23A) and IFNβ (IFNB1) compared to non-cytolytic and Ply-deficient D39 mutants, but had no effect in the A0229467 background. The impact of Ply on the immune response to the pneumococcus is highly dependent on the strain background, thus emphasising the importance of the interaction between specific virulence factors and other components of the genetic background of this organism.

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Ply-dependent induction of IL-1β release requires cytolytic activity.Differentiated THP-1 cells were co-incubated with bacteria at a 1∶10 ratio for 16 h. Supernatants were collected and IL-1β levels measured by ELISA. The mean quantity of IL-1β detected from resting samples is set at a relative value of 1 (dashed line), with all data points plotted as fold-change from this value. Error bars indicate the mean with standard deviation. Significant increases in IL-1β levels are highlighted, *** = P<0.001, n = 4.
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pone-0103625-g003: Ply-dependent induction of IL-1β release requires cytolytic activity.Differentiated THP-1 cells were co-incubated with bacteria at a 1∶10 ratio for 16 h. Supernatants were collected and IL-1β levels measured by ELISA. The mean quantity of IL-1β detected from resting samples is set at a relative value of 1 (dashed line), with all data points plotted as fold-change from this value. Error bars indicate the mean with standard deviation. Significant increases in IL-1β levels are highlighted, *** = P<0.001, n = 4.

Mentions: Table 1 lists the isogenic Ply mutants that were used in this study and include mutants in both the D39 and A0229467 backgrounds. All 8 strains were cultured in C+Y broth, and aliquots were taken for western blot and hemolytic activity analyses. Quantitative western blotting of equal quantities of total lysate supernatant protein confirmed that there were no significant differences in the amounts of Ply protein produced by D39, D39::Ply306, D39::PlyL460D, A0229467, A0229467::PlyD39 and A0229467::PlyL460D (Fig. 2B). Ply was not detected in either the D39ΔPly or A0229467ΔPly samples (Fig. 2A). Hemolysis assays were used to determine the specific hemolytic activity of equal quantities of total lysate supernatant protein of each strain. D39 and A0229467::PlyD39 exhibited hemolytic activity at approximately 3×103 and 2×103 HU/mg lysate supernatant protein, respectively (Table 1). Hemolytic activity was below the limit of detection (20 HU/mg lysate supernatant protein) for the remaining six strains, which confirms that the Ply306-expressing strains and PlyL460D-expressing strains are essentially non-hemolytic, as previously described [25], [32]. To confirm that lytic Ply (PlyD39) induces IL-1β release when expressed in either the D39 or A0229467 background, differentiated THP-1 cells were co-incubated for 16 h separately with all eight strains listed in Table 2. Approximately 15.3 and 12.7 fold more released IL-1β was detected in the presence of wild-type D39 and A0229467::PlyD39 compared to the resting control, respectively (Fig. 3). In contrast, there was no difference in the amount of IL-1β detected between the remaining six samples and the resting control. Therefore, Ply-dependent IL-1β release is associated with cytolytic activity of the toxin in a strain-independent manner, and confirms for the first time using isogenic strains that Ply-dependent activation of the NLRP3 inflammasome requires cytolytic activity in a strain-independent fashion.


The impact of pneumolysin on the macrophage response to Streptococcus pneumoniae is strain-dependent.

Harvey RM, Hughes CE, Paton AW, Trappetti C, Tweten RK, Paton JC - PLoS ONE (2014)

Ply-dependent induction of IL-1β release requires cytolytic activity.Differentiated THP-1 cells were co-incubated with bacteria at a 1∶10 ratio for 16 h. Supernatants were collected and IL-1β levels measured by ELISA. The mean quantity of IL-1β detected from resting samples is set at a relative value of 1 (dashed line), with all data points plotted as fold-change from this value. Error bars indicate the mean with standard deviation. Significant increases in IL-1β levels are highlighted, *** = P<0.001, n = 4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126675&req=5

