Supplementary Material for: The M1 Protein of <b><i>Streptococcus pyogenes</i></b> Triggers an Innate Uptake Mechanism into Polarized Human Endothelial Cells

Serotype M1 <i>Streptococcus pyogenes</i> is a major human pathogen associated with severe invasive diseases causing high morbidity and mortality. In a substantial number of cases, invasive disease develops in previously healthy individuals with no obvious port of entry. This has led to the hypothesis that the source of streptococci in these cases is a transient bacteraemia. This study focuses on the analysis of interaction of tissue-invasive serotype M1 <i>S. pyogenes</i> with human endothelial cells (EC) of the vascular system. We identify the M1 surface protein of <i>S. pyogenes</i> as the EC invasin which is recognised by polarized human blood EC, thereby triggering rapid, phagocytosis-like uptake of streptococci into polarized EC layers. Upon internalization, the M1 <i>S. pyogenes</i> serotype is incorporated into phagosomes which traffic via the endosomal/lysosomal pathway. However, some of the streptococci successfully evade this innate killing process and hereby mediate their escape into the cytoplasm of the host cell. The results of this study demonstrate that blood EC possess an efficient uptake mechanism for serotype M1 <i>S. pyogenes</i>. Despite efficient phagocytosis, streptococcal survival within EC constitutes one potential mechanism which favours intracellular persistence and thus facilitates continuous infection and dissemination from the primary side of infection into deep tissue

Supplementary Material for: LL-37 Triggers Formation of <b><i>Streptococcus pyogenes</i></b> Extracellular Vesicle-Like Structures with Immune Stimulatory Properties

Reports have shown that the antimicrobial peptide LL-37 is abundantly expressed but has limited bactericidal effect in <i>Streptococcus pyogenes</i> infections. At sub-inhibitory concentrations, LL-37 has been reported to alter virulence gene expression. Here, we explored the interaction of <i>S. pyogenes</i> strains with LL-37, focusing on bacterial growth, cell surface alterations and pro-inflammatory responses. Bioscreen turbidity measurements of strain 5448 cultured in the presence or absence of LL-37 confirmed the poor antimicrobial effect, and revealed a significant increase in turbidity of bacterial cultures exposed to sub-inhibitory concentrations of LL-37. However, this was not linked to increased bacterial counts. Electron microscopy of LL-37-exposed bacteria revealed the presence of vesicle-like structures on the bacterial surface. The vesicles stained positive for LL-37 and were released from the bacterial surface. Concentrated supernatants enriched in these structures had a broader protein content, including several virulence factors, compared to supernatants from untreated bacteria. The supernatants from LL-37-exposed bacteria were pro-inflammatory and elicited resistin and myeloperoxidase release from neutrophils. This is the first report on <i>S. pyogenes</i> extracellular vesicle-like structures formed at the bacterial surface in response to LL-37. The associated increased pro-inflammatory activity further implicates LL-37 as a potential factor involved in <i>S. pyogenes</i> pathogenesis

Supplementary Material for: Group A <b><i>Streptococcus</i></b> Modulates Host Inflammation by Manipulating Polymorphonuclear Leukocyte Cell Death Responses

Polymorphonuclear leukocyte (PMN) cell death strongly influences the resolution of inflammatory episodes, and may exacerbate adverse pathologies in response to infection. We investigated PMN cell death mechanisms following infection by virulent group A <i>Streptococcus</i> (GAS). Human PMNs were infected in vitro with a clinical, virulent GAS isolate and an avirulent derivative strain, and compared for phagocytosis, the production of reactive oxygen species (ROS), mitochondrial membrane depolarization and apoptotic markers. C57BL/6J mice were then infected, in order to observe the effects on murine PMNs in vivo. Human PMNs phagocytosed virulent GAS less efficiently, produced less ROS and underwent reduced mitochondrial membrane depolarization compared with phagocytosis of avirulent GAS. Morphological and biochemical analyses revealed that PMNs infected with avirulent GAS exhibited nuclear fragmentation and caspase-3 activation consistent with an anti-inflammatory apoptotic phenotype. Conversely, virulent GAS induced PMN vacuolization and plasma membrane permeabilization, leading to a necrotic form of cell death. Infection of the mice with virulent GAS engendered significantly higher systemic pro-inflammatory cytokine release and localized infiltration of murine PMNs, with cells associated with virulent GAS infection exhibiting reduced apoptotic potential. Avirulent GAS infection was associated with lower levels of proinflammatory cytokines and tissue PMN apoptosis. We propose that the differences in PMN cell death mechanisms influence the inflammatory responses to infection by GAS