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. 2017 Mar 17:7:44902.
doi: 10.1038/srep44902.

Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165

Sunil D Saroj  1 , Linda Holmer  1 , Júlia M Berengueras  1 , Ann-Beth Jonsson  1
Affiliations

Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165

Sunil D Saroj et al. Sci Rep. .

Abstract

Streptococcus pyogenes an adapted human pathogen asymptomatically colonizes the nasopharynx, among other polymicrobial communities. However, information on the events leading to the colonization and expression of virulence markers subject to interspecies and host-bacteria interactions are limited. The interference of acyl homoserine lactones (AHLs) with the hemolytic activity and viability of S. pyogenes M6 S165 was examined. AHLs, with fatty acid side chains ≥12 carbon atoms, inhibited hemolytic activity by downregulating the expression of the sag operon involved in the production of streptolysin S. Inhibitory AHLs upregulated the expression of transcriptional regulator LuxR. Electrophoretic mobility shift assays revealed the interaction of LuxR with the region upstream of sagA. AHL-mediated bactericidal activity observed at higher concentrations (mM range) was an energy-dependent process, constrained by the requirement of glucose and iron. Ferrichrome transporter FtsABCD facilitated transport of AHLs across the streptococcal membrane. The study demonstrates a previously unreported role for AHLs in S. pyogenes virulence.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Effect of oxo-AHL on the hemolytic activity of S. pyogenes.
(A) Hemolytic activity of S. pyogenes M6 S165 grown in the presence of different oxo-AHLs (20 μM). (B) Hemolytic activity of S. pyogenes M6 S165 co-incubated with different concentrations of oxo-C12-HSL. (C) S. pyogenes M6 S165 was grown in the presence of varying concentrations of oxo-C12-HSL, and the supernatant from the growth was analyzed for hemolytic activity. (D) Hemolytic activity of S. pyogenes M6 S165 in the presence of inhibitory oxo-AHLs during different growth stages. The hemolytic activity is measured in relative to caused by water.
Figure 2
Figure 2. Oxo-AHLs inhibit transcription of saga.
(A) Expression of sagA during co-incubation with inhibitory oxo-AHLs. (B) Expression of slo during co-incubation with inhibitory oxo-AHLs. (C) Luciferase assay to determine the effect of inhibitory oxo-AHLs on the promoter activity of sagA.
Figure 3
Figure 3. P. aeruginosa growth supernatant inhibits the hemolytic activity of S. pyogenes.
(A) Hemolytic activity of S. pyogenes M6 S165 grown in THB and CM obtained from growth of P. aeruginosa. (B) Expression of sagA in S. pyogenes M6 S165 grown in THB and CM obtained from growth of P. aeruginosa. (C) Luciferase assay to determine the effect of CM on the promoter activity of sagA.
Figure 4
Figure 4. Strain specificity in the inhibition of hemolytic activity due to oxo-AHLs.
(A) Hemolytic activity of two different strains of S. pyogenes in the presence of oxo-AHLS. (B) Expression of sagA in S. pyogenes M1 and M6 during co-incubation with oxo-C12-HSL. (C) Expression of slo in S. pyogenes M1 and M6 during co-incubation with oxo-C12-HSL.
Figure 5
Figure 5. Effect of oxo-AHLs on the transcriptional activity of luxR and luxS and the total intracellular iron content.
(A) Expression of luxR during co-incubation with different oxo-AHLs. (B) Expression of luxS during co-incubation with different oxo-AHLs. (C) Total intracellular iron content during co-incubation with different oxo-AHLs.
Figure 6
Figure 6. Role of luxR in the oxo-AHL-mediated inhibition of SLS activity.
(A) Hemolytic activity of S. pyogenes M6 S165 and its luxR mutant grown in the presence of oxo-C12-HSL. (B) Expression of sagA in S. pyogenes M6 S165 and its luxR mutant grown in the presence of oxo-C12-HSL. (C) EMSA to demonstrate the binding of luxR to the promoter region of sagA. (D) EMSA to demonstrate the bindiding of luxR is specific to DNA from the promoter region of sagA and not affected by AHL. No gel shift for DNA from slo promoter is observed when incubated with oxo-C12-HSL (10 μM). Also, sagA promoter a gel shift was observed even in the absence of oxo-C12-HSL.
Figure 7
Figure 7. Bactericidal effect of oxo-AHLs.
(A) Disk diffusion assay to demonstrate the inhibition of S. pyogenes M6 S165 growth by the oxo-AHLs. (B) Effect of glucose and iron on the S. pyogenes M6 S165 growth inhibition by oxo-C12-HSL.
Figure 8
Figure 8. Role of the ferrichrome transporter in the oxo-AHL-mediated killing of S. pyogenes.
(A) Survival assay of S. pyogenes M6 S165 and its ferrichrome transporter mutants in the presence of oxo-C12-HSL. (B) Hemolytic activity of S. pyogenes M6 S165 and its ferrichrome transporter mutants grown in the presence of oxo-C12-HSL. (C) Expression of sagA in S. pyogenes M6 S165 and its ferrichrome transporter mutants grown in the presence of oxo-C12-HSL. (D) Bioassay to determine the intracellular accumulation of oxo-C12-HSL in S. pyogenes M6 S165 and its ferrichrome transporter mutants grown in the presence of oxo-C12-HSL.
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