intracellular pathogens

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)

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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

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

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 bacteriaper vesicle

-ultimate goal: destructionof bacteria by antimicrobialcompounds

How phagosomes kill bacteria


-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

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


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)


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)


• 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….



…to form membrane prepore of sufficient size

-helical bundles in prepore are converted into amphipathic transmembrane -hairpins…



…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

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

http://www.cbc.ca/news/health/story/2012/01/11/legionnaires-disease-hospitals-water-features.html

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


CFT-1: CF tracheal cell line (F508)LCFSN: complemented cell line (wt CFTR)

Attachment vs. Internalization



(30 min)

Internalization of different bacteria



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



S. aureus loses association with acidic vesicles over time



S. aureus loses association with acidic vesicles over time


CFT-1 (live)

LCFSN (live)

CFT-1 (dead)LCFSN (dead)


Model for S. aureus trafficking within a CF mutant cell line



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!

intracellular pathogens

Documents

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