Bai et al JImmunol 2018 Raw Data.xlsx (23.13 kB)

Complete dataset for figures 1-6 of "Perforin-2 Breaches the Envelope of Phagocytosed Bacteria..." Bai et al. JImmunol 2018.

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posted on 10.07.2020 by George Munson, Fangfang Bai
This dataset contains the raw data for figures 1 through 6 of 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

FIG1. Perforin-2 limits the survival of phagocytosed bacteria. PEM or peritoneal neutrophils were isolated from WT and Perforin-2 KO mice and stimulated with IFN-γ for 14 h prior to infection with (A and B) WT S. Typhimurium strain GPM2004 or (C and D) strain ST188 (sodCI::kan). Intracellular bacteria were enumerated after gentamicin treatment eliminate extracellular bacteria.

FIG2. Perforin-2 is not required for ROS production in phagocytes. WT and Perforin-2 KO peritoneal (A and B) macrophages and (C and D) neutrophils were stimulated with PMA or LPS to elicit ROS production; detected by luminol-based chemiluminescence. (E) Phagocytic ROS production of Perforin-2 WT or KO neutrophils induced with PMA was detected after phagocytosis of luminol-coupled beads. As indicated, some cells were also treated with DPI, an inhibitor of the phagocytic NAPDH oxidase. ROS activity is reported as relative light units (RLUs).

FIG3. SodCII is functional in the absence of Perforin-2. PEM or peritoneal neutrophils were isolated from WT and Perforin-2 KO mice and stimulated with IFN-γ for 14 h prior to infection with S. Typhimurium strain (A and B) ST189 (∆sodCI sodCII::kan) or (C and D) GPM2008 (∆sodCI sodCII::kan sodCII+). Intracellular bacteria were enumerated after gentamicin treatment eliminate extracellular bacteria.

FIG4. Inhibition of ROS production allows Salmonella to proliferate intracellularly. PEMs from WT and Perforin-2 KO mice were stimulated with IFN-γ 14 h prior to infection. As indicated, some cells were also treated with DPI 30 min before infection with (A) WT S. Typhimurium strain GPM2004, (B) strain ST188 (sodCI::kan), or (C) S. Typhimurium strain ST189 (ΔsodCI sodCII::kan). Intracellular bacteria were enumerated after gentamicin treatment eliminate extracellular bacteria.

FIG5. Perforin-2 facilitates the degradation of antigens enclosed within the bacterial envelope. PEMs isolated from Perforin-2 WT and KO mice were infected with S. Typhimurium expressing SodCII-FLAG. After 18 h, the phagocytosed bacteria were recovered, and the indicated antigens were detected by Western blot. Similar infection experiments were conducted with BMDM, except the phagocytosed bacteria were recovered at 1, 3, and 6 h post phagocytosis.

FIG6. SodCII is functional in Perforin-2 KO but not WT mice. WT and Perforin-2 KO mice were inoculated by i.p. injection with S. Typhimurium WT strain GPM2004 and (A) ΔsodCI::kan strain ST188, (B) ΔsodCI::kan strain ST188b, (C) ΔsodCI sodCII::kan strain ST189, or (D) ΔsodCI sodCII::kan sodCII+ strain GPM2008 at a 1:1 ratio. Organs were harvested 4 d postinfection, and strains were enumerated on selective media. CI are derived from the ratios of mutant strains to WT strains, with compensation for any differences in inocula.

Refer to the associated publication for methodology and additional details.

Publication Abstract:
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.

Funding

Killing of intracellular bacteria by Perforin-2

National Institute of Allergy and Infectious Diseases

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  • AI - National Institute of Allergy and Infectious Diseases (NIAID)

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