Lecture overview

Fungal pathogens introduction Immune defences against fungi

Immune evasion by fungi

Immune evasion by fungi

Dr Ruth Ashbee Principal Clinical Scientist Mycology Reference Centre Leeds General Infirmary
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Fungal pathogens
Fungi are eukaryotes Most fungi can only cause systemic disease in people who are immunocompromised (opportunistic); a few fungi can cause systemic disease in healthy people (true pathogens) Antifungal drugs are often toxic due to nonspecificity of their targets (i.e. humans often have the same/similar targets) Yeasts: Candida, Cryptococcus Moulds: Aspergillus, Fusarium Dimorphic: Histoplasma, Coccidioides, Blastomyces,

Fungal pathogens
PATHOGENIC FUNGI
Pneumocystis jiroveci

Moulds Dimorphic

Yeasts

Paracoccidioides

Fungal pathogens

Innate host defences

Skin and mucosal surfaces
skin integrity, antimicrobial peptides, secretions mucociliary escalator urinary tract

Commensal population
established populations in niches

pH
skin pH (sweat & sebaceous secretions); vaginal pH, gastric pH

Complement cascade
classical pathway (immune complexes) lectin pathway (terminal mannose residues) alterative pathway (LPS, yeast cell wall)

Phagocytes
neutrophils macrophages dendritic cells

Innate immune responses

Fungus invading tissue
Pathogen associated molecular patterns (PAMP), e.g. -glucans, galactomannan, mannan Pattern recognition receptors (PRR), e.g. Toll-like receptors, dectins, pentraxin 3 T-cell receptor Antibody

Innate immune responses

Fungus invading tissue
Pathogen associated molecular patterns (PAMP), e.g. -glucans, galactomannan, mannan Pattern recognition receptors (PRR), e.g. Toll-like receptors, dectins, pentraxin 3 T-cell receptor Antibody

Neutro

Macro
Immature dendritic cell

Neutro

Macro

Superoxide

TNF-, IFN-, IL-1, IL-6, IL-12

Draining lymph node

TH1

IFN- Respiratory burst Granule release

including defensins, elastase, myeloperoxidase and reactive oxygen species

INFLAMMATORY RESPONSE
IL-10, IL-4, IL-5

Superoxide
produced by NADPH oxidase complex

H 2O 2

HOCl

Treg

Dendritic cell

Plasma cell

B-cell
TH2

Adaptive host defences

T lymphocytes (CD4, CD8)
cytokine production some killing regulation of immune response

Role of the phagocyte in fungal infection

Neutropenia is the main predisposing factor for invasive fungal infections Functional problems with neutrophils (e.g. Chronic granulomatous disease) predisposes to fungal infections Neutropenic patients who are treated with antifungals often dont respond until their neutrophil count recovers

B lymphocytes
antibody production

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Pattern recognition receptors (PRR)

PRRs (TOLL-like receptors, Ctype lectins e.g. Dectin 1) on host cells interact with polysaccharides (e.g. chitin, glucans) and mannosylated proteins of the fungal cell wall. Dectin 1 recognises -1-3glucan, which triggers multiple signalling pathways via Syk kinase and NF-B. Results in
initiation of respiratory burst and enhanced phagocytosis production of IL-12 and TNF induction of humoral immunity stimulation of cytotoxic T cells
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Modulation of inflammatory signals

TLR 2 activation leads to Th2 response, production of IL4, IL5 and IL10, a humoral immune response and lack of effective antifungal response. TLR4 activation leads to a Th1 response, production of proinflammatory cytokines and a good antifungal response
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Modulation of inflammatory signals

C. albicans and A. fumigatus activate the TLR2 and hence bias the response to Th2 Polysaccharide capsule of Cryptococcus induces IL10 and biases towards Th2 response Blastomyces dermatitidis has a surface adhesin which limits release of TNF- by macrophages

Masking of PAMPS (-glucans)

Organism

Masking strategy Mannoprotein* Hydrophobin layer on conidia Polysaccharide capsule Switch to -linked glucans in yeast phase

C. albicans Aspergillus fumigatus Cryptococcus neoformans Histoplasma capsulatum Paracoccidioides brasiliensis Blastomyces dermatitidis

* Antifungal treatment may reverse this by unmasking -1-3-glucan

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Phagocytosis

Phagocytosis of fungal spores

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Antiphagocytic mechanisms
Cell size: too big to be taken up Hyphal forms of Candida albicans and Aspergillus fumigatus arent internalised efficiently Polysaccharide capsule of Cryptococcus Titan or Giant cells of Cryptococcus (10x size of normal yeasts) Metabolites produced by C. albicans inhibit uptake by macrophages by interfering with cytoskeleton
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Capsule of Cryptococcus
Blocks opsonic effect of complement and antibodies Negative charge of capsule repels negatively charged host cells

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Titan/Giant cells of Cryptococcus

Intraphagocytic survival
Histoplasma capsulatum can survive within phagocytes and replicate in macrophages modulates pH to minimise activity of enzymes

