A phosphatidylinositol 3-kinase effector alters phagosomal maturation to promote intracellular growth of Francisella

. Author manuscript; available in PMC: 2019 Aug 8.

Published in final edited form as: Cell Host Microbe. 2018 Jul 26;24(2):285–295.e8. doi: 10.1016/j.chom.2018.07.003

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-FTN_0131	this paper	N/A
Mouse polyclonal anti-EEA1	Abcam	Cat#Ab7051; RRID:AB_1603734; Lot#GR278239–1
Mouse polyclonal anti-LAMP1	Abcam	Cat#Ab25630; RRID:AB_470708; Lot#GR905059–2
Mouse anti-penta-His HRP conjugated	Qiagen	Cat#34460; Lot#151045877
Rabbit polyclonal anti-Calnexin	Cell Signaling Technology	Cat#2433S; RRID:AB_2243887; Lot#4
Rabbit polyclonal anti-PMP70	ThermoFisher Scientific	Cat#PA1–650; RRID:AB_221991
Rabbit polyclonal anti-TGN46	Abcam	Cat#Ab50595; RRID:AB_2203289
Goat anti-Rabbit HRP conjugated	Sigma Aldrich	Cat#A6154; RRID:AB_258284; Lot#SLBP3451V
Alexa 488-conjugated goat anti-rabbit IgG	Invitrogen	Cat#A21202; RRID:AB_141607; Lot# 1796361
Alexa 488-conjugated donkey anti-mouse IgG	Invitrogen	Cat#A11008; RRID:AB_13165; Lot# 1937184
Alexa 594-conjugated donkey anti-mouse IgG	Invitrogen	Cat#R37115; RRID:AB_2556543
Bacterial and Virus Strains
Francisella novicida U112	Manoil lab	N/A
F. novicida ∆dotU	Eshraghi et al., 2016	N/A
F. novicida ∆pdpC	Eshraghi et al., 2016	N/A
F. novicida ∆pdpD	Eshraghi et al., 2016	N/A
F. novicida ∆opiA	Eshraghi et al., 2016	N/A
F. novicida ∆opiB	Eshraghi et al., 2016	N/A
F. novicida ∆pdpC ∆opiA	This paper	N/A
F. novicida ∆pdpD ∆opiA	This paper	N/A
F. novicida ∆opiB ∆opiA	This paper	N/A
F. novicida ∆opiB ∆pdpC	This paper	N/A
F. novicida ∆pdpC ∆opiA + Tn7:opiA	This paper	N/A
F. novicida ∆pdpC ∆opiA + Tn7:opiAH261A	This paper	N/A
F. novicida + Tn7:Pbfr-sfGFP	This paper	N/A
F. novicida ∆pdpC + Tn7:Pbfr-sfGFP	This paper	N/A
F. novicida ∆opiA + Tn7:Pbfr-sfGFP	This paper	N/A
F. novicida ∆pdpC ∆opiA + Tn7:Pbfr-sfGFP	This paper	N/A
F. novicida MFN245	Manoil lab	N/A
Escherichia coli strain DH5α	Thermo Fisher Scientific	Cat#18258012
Escherichia coli strain BL21	EMD Millipore	Cat#69450
Saccharomyces cerevisiae BY4742 MATalpha his3∆1 leu2∆0 lys2∆0 ura3∆0	Yeast Knockout Collection	N/A
S. cerevisiae BY4742 MATalpha his3∆1 leu2∆0 lys2∆0 ura3∆0 vps34∆::KanMX4	Yeast Knockout Collection	N/A
S. cerevisiae BY4742 MATalpha his3∆1 leu2∆0 lys2∆0 ura3∆0 vps30∆::KanMX4	Yeast Knockout Collection	N/A
Chemicals, Peptides, and Recombinant Proteins
Dulbecco’s Modified Eagle’s Medium	Corning	Cat#15013
Dulbecco’s Modified Eagle’s Medium with L-gluatamine and sodium pyruvate	Corning	Cat#10014
Fetal Bovine Serum	Atlanta biologicals	Cat#S11550; Lot#K16116
Fetal Bovine Serum	Gibco, Life Technologies
Fetal Bovine Serum	Invitrogen
Wortmannin	Santa Cruz Biotechnology	Cat#sc3505B
Doxycycline hyclate	Sigma-Aldrich	Cat#D9891
Hygromycin B	Research Products International	Cat# H75020
ProLong Diamond Antifade Moutant with DAPI	Thermo Fisher Scientific	Cat#P36962
Mowiol	Calbiochem
Biotin Phosphatidylinositol 3-phosphate	Echelon	Cat#C-03B6
