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Intracellular Pathogens. MIC4124 Thien-Fah Mah, PhD Department of Biochemistry, Microbiology and Immunology. Dr. Mah’s path to U of O. 1. B.Sc in Microbiology, UBC, Vancouver, BC 2. Three years of work (lab, library, teaching) and travel - PowerPoint PPT Presentation
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Intracellular Pathogens
MIC4124
Thien-Fah Mah, PhDDepartment of Biochemistry, Microbiology and Immunology
Dr. Mah’s path to U of O
http://www.worldmapsonline.com/classroommaps/ECNorthAmericaPhys.jpg
1. B.Sc in Microbiology, UBC, Vancouver, BC2. Three years of work (lab, library, teaching) and travel3a. M.Sc. in Molecular and Medical Genetics, U of Toronto3b. Ph. D in Molecular and Medical Genetics, U of Toronto4. Postdoctoral training at Dartmouth Medical School, Hanover, NH5. Assistant Professor, U of Ottawa (2005)
1
3 45
Dr. Desjardin’s path to the Ottawa Hospital
• 1985-1989: BSc with honours Biology - University of Ottawa
• 1989-1996: Graduate studies - PhD in Microbiology (used to be Dept of Micro and Immuno) U of Ottawa
• 1996-1998: Clinical Microbiology residency fellowship: Strong Memorial Hospital, Rochester New York
• 1998-2002: Director Microbiology Laboratory - Binghamton General Hospital, Binghamton New York
• 2002: Board Certification examination - Diplomat American Board of Medical Microbiology and Fellow of the Canadian College of Microbiology
• 2002: I'm back in Ottawa as a Clinical Microbiologist
What is an intracellular pathogen?
• A microbe that is capable of causing damage to its host that spends at least part of its lifecycle within a host cell or host cell vacuole– ability to replicate within the host cell
– ability to persist within the host cell
Why be an intracellular pathogen?
• Life outside a cell is harsh– Low pH, shear stress due to circulatory system,
complement, antibodies, macrophages, T cells
• Bacteria can hide out and replicate inside the cell cytoplasm or inside vacuoles (phagosomes) within cells
– Bacteria can invade into underlying tissue
Some notable intracellular pathogens• Coxiella burnetii - Q fever (highly contagious- only a single
bacterium required for infection; flu-like symptoms can progress to pneumonia and acute respiratory distress syndrome (ARDS) and death)
• Legionella pneumophila* - Legionnaires disease• Listeria monocytogenes* – Listeriosis• Mycobacterium tuberculosis* – Tuberculosis• Rickettsia prowazekii – epidemic typhus (occurs after wars and
natural disasters; headache, fever, chills, delirium, death)• Salmonella enterica* – gastroenteritis and typhoid fever• Shigella flexneri – shigellosis (bloody diarrhea and fever,
usually resolves within a week)
Entry into a cell
• by phagocytosis (cell-eating)– by professional phagocytes like neutrophils,
macrophages and dendritic cells
• by induced uptake– by cells that are not normally phagocytic
(endothelial and epithelial cells)
http://www.nature.com/nri/journal/v8/n2/full/nri2240.html
Receptor mediated, actin-driven process whereby foreign objects larger than 0.5 m are internalized and degraded.
Phagocytosis
Phagocytosis animation
http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__phagocytosis.html
Induced uptake
Knodler, LA et al 2001 Nat Rev Mol Cell Biol 2(8): 578-588
Host actin
• primary host determinant of cell shape and cytoplasmic structure
• Rho family of GTPases (Rho, Rac, Cdc42) is important for actin organization
• bacteria manipulate the actin cytoskeleton to induce or prevent phagocytosis
Actin cytoskeleton
http://scienceblogs.com/transcript/2007/01/cytoskeleton.php
Antiphagocytic action of Y. pestis Yop proteins
Cornelis, GR 2002 Nature Rev Mol Cell Biol 3:742-52
Persistence
• Two possible mechanisms allowing pathogens to persist within the host cell:– Modification of endocytic pathway
• Salmonella enterica• Mycobacterium tuberculosis• Legionella pneumophila
– Escape from endocytic vesicle (phagosome)• Listeria monocytogenes• Maybe M. tuberculosis?• Maybe Staphylococcus aureus?