pone-0103625-g003: Ply-dependent induction of IL-1β release requires cytolytic activity.Differentiated THP-1 cells were co-incubated with bacteria at a 1∶10 ratio for 16 h. Supernatants were collected and IL-1β levels measured by ELISA. The mean quantity of IL-1β detected from resting samples is set at a relative value of 1 (dashed line), with all data points plotted as fold-change from this value. Error bars indicate the mean with standard deviation. Significant increases in IL-1β levels are highlighted, *** = P<0.001, n = 4.
Mentions: Table 1 lists the isogenic Ply mutants that were used in this study and include mutants in both the D39 and A0229467 backgrounds. All 8 strains were cultured in C+Y broth, and aliquots were taken for western blot and hemolytic activity analyses. Quantitative western blotting of equal quantities of total lysate supernatant protein confirmed that there were no significant differences in the amounts of Ply protein produced by D39, D39::Ply306, D39::PlyL460D, A0229467, A0229467::PlyD39 and A0229467::PlyL460D (Fig. 2B). Ply was not detected in either the D39ΔPly or A0229467ΔPly samples (Fig. 2A). Hemolysis assays were used to determine the specific hemolytic activity of equal quantities of total lysate supernatant protein of each strain. D39 and A0229467::PlyD39 exhibited hemolytic activity at approximately 3×103 and 2×103 HU/mg lysate supernatant protein, respectively (Table 1). Hemolytic activity was below the limit of detection (20 HU/mg lysate supernatant protein) for the remaining six strains, which confirms that the Ply306-expressing strains and PlyL460D-expressing strains are essentially non-hemolytic, as previously described [25], [32]. To confirm that lytic Ply (PlyD39) induces IL-1β release when expressed in either the D39 or A0229467 background, differentiated THP-1 cells were co-incubated for 16 h separately with all eight strains listed in Table 2. Approximately 15.3 and 12.7 fold more released IL-1β was detected in the presence of wild-type D39 and A0229467::PlyD39 compared to the resting control, respectively (Fig. 3). In contrast, there was no difference in the amount of IL-1β detected between the remaining six samples and the resting control. Therefore, Ply-dependent IL-1β release is associated with cytolytic activity of the toxin in a strain-independent manner, and confirms for the first time using isogenic strains that Ply-dependent activation of the NLRP3 inflammasome requires cytolytic activity in a strain-independent fashion.

Bottom Line: In recent times a clinically significant and internationally successful serotype 1 ST306 clone has been found to express a non-cytolytic variant of Ply (Ply306).Strains that expressed cytolytic Ply were found to induce a significant increase in IL-1β release from macrophage-like cells compared to the non-cytolytic and Ply-deficient strains in a background-independent manner, confirming the requirement for pore formation in the Ply-dependent activation of the NLRP3 inflammasome.However, cytolytic activity in the D39 background was found to induce increased expression of the genes encoding GM-CSF (CSF2), p19 subunit of IL-23 (IL23A) and IFNβ (IFNB1) compared to non-cytolytic and Ply-deficient D39 mutants, but had no effect in the A0229467 background.

View Article: PubMed Central - PubMed

Affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia.

ABSTRACT
Streptococcus pneumoniae is the world's leading cause of pneumonia, bacteremia, meningitis and otitis media. A major pneumococcal virulence factor is the cholesterol-dependent cytolysin, which has the defining property of forming pores in cholesterol-containing membranes. In recent times a clinically significant and internationally successful serotype 1 ST306 clone has been found to express a non-cytolytic variant of Ply (Ply306). However, while the pneumococcus is a naturally transformable organism, strains of the ST306 clonal group have to date been virtually impossible to transform, severely restricting efforts to understand the role of non-cytolytic Ply in the success of this clone. In this study isogenic Ply mutants were constructed in the D39 background and for the first time in the ST306 background (A0229467) to enable direct comparisons between Ply variants for their impact on the immune response in a macrophage-like cell line. Strains that expressed cytolytic Ply were found to induce a significant increase in IL-1β release from macrophage-like cells compared to the non-cytolytic and Ply-deficient strains in a background-independent manner, confirming the requirement for pore formation in the Ply-dependent activation of the NLRP3 inflammasome. However, cytolytic activity in the D39 background was found to induce increased expression of the genes encoding GM-CSF (CSF2), p19 subunit of IL-23 (IL23A) and IFNβ (IFNB1) compared to non-cytolytic and Ply-deficient D39 mutants, but had no effect in the A0229467 background. The impact of Ply on the immune response to the pneumococcus is highly dependent on the strain background, thus emphasising the importance of the interaction between specific virulence factors and other components of the genetic background of this organism.

Show MeSH
Related in: MedlinePlus