Intracellular Pathogens
MIC4124
Thien-Fah Mah, PhD
Department of Biochemistry, Microbiology and Immunology
Dr. Mah’s path to U of O
1
35 4
1. B.Sc in Microbiology, UBC, Vancouver, BC
2. Three years of work (lab, library, teaching)
and travel
3a. M.Sc. in Molecular and Medical Genetics,
U of Toronto
3b. Ph. D in Molecular and Medical Genetics,
U of Toronto
4. Postdoctoral training at Dartmouth Medical
School, Hanover, NH
5. Assistant Professor, U of Ottawa (2005)
http://www.worldmapsonline.com/classroommaps/ECNorthAmericaPhys.jpg
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)
Phagocytosis
Receptor mediated, actin-driven process whereby foreign objects larger than
0.5 mm are internalized and degraded.
http://www.nature.com/nri/journal/v8/n2/full/nri2240.html
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
-membrane is derived from
host plasma membrane
-single or multiple bacteria
per vesicle
-ultimate goal: destruction
of bacteria by antimicrobial
compounds
Haas, A 2007 Traffic 8:311-330
How phagosomes kill bacteria
-nicotinamide adenine dinucleotide
phosphate (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
Haas, A 2007 Traffic 8:311-330
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
-Monolayer of epithelial cells infected with S. aureus at MOI 10:1
-V-ATPase labelled with antibody
-DNA stained with To-Pro-3
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
Acidification of a phagosome
-measurement of pH following internalization of a bead
-concanamycin A inhibits V-ATPase
Rohde, K et al 2007 Immunol Rev 219:37-54
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
Step I: using SPI-1 type III secretion system
-injection of effectors that mediate uptake
Step II: SPI-2 effectors mediate formation of
Salmonella-containing vesicles (SCVs)
Knodler, LA et al 2001 Nat Rev Mol Cell Biol 2(8): 578-588
http://www.hhmi.org/biointeractiv
e/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 vivo
Rajashekar 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 vivo
Rajashekar R et al 2008 Traffic
Salmonella genes important for intracellular life
Modulation of host cytoskeleton
(SPI-1)
SopB-activates Cdc42 and Rho
SopE-activates Cdc42, Rac and Rho
SopE2-activates Cdc42, Rac and Rho
SipA-actin nucleation
SipC-actin nucleation
Formation and
Maintenance of SCV (SPI-2)
SifA-Sif formation
SopD2- Sif formation
SseJ- maintains SCV integrity
Steele-Mortimer,Finlay Cell. Micro. (1999) 1(1), 33-49
Haraga A et al 2008 Nat Rev Microbiol 6: 53-66
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
Intracellular M. tuberculosis
Granuloma
Russell 2007 Nat Rev Microbiol 5:39-47
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
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. tuberculosiscontaining phagosome
Van der Wel et al 2007 Cell 129:1287-98
Evidence of escape into cytosol
4 hours
96 hours
-green= bacteria
-red= LAMP-1
-blue=host cell DNA
-yellow=green and red merged
*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 hours
and no association with LAMP-1 (dots)
-escape into cytosol requires RD1 (regions of difference between pathogenic
and 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) cocolocalizes (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
Listeria monocytogenes
•
•
•
•
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
http://www.rps.psu.edu/indepth/graphics/knabel2.jpg
Genes required for L. monocytogenes to
invade and spread
L. monocytogenes avoids destruction by escaping into host cytosol
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
Genes required for survival in the host
• inlA and inlB (invasion-associated surface proteins
internalin A and B: mediate invasion of nonphagocytic 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 cholesteroldependent 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
Monomers bind cholesterol,
diffuse laterally….
Tweten 2005 Infect Immun 73:6199-6209
Mechanism of cholesterol-dependent cytolysin
pore formation
…to form membrane prepore
of sufficient size
a-helical bundles in prepore
are converted into amphipathic
transmembrane b-hairpins…
Tweten 2005 Infect Immun 73:6199-6209
Mechanism of cholesterol-dependent cytolysin
pore formation
…and the prepore collapses 40Å
such that specific domains are
inserted into the membrane
The end result is the formation of
a large b-barrel pore
Tweten 2005 Infect Immun 73:6199-6209
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
actin
profilin
Arp2/3
-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
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/legionnai
res-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
-L. pneumophila creates intravacuolar
niche for replication
-normal phagocytic pathway
-normal eukaryotic secretory
membrane system (from endoplasmic
reticulum [yellow], to golgi complex,
to the plasma membrane)
Isberg et al 2009 Nat Rev Micriobol 7:13-24
L. pneumophila modulates phagocytic pathway
-L. pneumophila creates intravacuolar
niche for replication
Isberg et al 2009 Nat Rev Micriobol 7:13-24
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)
-several Dot/Icm effectors associate with
the LCV and recruit host proteins (boxed) that
are involved in vesicle trafficking through
the secretory pathway
Isberg et al 2009 Nat Rev Microbiol 7:13-24
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
patients
Courtesy 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
CFT-1: CF tracheal cell line (DF508)
LCFSN: complemented cell line (wt CFTR)
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
Attachment vs. Internalization
(30 min)
CFT-1: CF tracheal cell line (DF508)
LCFSN: complemented cell line (wt CFTR)
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
Internalization of different bacteria
CFT-1: CF tracheal cell line (DF508)
LCFSN: complemented cell line (wt CFTR)
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
S. aureus loses association with LAMP-1
and LAMP-2 over time
CFT-1: CF tracheal cell line (DF508)
LCFSN: complemented cell line (wt CFTR)
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
S. aureus loses
association with LAMP-1
and LAMP-2 over time
CFT-1: CF tracheal cell line (DF508)
LCFSN: complemented cell line (wt CFTR)
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
S. aureus loses association with
acidic vesicles over time
CFT-1: CF tracheal cell line (DF508)
LCFSN: complemented cell line (wt CFTR)
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
S. aureus loses association with
acidic vesicles over time
CFT-1 (dead)
LCFSN (dead)
LCFSN (live)
CFT-1 (live)
CFT-1: CF tracheal cell line (DF508)
LCFSN: complemented cell line (wt CFTR)
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
Model for S. aureus trafficking
within a CF mutant cell line
CFT-1: CF tracheal cell line (DF508)
LCFSN: complemented cell line (wt CFTR)
Jarry, TM and Cheung, AL 2006 Infect Immun 74 (5):2568-2577
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!