Endocytic and exocytic pathways
http://www.landesbioscience.com/curie/chapter/4274/
Endocytic (phagocytic) pathway
Haas, A 2007 Traffic 8:311-330
Endocytic (phagocytic) pathway
Haas, A 2007 Traffic 8:311-330
Phagosomal Maturation• phagosomes mature due to sequential addition
of cellular proteins delivered to them by fusion with different endosomal compartments
• anything left in the phagosomes will be destroyed
• bacteria need to either – escape before the phagosome environment becomes
bactericidal– modify the phagosomal maturation process
Bacteria inside phagosomes
Haas, A 2007 Traffic 8:311-330
-membrane is derived from host plasma membrane
-single or multiple bacteriaper vesicle
-ultimate goal: destructionof bacteria by antimicrobialcompounds
How phagosomes kill bacteria
Haas, A 2007 Traffic 8:311-330
-nicotinamide adenine dinucleotidephosphate (NADPH) oxidase complex produces superoxide radicals from oxygen
-nitric oxide synthetase produces NO radicals
-vacuolar ATPase pumps protons into the phagosome in reduce pH to ~4.5
-fusion with lysosomes results in delivery of various hydrolases that digest most biological macromolecules
Ways to study bacterial-phagosomal interactions
• Direct microscopic observation• Characterize the vacuoles that contain bacteria
using immunofluorescence colocalization– correlate the presence and absence of various host
markers with the presence of the bacterium
• Directly measure physiological parameters of the phagosome– pH– hydrolytic activities
Colocalization of V-ATPase with bacterial DNA
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
-Monolayer of epithelial cells infected with S. aureus at MOI 10:1-V-ATPase labelled with antibody-DNA stained with To-Pro-3
Acidification of a phagosome
Rohde, K et al 2007 Immunol Rev 219:37-54
-measurement of pH following internalization of a bead
-concanamycin A inhibits V-ATPase
Salmonella enterica
• Gram negative pathogen– gastroenteritis (self-limiting, mild) to typhoid fever
(fatal)
• Virulence factors mainly located on one of two Salmonella pathogenicity islands, SPI-1 or SPI-2– encode type III secretion systems important for
invasion of non-phagocytic cells and bacterial replication in phagosomes
Pathogenicity Islands• Large genomic regions that are present in
pathogens but absent in their non-pathogenic counterparts
• % GC content quite different from surrounding DNA (suggests that DNA acquired from different organism)
• Found in both gram negative and gram positive bacteria
• Carry virulence genes• Carry factors that promote mobility (integrases,
transposases, IS elements)
SPI-1 and SPI-2
Hansen-Wester and Hensel 2001 Microbes Infect 3: 549-559
Intracellular S. enterica
Knodler, LA et al 2001 Nat Rev Mol Cell Biol 2(8): 578-588
Step I: using SPI-1 type III secretion system-injection of effectors that mediate uptake
Step II: SPI-2 effectors mediate formation ofSalmonella-containing vesicles (SCVs)
http://www.hhmi.org/biointeractive/disease/salmonella.html
Salmonella-containing vesicle (SCV)
• can persist for hours to days• formation requires genes from SPI-
2• characterized by several endosomal
markers– EEA1 (early endosomal marker)
– Rab5 and Rab11 (middle)
– Rab 7, LAMP1, LAMP2, LAMP3, V-ATPase (late)
– low pH (indicative of lysosomal fusion)
– transient?
