Western blots of Salmonella recovered from wild-type and Perforin-2 -/- bone marrow derived macrophages
2020-07-10T20:35:04Z (GMT) by
This series of Western blots demonstrate that the pore-forming protein Perforin-2 (MPEG1) is active within phagosomes and allows proteases to cross the bacterial envelope to digest periplasmic SodCII. In contrast, digestion of the surface antigen flagellin is independent of Perforin-2. Cytoplasmic DnaK and host beta-actin were included as controls. For each antigen three independent Western blots are provided. Lanes MW) molecular weight ladder, 1) bacteria pre-phagocytosis, 2) one hour after phagocytosis by WT BMDMs, 3) one hour after phagocytosis by Perforin-2 -/- BMDMs, 4) three hours after phagocytosis by WT BMDMs, 5) three hours after phagocytosis by Perforin-2 -/- BMDMs, 6) six hours after phagocytosis by WT BMDMs, 7) six hours after phagocytosis by Perforin-2 -/- BMDMs. Refer to the associated citation for complete methodology and additional details.
Supports the publication: Perforin-2 Breaches the Envelope of Phagocytosed Bacteria Allowing Antimicrobial Effectors Access to Intracellular Targets, Fangfang Bai, Ryan M. McCormack, Suzanne Hower, Gregory V. Plano, Mathias G. Lichtenheld and George P. Munson, J Immunol November 1, 2018, 201 (9) 2710-2720, https://doi.org/10.4049/jimmunol.1800365
Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study, we used Salmonella enterica serovar Typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2’s bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of Ags contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface Ag was found to be independent of Perforin-2. Consistent with our in vitro results, a protease-sensitive, periplasmic superoxide dismutase (SodCII) contributed to the virulence of S. Typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate, our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria, facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial cell.