Host-Pathogen Interaction

Sites of microbial infectionSites of microbial infection

Skin

Lung

Conjunctiva of eye

Scratch, injury

Insect bite

Mouth

Digestivetract

Urogenitaltract

Anus

Pores, hair Follicles,Sweat glands

Pathogenic microbes

Normal non-pathogenic microflora

Pathogens can be successful Pathogens can be successful

in causing an infectionin causing an infection

They can They can attachattach to and to and penetratepenetrate body surfaces body surfaces

Ex: Ex: Schistosoma mansoniSchistosoma mansoni

Pathogens can be Pathogens can be introducedintroduced by a by a biting insectbiting insect

Ex: Ex: Malaria, Malaria, LeishmaniaLeishmania, virus, bacteria, virus, bacteria

Pathogens can Pathogens can take advantagetake advantage of of preliminarypreliminary damagedamage (wound, respiratory tract (wound, respiratory tract damage)damage)

Ex: Ex: Trypanosoma cruziTrypanosoma cruzi

Vector: Reduviid bugs (Vector: Reduviid bugs (TriatomaTriatoma & & RhodniusRhodnius))

a.k.a. Kissing buga.k.a. Kissing bug

However, the Immune SystemHowever, the Immune Systemhas has NaturalNatural, , ConstitutiveConstitutive Microbial SensorsMicrobial Sensors Utilized to RaUtilized to Rapidly React to Invasive Pathogens.pidly React to Invasive Pathogens.

They are part of the Innate Immune Response and They are part of the Innate Immune Response and Play Pivotal Role in the Development of the Play Pivotal Role in the Development of the

Adaptive Immune Response.Adaptive Immune Response.

TLR RolesTLR Roles

O’Neill, Luke A.J. “Immunity’s Early-Warning System”. Scientific American, Jan (2005), 38-45.

O’Neill, Luke A.J. “Immunity’s Early-Warning System”. Scientific American, Jan (2005), 38-45.

TLR1TLR1

TLR2TLR2

TLR4TLR4 TLR3TLR3TLR5TLR5 TLR9TLR9

PGNPGNUnmethylateUnmethylate

ddCpG DNACpG DNA

LPSLPSdsRNAdsRNA

FlagellFlagellinin

Phagocytic

receptor

MicroorganiMicroorganismssms Antigen Presenting CellAntigen Presenting Cell

Naive Naive T T

CellCell

CD28B.7

TCRMHC

NF-NF-BB

Cytokines (IL-1, IL-6, IL-12, TNF)Chemokines (MIP-2, MIP-1/)

PeptidePeptide

LipopeptidesLipopeptides

Phagosome

TLR2TLR2

Activation of adaptive Activation of adaptive immunity by innate immunity by innate

immunityimmunity

Co-stimulatorymolecules

Rapid crystalline (0.7-0.9 m)Size and Shape similar to PfHZ(see Inset). Bar size is 200 nm.

PfHZ sPLHZ

Hemozoin: A Malarial Metabolic WasteHemozoin: A Malarial Metabolic Waste

Intracellular Microbial SensorsIntracellular Microbial Sensors

Toll-like receptors:Toll-like receptors: Membrane Receptor that Sense Membrane Receptor that Sense ExtracellularExtracellular Microbes Microbesand within and within phagosome/endosome.phagosome/endosome.

NOD-Like Receptors, RIG and MDA:NOD-Like Receptors, RIG and MDA:IntracellularIntracellular Microbial Sensors Microbial Sensors Are Are CytoplasmicCytoplasmic surveillance proteins with surveillance proteins with CARD domainCARD domain

NOD proteinsNOD proteins (Nucleotide-binding oligomerisation domain)(Nucleotide-binding oligomerisation domain) Two membersTwo members: NOD-1 and NOD-2: NOD-1 and NOD-2

RIG-1RIG-1 (Retinoid-induced gene 1)(Retinoid-induced gene 1)

MDAMDA (Melanoma differentiation-associated gene)(Melanoma differentiation-associated gene)

CARD domainCARD domain

CARDCARD ((CCaspase-aspase-AActivating and ctivating and RRecruitment ecruitment DDomain)omain)

Found in some caspase proteinsFound in some caspase proteins

Mediates Mediates protein-proteinprotein-protein interaction interaction

Bouchier-Hayes L and Martin S.J., EMBO (2002)