Steele-Mortimer,Finlay Cell. Micro. (1999) 1(1), 33-49
Salmonella-induced filaments (Sifs)
• 4-6 hours after SCV formation, see salmonella-induced filaments (sifs)
• not sure what their function in pathogenesis is
• not sure what effectors do
• Sif formation not yet shown in vivoRajashekar R et al 2008 Traffic
Salmonella-induced filaments (Sifs)
• 4-6 hours after SCV formation, see salmonella-induced filaments (sifs)
• not sure what their function in pathogenesis is
• not sure what effectors do
• Sif formation not yet shown in vivoRajashekar R et al 2008 Traffic
Steele-Mortimer,Finlay Cell. Micro. (1999) 1(1), 33-49
Modulation of host cytoskeleton (SPI-1)SopB-activates Cdc42 and RhoSopE-activates Cdc42, Rac and RhoSopE2-activates Cdc42, Rac and RhoSipA-actin nucleationSipC-actin nucleation
Formation and Maintenance of SCV (SPI-2)SifA-Sif formationSopD2- Sif formationSseJ- maintains SCV integrity
Haraga A et al 2008 Nat Rev Microbiol 6: 53-66
Salmonella genes important for intracellular life
Mycobacterium tuberculosis
• Gram positive• Reservoir: humans• Causes tuberculosis, spread by aerosols• Enters host macrophages and subverts normal
phagosome maturation• Persists in a granuloma
http://www.sunysb.edu/icbdd/images/infectious2.jpg
Infected macrophages and the granuloma
Russell 2007 Nat Rev Microbiol 5:39-47
Intracellular M. tuberculosis
Granuloma
M. tuberculosis Phagosome
• M. tuberculosis– Blocks rab conversion– Retains characteristics
of early endosome (Rab5 and EEA-1)
– Phagosome pH only reaches pH 6.5
Adapted from Via and Deretic 1997 JBC 272:13326-13331
Rab conversion block
-Rab5 (early) and Rab7 (late) are GTPases involved in endosome maturation
-Rab conversion occurs when Rab5 is replaced with Rab7
Deretic, V. et al 2006 Cell Microbiol 8:719-727
Rab conversion block
Mycobacterial factors that cause the block:– Liparabinomannan (LAM) is a lipid (part of
mycobacterial cell wall) that prevents the increase of cytosolic [Ca2+] that normally accompanies phagocytosis (and this affects the recruitment of a Rab5 effector)
– Phosphatidylinositol mannoside (PIM)- mechanism unknown
– SapM is a PI3P phosphatase *
*PI3P is important for proper membrane trafficking through the endosomal pathway
Recent observations suggest that M. tuberculosis can escape into the cytosol
Van der Wel et al 2007 Cell 129:1287-98
Direct microscopic observations suggested that M. tuberculosis was not blocking phagosomal maturation at early stages….
-M. tuberculosis in dendritic cells at 2 hours
-bacteria (star) colocalize with LAMP-1 (dot)(late endosomal marker)
-lysosomes fuse with M. tuberculosis-containing phagosome
Evidence of escape into cytosol
-green= bacteria-red= LAMP-1-blue=host cell DNA-yellow=green and red merged
4 hours
96 hours
*also counted number of bacterial cells at 4 hours and 96 hours and saw increase
Van der Wel et al 2007 Cell 129:1287-98
More evidence for escape into cytosol
-no phagosomal membrane surrounding M. tuberculosis at 96 hoursand no association with LAMP-1 (dots)-escape into cytosol requires RD1 (regions of difference between pathogenicand non-pathogenic mycobacteria) genes (contains ESAT/type VII secretion system)
Van der Wel et al 2007 Cell 129:1287-98
Non-pathogenic mycobacterium does not escape into cytosol
-M. bovis (vaccine strain) (green) co-colocalizes (yellow) with LAMP-1 (red)-host cell DNA (blue)
-evidence of phagosomal membrane surrounding bacteria-association with LAMP-1 (dots)
Van der Wel et al 2007 Cell 129:1287-98
• Gram positive
• Ubiquitous in the environment
• Intracellular pathogen
• Causes listeriosis – acquired by ingesting contaminated food– mainly affects the elderly, the very young,
immunocompromised individuals and pregnant women
– results in gastoentiritis, meningitis, encephalitis, septicaemia and death in 25-30% of cases
Listeria monocytogenes
http://www.rps.psu.edu/indepth/graphics/knabel2.jpg
Genes required for L. monocytogenes to invade and spread
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VPN-4NNN0J0-6&_user=1067359&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=1067359&md5=30ef1ede32268634f516a8778f263c32
L. monocytogenes avoids destruction by escaping into host cytosol
Genes required for survival in the host
• inlA and inlB (invasion-associated surface proteins internalin A and B: mediate invasion of non-phagocytic cells)
• hly (listeriolysin O or LLO: pore-forming toxin)• plcA and plcB (phospholipase C enzymes: phagocytic
vacuole disruption)• actA (actin polymerization)• mpl (metalloprotease required for PlcB maturation)• hpt (hexose phosphate transporter important for rapid
growth in host cytosol)
LLO
• a primary virulence factor facilitating escape from the phagosome and cell-to-cell spread
• member of pore-forming cholesterol-dependent cytolysin family (C. perfringens, S. pyogenes, B. anthracis)
• active at pH 5.5 (therefore active in phagosome and not in cytosol)
Mechanism of cholesterol-dependent cytolysin pore formation
Tweten 2005 Infect Immun 73:6199-6209
Monomers bind cholesterol,diffuse laterally….
