M1T1 group A streptococcal pili promote epithelial colonization but diminish systemic virulence through neutrophil extracellular entrapment

Abstract

Group A Streptococcus is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Cell wall anchored pili were recently described in several species of pathogenic streptococci, and in the case of GAS, these surface appendages were demonstrated to facilitate epithelial cell adherence. Here we use targeted mutagenesis to evaluate the contribution of pilus expression to virulence of the globally disseminated M1T1 GAS clone, the leading agent of both GAS pharyngitis and severe invasive infections. We confirm that pilus expression promotes GAS adherence to pharyngeal cells, keratinocytes, and skin. However, in contrast to findings reported for group B streptococcal and pneumococcal pili, we observe that pilus expression reduces GAS virulence in murine models of necrotizing fasciitis, pneumonia and sepsis, while decreasing GAS survival in human blood. Further analysis indicated the systemic virulence attenuation associated with pilus expression was not related to differences in phagocytic uptake, complement deposition or cathelicidin antimicrobial peptide sensitivity. Rather, GAS pili were found to induce neutrophil IL-8 production, promote neutrophil transcytosis of endothelial cells, and increase neutrophil release of DNA-based extracellular traps, ultimately promoting GAS entrapment and killing within these structures.

Fig. 1

Targeted deletion of the spy0128 gene eliminates pilus expression and reduces GAS adherence to human epithelial cells and mouse skin. a Green fluorescent immunostaining with anti-T1 antigen (Spy0218) antisera is positive for WT GAS, negative for the isogenic ΔPil mutant, and restored in the ΔPil + Pil complemented strain. b GAS WT and ΔPil + Pil complemented strains are more adherent to keratinocytes and epithelial cells compared with the ΔPil mutant, *P < 0.05. c GAS WT outcompetes ΔPil in adherence to mouse skin; circles represent the ratio of GAS WT CFU to ΔPil mutant CFU recovered for individual skin lesions, bar represents the mean. In vivo experiments were repeated three times, representative experiment shown

Fig. 2

Deletion of GAS pilus expression increases virulence in a mouse model of necrotizing fasciitis. a Subcutaneous infection with the ΔPil mutant generates larger skin lesions compared with GAS WT at 48 h (P = 0.014) and 72 h (P = 0.013); representative mouse from these experiments shown in b. c The ΔPil + Pil complemented mutant produces smaller skin lesions than the ΔPil mutant at 72 h (P = 0.011). Eight mice were used per group and all experiments were repeated three times with similar results

Fig. 3

Deletion of GAS pilus expression increases virulence in mouse models of pneumonia and sepsis. a Mice infected intranasally with GAS WT had significantly fewer bacteria in their lungs at 24 and 48 h. b Representative hematoxylin–eosin-staining of lung sections demonstrates more histopathological damage in ΔPil lungs vs. WT-infected lungs at 24 and 48 h. c Mice infected intravenously with the ΔPil mutant died an average of 72 h after infection compared with 96 h after infection for WT-infected mice. (P = 0.002). The survival curve was generated from pooling data from two experiments. At least three mice were used per group and all experiments were repeated three times

Fig. 4

Pilus expression in GAS leads to increased susceptibility to whole blood killing and stimulates increased IL-8 release and transendothelial migration by human neutrophils. a The ΔPil mutant had increased survival in human whole blood compared with WT GAS (P = 0.0001) or the ΔPil + Pil complemented mutant (P = 0.016). b No difference in survival between piliated and non-piliated GAS strains in a macrophage killing assay. c No difference in surface deposition of complement component C3b in piliated vs. non-piliated GAS. d Human neutrophils phagocytose FITC labeled GAS WT, ΔPil mutant, and ΔPil + Pil complemented mutant strains with equivalent efficiency. e Increased secretion of IL-8 by neutrophils exposed to WT GAS vs. the ΔPil mutant (P = 0.009). f Increased migration of neutrophils through an endothelial monolayer in response to WT GAS compared with the isogenic ΔPil mutant (P = 0.03). All experiments were performed in triplicate and were repeated three times; representative experiment shown

Fig. 5

GAS pilus expression stimulates neutrophil extracellular trap (NET) release and increases GAS entrapment and killing within NETs. a WT GAS (3.6 fold) and ΔPil + Pil complemented mutant (3.0 fold) induce more NETs than the ΔPil mutant (P < 0.0001). b Representative fluoresencent microscopic images of NETs (red arrows) induced by WT GAS, ΔPil mutant, ΔPil + Pil complemented mutant strains and c WT GAS and ΔPil + Pil complemented mutant strains are entrapped to a greater extent than the ΔPil mutant (P = 0.0001 and 0.003, respectively). d Representative images of FITC-labeled GAS adherent to NETs, with red arrows highlighting the green bacteria adherent to blue NETs. e WT GAS (P = 0.03) and the ΔPil + Pil complemented mutant (P = 0.006) are killed to a greater extent within NETs than the ΔPil mutant