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

Unique regulation of SclB - a novel collagen-like surface protein of Streptococcus pyogenes

Slipped-strand mispairing at sites containing so-called coding repeats (CRs) can lead to phase variation of surface proteins in Gram-negative bacteria. This mechanism, believed to contribute to virulence, has so far not been identified in a Gram-positive bacterium. In the genome of the Gram-positive human pathogen Streptococcus pyogenes, we identified pentanucleotide CRs within a putative signal sequence of an open reading frame (ORF) encoding a novel collagen-like surface protein, denoted SclB. In 12 S. pyogenes strains, the number of CRs in the sclB gene varied from three to 19, rendering the start codon in frame with the downstream ORF in four strains and out of frame in eight strains. A protein reacting with anti-SclB antibodies could only be solubilized from three strains, all containing an intact sclB gene. Variations in the number of CRs were observed within strains of the same M serotype and occurred during growth of S. pyogenes in fresh human blood, but not in medium. The SclB protein has a hypervariable N-terminal part, a collagen-like central part and a typical cell wall sorting sequence containing the LPXTGX motif. SclB is related to the collagen-like SclA and is, like SclA, involved in the adhesion of S. pyogenes bacteria to human cells. However, the Mga protein, known to upregulate sclA and several additional genes encoding virulence factors of S. pyogenes, downregulates sclB transcription. This observation and the potential of SclB to phase vary by slipped-strand mispairing emphasize the unique regulation of this novel S. pyogenes surface protein