Mechanism of cholesterol-dependent cytolysin pore formation
Tweten 2005 Infect Immun 73:6199-6209
…to form membrane prepore of sufficient size
-helical bundles in prepore are converted into amphipathic transmembrane -hairpins…
Mechanism of cholesterol-dependent cytolysin pore formation
Tweten 2005 Infect Immun 73:6199-6209
…and the prepore collapses 40Å such that specific domains areinserted into the membrane
The end result is the formation ofa large -barrel pore
ActA
• identified in a screen for bacteria deficient in cell to cell spreading
• required for actin-based motility
• bacterial surface protein that interacts with several host proteins
Actin comet tails• after a few hours of growth, host actin
filaments form on surface of bacteria
• polarization into a comet tail (cross-linked network of actin filaments)
Cameron LA et al 2000 Nat Rev Mol Cell Biol 1:110-119
Actin-Arp2/3-ActA interaction
-vasodilator-stimulated phosphoprotein (VASP) interacts with profilin (actin binding protein)-Arp2/3 is a 7 protein complex that nucleates actin
Cameron LA et al 2000 Nat Rev Mol Cell Biol 1:110-119
Arp2/3
actin profilin
Listeria with actin tails
http://mcb.berkeley.edu/labs/portnoy/
Theriot lab movies
• http://cmgm.stanford.edu/theriot/movies.htm
Actin-based motility in other systems
• Shigella flexneri
• Rickettsia species
• Vaccinia virus
• Enteropathogenic E. coli (pedestal formation)
Refs: Goldberg 2001 Microbiol Mol Biol Rev 65:595-626 Cameron et al 2000 Nat Rev Mol Cell Biol 1:110-119
Legionella pneumophila
• Gram negative• Found as parasite of freshwater protozoa• Infects alveolar macrophages and causes severe
pneumonia• Legionnaires disease
– Outbreak at a war vet convention in Philadelphia 1976 (34 deaths)
– isolated bacterium from cooling tower that linked to the air conditioning system of the hotel
– Climate control changes implemented
Legionnaires disease today
• http://www.cbc.ca/news/health/story/2012/01/11/legionnaires-disease-hospitals-water-features.html
Intracellular L. pneumophila
• Legionella containing vacuole (LCV)– Does not fuse with late endocytic vesicles
• Resists lysosomal degradation• Associates with smooth vesicles and is decorated with
ribosomes• Is a replication niche
• Smooth vesicles are derived from ER– Unique biomarkers for ER– Believed that this is a source of polypeptides (energy
for bacterial replication)– Camouflage?
L. pneumophila modulates phagocytic pathway
Isberg et al 2009 Nat Rev Micriobol 7:13-24
-normal phagocytic pathway-normal eukaryotic secretory membrane system (from endoplasmic reticulum [yellow], to golgi complex,to the plasma membrane)
-L. pneumophila creates intravacuolar niche for replication
L. pneumophila modulates phagocytic pathway
Isberg et al 2009 Nat Rev Micriobol 7:13-24
-L. pneumophila creates intravacuolar niche for replication
Type IV secretion: Dot/Icm gene locus mediates intracellular survival
• Genetic screen identified mutants defective in intracellular replication
• Dot/Icm: defect in organelle trafficking/intracellular multiplication genes
• Type IV secretion system required for delivery of proteins across the host cell membrane (phagosome membrane)
Dot/Icm secretion apparatus
Isberg et al 2009 Nat Rev Microbiol 7:13-24
Legionella containing vacuole (LCV)
Isberg et al 2009 Nat Rev Microbiol 7:13-24
-several Dot/Icm effectors associate withthe LCV and recruit host proteins (boxed) thatare involved in vesicle trafficking throughthe secretory pathway
Type IV effectors• A growing number of proteins (effectors) are
substrates for the Dot/Icm associated type IV secretion system (30 +) but functions not well understood– Some proposed to promote phagocytosis– Others prevent fusion with normal endocytic vesicles– Some of eukaryotic resemblance suggesting roles in
mimicry– Acquisition of eukaryotic genes?