Helicase domain

Helicase domain

CARD CARD

RIG-I

MDA-5/HelicardCARDCARD

1

1

925

1025

CARD1 540IPS-1/Cardif/VISA/MAVS

************

************

NOD protein structureNOD protein structure

NOD-1

NOD-2

CARD NBD LRRs

N-term

N-term C-term

C-term

CARD CARD NBD LRRs

CARD (Caspase-activating and recruitment domain)Caspase-activating and recruitment domain)

NBD (Nucleotide binding Domain)Nucleotide binding Domain)

LRRs (Leucine-Rich Repeats)Leucine-Rich Repeats)

NOD ligands: PeptidoglycanNOD ligands: Peptidoglycan

Peptidoglycan (PGN)Peptidoglycan (PGN)

Major component of Major component of gram+ cell wallgram+ cell wall

Found in thin layer in Found in thin layer in periplasmic space of gram–periplasmic space of gram–

Glycan chainsGlycan chains alternating alternating GlcNAcGlcNAc and and MurNAcMurNAc

linked by peptide bridgelinked by peptide bridge

Philpott D.J. and Girardin S.E., Mol Imm (2004)

Meso-DAP

MDP

NOD signallingNOD signalling

IKK complex

IkB

NFkB

NFkB

IkB

Proteasome

PGNNOD2

NOD2

NOD1

NOD1

Transcription

RIP2

RIP2

RIP2

NOD-Like Receptors (NLRs)NOD-Like Receptors (NLRs)

NOD1/2NOD1/2 IPAF/NAIPIPAF/NAIP NALP3/ASCNALP3/ASC

BacteriaBacteria

PGNPGN PGN PGN DAP DAP (meso-diaminopimelic)(meso-diaminopimelic)

MDP MDP (muramyl dipeptide)(muramyl dipeptide) MDPMDPSalmonellaSalmonella FrancisellaFrancisellaLegionellaLegionella RNA RNA (PAMP)(PAMP)

FlagellinFlagellin ToxinsToxins

Danger-Associated Host ComponentsDanger-Associated Host Components

Uric Acid CrystalsUric Acid CrystalsHemozoin ???Hemozoin ???(metabolic waste)(metabolic waste)

NOD-1

NOD-2

Meylan et al. Nature July 2006Meylan et al. Nature July 2006

NOD-2 and Crohn’s diseaseNOD-2 and Crohn’s disease

Abnormal NOD-2 expression correlates Abnormal NOD-2 expression correlates with defective epithelial defensewith defective epithelial defense

NOD-2NOD-2 expressed in expressed in Paneth cellsPaneth cells of intestine of intestine

Enteric bacteriaEnteric bacteria induce induce -defensin-defensin through NOD-2 to through NOD-2 to kill luminal microbes.kill luminal microbes.

Clinical evidence:Clinical evidence: CD patients have decreased CD patients have decreased -defensin expression in Paneth cells-defensin expression in Paneth cells

NOD-Like Receptors, RIG and MDANOD-Like Receptors, RIG and MDA

IntracellularIntracellular Microbial Sensors Microbial Sensors

That can detect bacterial and viral ligandsThat can detect bacterial and viral ligands

NODNOD detects PGN of several detects PGN of several bacteriabacteria

Inflammatory responseInflammatory response & & Cell DeathCell Death (IPAF/NAIP)(IPAF/NAIP)

RIG-1/MDA-5RIG-1/MDA-5 detects dsRNA of detects dsRNA of virusvirus

Modulation of Modulation of IFN signallingIFN signalling to control virus to control virus

SummarySummary

Anti-microbial Functions Anti-microbial Functions of Phagocytic Cells of Phagocytic Cells

Upon Receptor-Mediated recognitionUpon Receptor-Mediated recognition

Phagocytosis and InternalizationPhagocytosis and Internalization

Phagosome BiogenesisPhagosome Biogenesis

Anti-Microbial ProductsAnti-Microbial Products

Pro-Inflammatory Response (last lecture)Pro-Inflammatory Response (last lecture)

Major Players Major Players

Polymorphonuclear leukocytes (Polymorphonuclear leukocytes (PMNsPMNs) / neutrophils) / neutrophils

Abundant, short livedAbundant, short lived Elevated number indicate infectionElevated number indicate infection