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Outgrowth from phagocytes

C. glabrata recently been shown to survive macrophage phagocytosis and replicate intracellularly
Doesnt cause apoptosis Inhibits proinflammatory cytokine production Suppresses ROS production Alters phagosome maturation Replicates in non-acidic phagosome Escapes from macrophages by uncertain mechanisms

C. albicans can transform to hyphal phase, under the influence of carbon dioxide, pierce and kill the macrophage

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Expulsion from phagocytes

Cryptococcus can permeabilise the phagosomal membrane Ejected from the macrophage causing no damage to fungus or host during vomocytosis

Modulation of phagocytic killing mechanisms Phagocytes undergo respiratory burst and produce toxic metabolites:
Nitric oxide Hydrogen peroxide Superoxide anion Hypochlorous acid

Various fungi have ways to modulate the ability of these toxic metabolites to kill

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Inhibition of nitric oxide production

Occurs by several fungi mechanisms unclear for some C. albicans produces detoxification enzymes (e.g. catalases, superoxide dismutase (SOD)) C. posadasii* secretes an uncharacterised factor which may mediate by down-regulation of inducible NO synthase mRNA in macrophages C. neoformans produces many enzymes (4 catalases, 2 SODs and many others) and can also enlarge capsule
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Melanin
High MW, hydrophobic pigments Produced by organisms in all biological kingdoms Extremely resistant need boiling in acid to dissolve! Provide pigment in many organisms humans, insects, fungi In fungi, produced via phenoloxidases Associated with virulence in fungi

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Melanin in Cryptococcus

Melanin: protection against host defences

Effect
Phagocytosis

Organism
C. neoformans P. brasiliensis S. schenckii F. pedrosoi

Magnitude (%)*
7 (in vivo) 27 (in vitro) 9 50 36 31 18 >30 27 22 22 75 20 22

Killing by host cell

C. neoformans A. fumigatus P. brasiliensis S. schenckii F. pedrosoi

Oxidants

C. neoformans Aspergillus spp S. schenckii

Microbial peptides

C. neoformans

* Maximum protection for organisms with melanin cf those without

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Complement cascade
Classical Pathway: Ag-Ab complexes C1q, C1r, C1s C4 and C2 Lectin Pathway: Carbohydrates MBL, MASPs, C4 and C2 C3 convertase C3a C3 C3b Amplification step C5 convertase C5a C5 C5b C5b-9 Membrane attack complex Lysis of some pathogens and cells Potent anaphylatoxin Chemotaxis Phagocyte activation Generation of oxygen radicals T-cell activation and survival Alternative Pathway e.g LPS C3, FB FD

Inhibition of the complement cascade

Binding of complement components
Mast cell activation Chemotaxis Phagocyte activation Generation of oxygen radicals T-cell activation and survival Opsonin

Secretion of antiphagocytic protein 1 (App1) protein by Cryptococcus which binds to complement receptors 2 and 3, so inhibiting complement-mediated uptake of Cryptococcus C. albicans binds several complement regulatory factors, including Factor H and C4b-binding protein (C4BP) thus inhibiting complement activity. C. albicans produces Pra1 (pH-regulated antigen) which binds plasminogen, Factor H and Factor H-like protein. This mediates complement evasion and extracellular degradation

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Inhibition of the complement cascade

Binding of complement components Other fungi also bind complement regulators: Aspergillus fumigatus conidia Paracoccidioides brasiliensis Cryptococcus Pneumocystis jiroveci

Inhibition of the complement cascade

Degradation of complement components C. albicans produces Secreted Aspartyl Proteases (SAPs) which can degrade complement proteins (C3b, C4b and C5) and extracellular matrix proteins. Asp fumigatus also produces a secreted serine protease (Alp1) which degrades C3b, C4b and C5

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Summary
Mechanisms used by fungi to evade host defences:
Shielding of PAMPs Ability to live and multiply in phagocytes Outgrowth from phagocytes Modulation of phagocytic killing mechanisms Role of melanin in immune protection Inhibition of complement cascade Degradation of complement proteins

References
GD Brown (2011) Innate antifungal immunity: the key role of phagocytes. Ann Rev Immunol 29:1-21 LA Chai et al (2009) Fungal strategies for overcoming host innate immune response. Med Mycol 47:227-236 JR Collette & MC Lorenz (2011) Mechanisms of immune evasion in fungal pathogens Curr Opin Micro (in press) L Romani (2004) Immunity to fungal infections Nature Rev Immunol 4:113 K Seider et al (2010) Interaction of pathogenic yeasts with phagocytes: survival, persistence and escape. Curr Opin Microbiol (2010) 13: 392400 K Seider et al (2011) The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation. J Immunol (in press) PF Zipfel et al (2011) Immune escape of the human facultatitve pathogenic yeast Candida albicans: The many faces of the Candida Pra1 protein. Int J Med Microbiol 301:423-430
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