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. 2009;1(3):202-14.
doi: 10.1159/000203645. Epub 2009 Feb 20.

M1 protein allows Group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition

Xavier Lauth  1 , Maren von Köckritz-Blickwede, Case W McNamara, Sandra Myskowski, Annelies S Zinkernagel, Bernard Beall, Partho Ghosh, Richard L Gallo, Victor Nizet
Affiliations

M1 protein allows Group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition

Xavier Lauth et al. J Innate Immun. 2009.

Abstract

M1 protein contributes to Group A Streptococcus (GAS) systemic virulence by interfering with phagocytosis and through proinflammatory activities when released from the cell surface. Here we identify a novel role of M1 protein in the stimulation of neutrophil and mast cell extracellular trap formation, yet also subsequent survival of the pathogen within these DNA-based innate defense structures. Targeted mutagenesis and heterologous expression studies demonstrate M1 protein promotes resistance to the human cathelicidin antimicrobial peptide LL-37, an important effector of bacterial killing within such phagocyte extracellular traps. Studies with purified recombinant protein fragments mapped the inhibition of cathelicidin killing to the M1 hypervariable N-terminal domain. A survey of GAS clinical isolates found that strains from patients with necrotizing fasciitis or toxic shock syndrome were significantly more likely to be resistant to cathelicidin than GAS M types not associated with invasive disease; M1 isolates were uniformly resistant. We conclude increased resistance to host cathelicidin and killing within phagocyte extracellular traps contribute to the propensity of M1 GAS strains to produce invasive infections.

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Figures

Fig. 1
Fig. 1
GAS M1 protein is necessary and sufficient to promote the induction of NETs and MCETs. Representative fluorescent images of neutrophils and NETs (a) and MCs and MCETs (b), stained with Live/Dead viability/cytotoxicity kit for mammalian cells after exposure to wild-type (WT) and isogenic ΔM1 mutant GAS. PMA was used as a positive control to stimulate extracellular trap formation. Note the distinctly reduced NET and MCET formation with the ΔM1 mutant strain. Scale bars = 25 μm. c Quantitative enumerations of NETs or MCETs per field of view containing approximately 150 cells. Decreased trip induction was observed upon deletion of M1 in the GAS wild-type (WT) strain, while increased induction was observed upon heterologous expression of M1 in M49 GAS or L. lactis. Experiments were performed in triplicate and repeated 3 times with similar results. One representative experiment is shown ± standard deviation. * p < 0.05 by t test.
Fig. 2
Fig. 2
GAS M1 protein is necessary and sufficient to promote bacterial resistance to killing in NETs. a M1 protein contributes to bacterial survival in a total (intracellular + extracellular) neutrophil killing assay. b M1 protein promotes bacterial resistance to neutrophil extracellular killing when phagocytic uptake is inhibited with cytochalasin D (10 μg/ml). c Neutrophils were pre-stimulated with 25 nM PMA for 4 h before infection to induce maximal trap formation and to avoid phagocytosis. Note that M1 contributes to GAS survival and increases L. lactis survival upon 30 min co-incubation with NETs. As control, the cells were treated with 100 U/ml DNase to disrupt extracellular trap formation before infection, which completely abolished the killing of bacteria by extracellular traps. Experiments were performed in triplicate and repeated 3 times with similar results. One representative result is shown ± standard deviation. * p < 0.05 by t test. d Representative fluorescent image of viable (green) versus dead (red) GAS entrapped by NETs (DAPI stained, blue) as determined by Live/Dead BacLightTM Bacterial Viability assay. Scale bars = 10 μm. Note that M1 contributes to GAS survival within NETs. WT = Wild type.
Fig. 3
Fig. 3
M1 protein contributes to cathelicidin resistance. a Killing kinetics for LL-37 and CRAMP against M1 wild-type (WT) or ΔM1 mutant GAS. Growth index = CFU at specified time point/CFU in initial inoculum. b Sensitivity testing to mCRAMP of isogenic M1 mutant and strains heterologously expressing M1 protein show the peptide is necessary and sufficient to promote increased cathelicidin resistance. c Complementation with an M1 protein expression plasmid promotes survival of the M1 mutant significantly greater than complementation with M49 protein. d Reduced killing of M49 GAS expressing M1 protein by LL-37 and mCRAMP. Experiments were performed in triplicate and repeated 3 times with similar results. One representative result is shown ± standard deviation.
Fig. 4
Fig. 4
The cathelicidin inhibitory activity of M1 protein maps to its N-terminal domain. a Recombinant M1 soluble protein constructs used in these studies. b–d Exogenous M1 N-terminal protein fragment reverses increased sensitivity of ΔM1 GAS mutant to LL-37 and blocks LL-37 killing of E. coli. Experiments were performed in triplicate and repeated three times with similar results. One representative result is shown ± standard deviation. WT = Wild type. e Western blot analysis shows that the M1 N-terminal protein fragment depletes LL-37 from solution.
Fig. 5
Fig. 5
M1 and other invasive isolates of GAS show increased resistance to cathelicidin compared to colonizing strains. Sensitivity or resistance of GAS clinical isolates to inhibition by the human cathelicidin LL-37 is reported. M protein (emm) genotypes are indicated within individual circles. MIC testing was performed 3–4 times for each isolate.
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