MSJ767327_supplementary_data – Supplemental material for Evidence of <i>Clostridium perfringens</i> epsilon toxin associated with multiple sclerosis

<p>Supplemental material, MSJ767327_supplementary_data for Evidence of <i>Clostridium perfringens</i> epsilon toxin associated with multiple sclerosis by Sariqa Wagley, Monika Bokori-Brown, Helen Morcrette, Andrea Malaspina, Caroline D’Arcy, Sharmilee Gnanapavan, Nicholas Lewis, Michel R Popoff, Dominika Raciborska, Richard Nicholas, Ben Turner and Richard W Titball in Multiple Sclerosis Journal</p

Macroautophagy is essential for killing of intracellular <i>Burkholderia pseudomallei</i> in human neutrophils

<div><p>Neutrophils play a key role in the control of <i>Burkholderia pseudomallei</i>, the pathogen that causes melioidosis. Here, we show that survival of intracellular <i>B. pseudomallei</i> was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in <i>B. pseudomallei</i>-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that <i>B. pseudomallei</i>-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of <i>B. pseudomallei</i>-infected neutrophils at 3 h revealed <i>B. pseudomallei</i>-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a <i>B. pseudomallei</i> mutant lacking the <i>Burkholderia</i> secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to <i>B. pseudomallei</i>-containing phagosomes in a Type III secretion system-dependent manner.</p></div