Legionella pneumophila: an intracellular pathogen of phagocytes Prof. Craig Roy

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1 an intracellular pathogen of phagocytes Section of Microbial Pathogenesis, Yale University School of Medicine 1 Legionella pneumophila Gram-negative bacterium Facultative intracellular pathogen Protozoa are natural hosts Aerosols can result in human infection Replication in macrophages Legionnaires' disease 2 Horwitz and colleagues define the Legionella lifecycle 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 The Shuman and Isberg laboratories independently identify the dot and icm genes Andrea Marra: Complementing an icm defect restores the ability of Legionella 25d to avoid fusion with lysosomes Karen Berger: The dota gene is essential for intracellular replication and avoiding lysosome fusion Joe Vogel and Gil Segal sequence a total of 24 dot and icm genes 4 The dot and icm genes define a new type IV secretion system Host cell plasma membrane Outer membrane Inner membrane Figure by Christie and Vogel A. tumefaciens virb type IVa L. pneumophila dot/icm type IVb 5 Why is the Dot/Icm system required for intracellular replication? Does Dot/Icm control nutrient acquisition into the vacuole? Does Dot/Icm poison the cell to make it generally permissive for bacterial replication? Does Dot/Icm interfere directly with vesicle transport? Are bacterial effector proteins translocated by the Dot/Icm system? 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Endocytic membrane transport Endocytic membrane transport 7 The Dot/Icm system mediates rapid evasion of endocytic maturation 100% wild type dot/icm mutants hagosomes ining positive Ph stai 75% 50% 25% 5 min 15 min 30 min Early Endocytic Compartments Rab7 - LAMP-1 - Rab7 + LAMP-1-5 min 15 min 30 min Late Endocytic Compartments Rab7 + LAMP-1 + Rab7 - LAMP Understanding Dot/Icm function through coinfection of macrophages Wild type bacteria do not produce GFP dota mutants produce GFP Macrophages coinfected with wild type and dota mutants dota mutant transdominant over wild type Vacuoles traffic independently Wild type transdominant over dota mutant Joern Coers and Catherine Monahan 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 The Dot/Icm system controls transport of the Legionella-containing vacuole All bacteria dota mutant Merge-DIC All bacteria + nucleus dota mutant Merge 10 Legionella subverts vesicular transport between the endoplasmic reticulum and Golgi Inhibiting Golgi assembly and membrane transport in the late secretory pathway had no effect on Legionella replication Inhibiting membrane transport from the ER interfered with Legionella replication X X X Inhibiting membrane transport in the endocytic pathway enhanced Legionella replication Jonathan Kagan 11 Legionella replication requires vesicular transport out of the endoplasmic reticulum 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 ARF function is required for ER remodeling of the Legionella-containing vacuole ER labeled with YFP-KDEL No inhibition of ARF ER labeled with YFP-KDEL ARF function inhibited with Brefeldin A 13 ER-derived vesicles associate rapidly with the Legionella-containing vacuole 5 min post infection 15 min post infection Lew Tilney and Cam Robinson 14 Legionella utilize host ases for biogenesis of a vacuole that supports replication 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 The Dot/Icm system modulates Arf Legionella ARF1-GFP Merge dota wild type Jon Kagan 16 The Dot/Icm system modulates Rab1 GFP-Rab1a DNA GFP-Rab1a DNA anti-rab1b DNA anti-rab1b DNA Jon Kagan 17 Control of Arf and Rab ases cytosolic Arf1 GEF membrane associated Arf1 Effectors Coat proteins -COPI - AP-1 - AP-3 - GGAs Lipid modification enzymes -PLD -PI 4 K -PI 4 P 5 K Pi GAP Others - Arfaptin/POR1 - Arfophilins/FIPs - FAPPs 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Can we identify Legionella proteins that modulate Arf and Rab1 function? Arf1 Putative Dot/Icm effector proteins Rab1 19 Identification of ralf (Recruitment of Arf to the Legionella phagosome) ralf wild type Legionella ARF1-GFP Hiroki Nagai ARF1-GFP 20 RalF is present on the cytosolic surface of the Legionella-containing vacuole anti-ralf Legionella anti-ralf Legionella anti-ralf Legionella anti-ralf Legionella 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 RalF is delivered into host cells by the Dot/Icm system upon uptake DNA anti-legionella (extracellular) ARF1 22 RalF defines a bacterial member of the Sec7 family of Arf exchange factors L. pneumophila RalF protein S. cerevisiae Sec7p Sec7 Domain RalF + Arf1 RalF + Arf6 RalF + Arf5 Richard Kahn 23 Activation of Arf by Legionella cytosolic RalF membrane associated Arf1 Arf1 Legionella Pi GAP Vacuole 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 Can Legionella modulate the function of other host ases? 25 Identification of DrrA: a Legionella effector protein that modulates Rab1 activity Legionella transposon insertion mutants Pick individual mutants Array mutants in 96-well dish 96-pin replicator Infect macrophages in 96-well glass-bottom dish Takahiro Murata Fix and stain for Rab1 Screened 3948 mutants Re-screened 441 mutants Rab defects 119 mutants Dot + /Rab1-9 mutants Score Rab1 recruitment Clean deletion 1 mutant by microscopy 26 DrrA is a novel protein that functions as a guanine nucleotide exchange factor for Rab1 cytosolic DrrA (SidM) membrane associated Rab1 Rab1 Legionella GAP Pi Vacuole 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 Does Legionella have other effectors that target Rab1 specifically?? DrrA?? Alyssa Ingmundson 28 DrrA does not require other Legionella effectors to localize and activate Rab1 GFP-DrrA Total Internal Reflection Fluorescence Microscopy (TIRFM) of plasma membrane 29 DrrA is able to dissociate GDI from Rab1 Pull-down Rab1-GDI complex from transfected cells Add purified DrrA Immunoblot supernatant to measure GDI release 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 DrrA has GDI displacement activity 31 DrrA modulates Rab1 function by removing GDI and stimulated for exchange DrrA (SidM) GDI Rab1 GDI Rab1 Legionella GAP Pi 32 LepB interacts specifically with Rab1-33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 LepB interacts specifically with Rab1- (2) 34 LepB is a Rab1 GAP 35 Control of Rab1 membrane cycling by Legionella effector proteins Plasma membrane DrrA Rab1-GDI GDI Pi LepB Pi ER exit sites 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 Legionella: the greatest cell biologist on Earth! Phagocytosis Trafficking Inhibit host defenses Estimated 150 effectors Egress Cell survival Modulate host transcription/ translation The screen versions of these slides have full details of copyright and acknowledgements 13