p110α/p85α	Promega	Cat#V1721
PIK4B	Echelon	Cat#P32–10G
PIP5K1C	SignalChem	Cat#P16–102CG-05
PI(3)P:PS liposomes	Promega	Cat#V1711
PI(4,5)P₂:PS liposomes	Promega	Cat#V1701
PI(4)P:PS lipid substrate	SignalChem	Cat#P427–59
Radian HRP substrate	Azure biosystems	Cat#AC2101
Critical Commercial Assays
Class III PI3K Elisa Kit	Echelon	Cat#K-3000
PI 4-Kinase Activity Assay	Echelon	Cat#K-4000K
PI3-Kinase Activity Assay	Echelon	Cat#K-1000s
PIP strip	Echelon	Cat#P-6001
Lipofectamine 3000	Thermo Fisher Scientific	Cat#L30000015
Experimental Models: Cell Lines
Mouse: RAW 264.7 (TIB-71)	ATCC	TIB-71; RRID:CVCL_0493
Human: HeLa cells (CCL-2)	ATCC	CCL-2; RRID:CVCL_0030
HEK293T/17	ATCC	CRL-11268; RRID:CVCL_1926
Experimental Models: Organisms/Strains
Mice: 8–10 week old Ffmale C57BL/6	Jackson Laboratories (Bar Harbor, ME)	Cat# 000664
Mouse: Bone Marrow Derived Macrophages (BMMs) from C57BL/6NHsd mice	Envigo	Cat# 4403F
Oligonucleotides
Primers used in this study are listed in Table S1	This paper	N/A
Recombinant DNA
pET28b-opiA	This paper	N/A
pET28b-opiA^H261A	This paper	N/A
pET28b-legA5	This paper	N/A
pET28b-legA5^H178A	This paper	N/A
pEX18-pheS-km	Eshraghi et al., 2016	N/A
pEX18-pheS-km-∆opiA	Eshraghi et al., 2016	N/A
pEX18-pheS-km-∆opiB	Eshraghi et al., 2016	N/A
pEX18-pheS-km-∆pdpC	Eshraghi et al., 2016	N/A
pEX18-pheS-km-∆pdpD	Eshraghi et al., 2016	N/A
pEX18-pheS-km-∆dotU	Eshraghi et al., 2016	N/A
pMP720	LoVullo et al., 2009	N/A
pMP749	LoVullo et al., 2009	N/A
pMP749-opiA_comp (5’ end+337bp, 3’ end +169bp)	This paper	N/A
pMP749-opiA^H261A_ comp (5’ end+337bp, 3’ end +169bp)	This paper	N/A
pMP749-Pbfr-sfGFP	This paper	N/A
pCM190	Gari et al., 1997	N/A
pCM190-opiA	This paper	N/A
pCM190-opiA^H261A	This paper	N/A
pCM190-mcm1	This paper	N/A
pEGFP-2xFYVE	Starr et al., 2012	N/A
pBABE- 2xFYVE-mCherry	Gift from Dr. Oliver Florey; Florey et al., 2015	N/A
pEGFP-mt target sequence from subunit VIII of human cytochrome oxidase	Gift from Dr. Suzanne Hoppins	N/A
pMSCVpuro	Clonetech	N/A
pMSCVpuro-opiA	This paper	N/A
pMSCVpuro-opiA^H261A	This paper	N/A
plenti_CMV_neo_mCherry-2xFYVE	Gift from Dr. Schink; Palibrk et al., 2014	N/A
pCDNA3-eGFP-rab7_T22N	Sun et al., 2010	Addgene Cat# 28048
pCDNA3	This study	N/A
pCMVdeltaR8.2	Gift from D. Trono	Addgene Cat# 12263
pMD2.G	Gift from D. Trono	Addgene Cat# 12259
Software and Algorithms
GraphPad Prism version 7.0 for Mac	GraphPad, Software, La Jolla, California, USA	www.graphpad.com; RRID:SCR_022798
ImageJ	NIH	Imagej.nih.gov; RRID:SCR_003070
Adobe Illustrator CC 2015	Adobe Systems Incorporated, San Jose, California, USA	www.adobe.com/products/illustrator; RRID:SCR_010279
Adobe Photoshop CS6	Adobe Systems Incorporated, San Jose, California, USA	www.adobe.com/products/photoshop; RRID:SCR_014199
Leica LAS AF Image Acquisition Software	University of Washington	RRID:SCR_013673
Geneious 10.0.9	Geneious, Software, Newark, New Jersey, USA	www.geneious.com RRID:SCR_010519
Other
1mL HisTrap FF NI-TA cartridge	GE Healthcare	Cat#17–5319-01