Supplementary Material for: M1 Protein-Dependent Intracellular Trafficking Promotes Persistence and Replication of <i>Streptococcus pyogenes</i> in Macrophages E.Hertzén L.Johansson R.Wallin H.Schmidt M.Kroll A.P.Rehn M.Kotb M.Mörgelin A.Norrby-Teglund 2010 <i>Streptococcus pyogenes</i> is an important human pathogen that causes a variety of diseases including life-threatening invasive diseases, such as toxic shock and deep tissue infections. Although <i>S. pyogenes</i> are classically considered extracellular pathogens, a clinical significance of an intracellular source has been emphasized. In patients with deep tissue infections, an intracellular reservoir of <i>S. pyogenes</i> within macrophages was shown to contribute to prolonged bacterial persistence. Here we demonstrate that intracellular survival of <i>S. pyogenes</i> in macrophages is associated with an M1 protein-dependent intracellular trafficking in the phagosomal-lysosomal pathway, which results in impaired fusion with lysosomes. The phagocytic vacuoles harbouring M1 protein-expressing bacteria not only served as a safe haven for the bacteria, but also as a replicating niche. An M1 protein-dependent modulation of macrophages was further supported by differences in NF-ĸB signalling between cells infected with either the wild-type or M1 protein-deficient strains, thereby indicating a suppressed inflammatory response when M1 protein was involved. Evidence of egress of bacteria out of their host cell and subsequent re-infection of new cells emphasize the importance of intracellular bacteria as a reservoir for dissemination of infection and continued tissue injury.