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

This dataset contains the raw data for figures 1 through 6 of the publication: <i>Perforin-2 Breaches the Envelope of Phagocytosed Bacteria Allowing Antimicrobial Effectors Access to Intracellular Targets, </i>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<br><b>FIG1</b>. 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.<br><b>FIG2</b>. 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).<br><b>FIG3</b>. 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.<br><b>FIG4</b>. 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.<br><b>FIG5</b>. 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. <br><b>FIG6</b>. 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. <br>Refer to the associated publication for methodology and additional details.<br><br>Publication Abstract:<br>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.<br>

本数据集包含发表论文《Perforin-2 破坏被吞噬细菌的包膜，使抗菌效应物得以进入胞内靶点》（作者：Fangfang Bai、Ryan M. McCormack、Suzanne Hower、Gregory V. Plano、Mathias G. Lichtenheld、George P. Munson，发表于《免疫学杂志》（*Journal of Immunology*），2018年11月1日，第201卷第9期，第2710-2720页，DOI: https://doi.org/10.4049/jimmunol.1800365）中图1至图6的原始数据。

**图1**：Perforin-2可限制被吞噬细菌的存活。从野生型（Wild Type, WT）和Perforin-2敲除（Knockout, KO）小鼠中分离腹膜巨噬细胞（PEM）或腹腔中性粒细胞，用干扰素-γ（IFN-γ）刺激14小时后，分别以（A、B组）野生型鼠伤寒沙门氏菌（*S. Typhimurium*）菌株GPM2004，或（C、D组）菌株ST188（sodCI::kan）进行感染。经庆大霉素处理清除胞外细菌后，对胞内细菌进行计数。

**图2**：吞噬细胞产生活性氧（Reactive Oxygen Species, ROS）不依赖Perforin-2。用佛波酯（PMA）或脂多糖（LPS）刺激WT与Perforin-2 KO小鼠的（A、B组）腹腔巨噬细胞及（C、D组）中性粒细胞，通过基于鲁米诺（luminol）的化学发光法检测ROS产生情况。（E组）对吞噬了鲁米诺偶联磁珠的中性粒细胞，经PMA诱导后检测其吞噬性ROS产生；如标注所示，部分细胞同时用二苯基碘鎓（DPI，吞噬性NAPDH氧化酶抑制剂）进行处理。ROS活性以相对光单位（Relative Light Units, RLUs）表示。

**图3**：缺失Perforin-2不影响SodCII的功能。从WT和Perforin-2 KO小鼠中分离PEM或腹腔中性粒细胞，用IFN-γ刺激14小时后，分别以（A、B组）鼠伤寒沙门氏菌菌株ST189（∆sodCI sodCII::kan）或（C、D组）GPM2008（∆sodCI sodCII::kan sodCII+）进行感染。经庆大霉素清除胞外细菌后，对胞内细菌进行计数。

**图4**：抑制ROS产生可使沙门氏菌实现胞内增殖。从WT和Perforin-2 KO小鼠中分离PEM，用IFN-γ刺激14小时后进行感染。如标注所示，部分细胞在感染前30分钟经DPI处理，分别感染（A组）野生型鼠伤寒沙门氏菌菌株GPM2004、（B组）菌株ST188（sodCI::kan）或（C组）鼠伤寒沙门氏菌菌株ST189（ΔsodCI sodCII::kan）。经庆大霉素清除胞外细菌后，对胞内细菌进行计数。

**图5**：Perforin-2可促进包裹于细菌包膜内的抗原降解。从Perforin-2 WT和KO小鼠中分离PEM，用表达SodCII-FLAG的鼠伤寒沙门氏菌进行感染。18小时后回收被吞噬的细菌，通过蛋白质印迹法（Western blot）检测指定抗原。采用骨髓来源巨噬细胞（Bone Marrow Derived Macrophage, BMDM）开展了类似的感染实验，仅在吞噬后1、3、6小时回收被吞噬的细菌。

**图6**：SodCII在Perforin-2 KO小鼠中具备功能，但在WT小鼠中无此效应。通过腹腔注射（i.p. injection）以1:1的比例，给WT和Perforin-2 KO小鼠接种鼠伤寒沙门氏菌野生型菌株GPM2004与（A组）ΔsodCI::kan菌株ST188、（B组）ΔsodCI::kan菌株ST188b、（C组）ΔsodCI sodCII::kan菌株ST189或（D组）ΔsodCI sodCII::kan sodCII+菌株GPM2008。感染4天后收获器官，在选择性培养基上对菌株进行计数。竞争指数（Competitive Index, CI）由突变菌株与野生型菌株的比值计算得到，并校正接种量的差异。

相关实验方法与更多细节请参阅该发表论文。

### 论文摘要
Perforin-2是MPEG1基因的编码产物，可在体内限制细菌病原体的扩散与播散。其在小鼠和人类吞噬细胞中高表达，缺失Perforin-2的巨噬细胞杀伤被吞噬细菌的能力受损。本研究以鼠伤寒沙门氏菌作为模型胞内病原菌，解析Perforin-2的杀菌活性机制。体外实验发现，Perforin-2可促进被吞噬细菌包膜内所含抗原的降解；与之相反，某一代表性表面抗原的降解则不依赖Perforin-2。与体外实验结果一致，一种对蛋白酶敏感的胞周超氧化物歧化酶（SodCII）在Perforin-2敲除小鼠中可增强鼠伤寒沙门氏菌的毒力，而在野生型小鼠中则无此效应。综上，本研究表明Perforin-2可破坏被吞噬细菌的包膜，促进蛋白酶及其他抗菌效应物向细菌胞内靶点递送。