Professional antigen presenting cellsProfessional antigen presenting cells

Dendritic cells / Langerhan cells (skin) Dendritic cells / Langerhan cells (skin) Monocytes (blood) Monocytes (blood) macrophagesmacrophages (motile or (motile or

stationary), which can be tissue specific stationary), which can be tissue specific Longer lived, lower frequencyLonger lived, lower frequency

Underhill and Ozinsky. Annu. Rev. Immunol. 2002

Formation of the phagolysosomeFormation of the phagolysosomeupon ingestion of a microbeupon ingestion of a microbe

Cellular Microbiology, 1999, 1(3):195-203

Phagosome maturationPhagosome maturation

Degradation in the phagolysosomeDegradation in the phagolysosome

Phagolysosome

Lysosome

Endosome

Bacteria degradation

Acid hydrolases

Phagocytosis and Anti-Microbial ProductsPhagocytosis and Anti-Microbial Products

LysosomesBacteria

Phagolysosome

NAPDHoxidase O2-

H2O2

Proteases

Ionic strength pH

Plasma membrane

Phagosome

Nitric Oxide

AP-1

AP-1

p38

MAPKs

LPS+TLR 4IL-1 + IL-1R1

SAPs/JNKs

TNF- + TNF-R1

GAS

IFN- + JAK1/2

IRF-1

ΚB

NF-ΚB

NF-ΚB

IKB

IFN- + JAK1/2

TNF- + TNF-R1

ISRE

STAT1

STAT2

IRF-9

IFN-/ + JAK/TYKInduction of iNOS

Expression

IL-6 + JAK1/2

NF-IL6

NF-IL6

ArginineCitruline

Heme

FAD

NADPH

gp91phoxp22phox

p47phox

p40phox

O2-O2

P67phox

Rac

RestingActive

PKC

PI3K

PKA

P

P

P P

MAPK

Reactions Between ROS and RNSReactions Between ROS and RNS

NO + ONO + O22-- NONO2 2 + ONOO+ ONOO--

NONOates

S-nitrothiols

Nitrite

Nitrous Acid

Chakravortty and Hensel. Microbes Infect. 2003.

Mechanisms of Microbicidal ActivityMechanisms of Microbicidal Activity

Polyunsaturated LipidsPolyunsaturated Lipids

DNADNA

Enzyme Function Enzyme Function Formation of Oxylipins disrupts membraneFormation of Oxylipins disrupts membrane

Deamination of nucleosidesDeamination of nucleosides

Abasic sitesAbasic sites

Strand breaksStrand breaks

Oxidization and Nitrosylation of SH groupsOxidization and Nitrosylation of SH groups

Nitrosylation of Tyrosine residuesNitrosylation of Tyrosine residues

Inactivation of metal ions at active siteInactivation of metal ions at active site

Depletion of antioxidantsDepletion of antioxidants

Evasion:Evasion:

““the act of physically escaping from something (an the act of physically escaping from something (an opponent or a pursuer or an unpleasant situation) by opponent or a pursuer or an unpleasant situation) by some adroit maneuver”some adroit maneuver”

““the act of physically escaping from something (an the act of physically escaping from something (an opponent or a pursuer or an unpleasant situation) by opponent or a pursuer or an unpleasant situation) by some adroit maneuver”some adroit maneuver”

Nature 3:11, 2002 editorial

•Passive

•Active

Bacterial defense against phagocytesBacterial defense against phagocytes

√ Down-regulate

√ Escape X Endosomal trafficking/

Phagosome maturation

X Defense factors

√ Induction of apoptosis X Uptake

Green: hostOrange: bacterial

EVASION MECHANISMS

MS150502 3

Phagocytosisresistance

Phagocytosissensitive

Wild type S735 Mutant 2A

Streptococcus suis type 2

Adapted from Charland et al.,1998

M. Olivier 0295 2

Protein phosphorylations

PKC

EXTINT

PTKJAK2

FMLP

PIP2

DAG

IP2

•H2O2,O2-

• IL-1•phagocytosis

Ca2+Influx

PLCG

Ca2+stores

Ca2+

IFN

STAT

(SHP-1)

Tyrosinephosphatase

[Ca2+]i

Ser⁄Thr phosphatasePMA

LPG

R

Ca2+

Channel

Cellular functions

Acidphosphatase

JAK1

•MHC Class II•c-fos expression

Host Signalling AlterationHost Signalling Alteration