ArfI – a host trafficking protein
• ArfI is a GTPase responsible for mediating vesicular transport between Golgi and ER– ArfI: inactive [GDP bound form]
and active [GTP bound form] –natural cycle
• Recruitment of ArfI to membranes requires GEF (guanine nucleotide exchange factor) which remove GDP to allow GTP binding
Type IV effector: RalF
• RalF– is a GEF (enzymatic activity)
– Stimulates ArfI activation promoting its fusion to the LCV
• ralF mutant is deficient in ArfI localization to the LCV
Type IV effectors
• Other effectors are involved– VipA and VipD block lysosomal fusion– LepA and LepB promote Legionella release -
dissemination– SdhA prevents apoptosis
• Key to intracellular survival (cell remains a viable unit to allow replication!)
– LidA binds Rab1-GTPase• Modulates vesicular pathway
Staphylococcus aureus
• gram-positive cocci• production of a large
number of secreted toxins• humans are major reservoir
(up to 40% are carriers)• major nosocomial pathogen• emergence of antibiotic
resistant strains -MRSA• pathogen of Cystic Fibrosis
patientsCourtesy of Rob Shanks, Dartmouth Medical school
Staphylococcus aureus Escapes More Efficiently from the Phagosome of a Cystic Fibrosis Bronchial
Epithelial Cell Line than from Its Normal Counterpart
Todd M. Jarry and Ambrose L. Cheung 2006
Infection and Immunity 74(5):2568-2577
S. aureus: intracellular pathogen?
• Rationale:– Originally thought to be exclusively extracellular
– Why it might not be: “although the role of S. aureus in the disease progression of CF remains unknown, the clinical observation of rapid recolonization soon after treatment suggests that S. aureus may persist inside host cells of the lung”*
*Jarry and Cheung 2006 Infect Immun 74:2568-2577
Hypothesis:
S. aureus is an intracellular pathogen
Experimental set up
• Invasion assays– Bacteria were added to host cells in tissue culture plates and
allowed to incubate for specific periods of time– Extracellular bacteria were killed with addition of antibiotic (only
intracellular bacteria will survive)– Host cells were lysed and bacterial cells were counted
• Immunofluorescence– Bacteria were added to host cells, as above– Cells were fixed and antibodies (to LAMP-1 or LAMP-2) were
added– Also utilized this technique to observe association with lysotracker
Intracellular replication
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)
Attachment vs. Internalization
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)
(30 min)
Internalization of different bacteria
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)
S. aureus loses association with LAMP-1 and LAMP-2 over time
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)
S. aureus loses association with LAMP-1 and LAMP-2 over time
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)
S. aureus loses association with acidic vesicles over time
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)
S. aureus loses association with acidic vesicles over time
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
CFT-1 (live)
LCFSN (live)
CFT-1 (dead)LCFSN (dead)
CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)
Model for S. aureus trafficking within a CF mutant cell line
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)
Is S. aureus an intracellular pathogen?
Did they prove or disprove their hypothesis?
Summary
• S. enterica: SCVs (early, middle and late markers), Sifs
• M. tuberculosis: Rab conversion block (early); possible escape into cytoplasm
• L. monocytogenes: Escapes from phagosome, invades other cells via actin-based motility
• L. pneumophila: LCV mimics host ER?• S. aureus: invades a CF cell line and escapes into
the cytoplasm.
Reminder: Final exam• April 21, 2012 from 9:30am-12:30pm in Desmarais (DMS) 1160 • Format:
– short answer (no multiple choice)
• It will cover the lectures from Feb. 15 (Bioterrorism) to April 4 (Intracellular Pathogens)
• I will also assign a paper (check Blackboard on April 13, 2012) to read before the exam and you will be asked to answer questions about it. You may NOT bring the paper or any notes to the exam.
• I will post a study guide on the Blackboard site on April 11, 2012. Please remember it is just a guide- anything that was addressed during the lectures is fair game
Good luck on your exams!