VIRULENCE FACTORS OF FUNGI

PRESENTED BY : DR. KUMAR VIKRAM (MD 3rd YR),IGIMS,Patna

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INTRODUCTION There are 100,000 species of fungi . These fungi inhabit different niches, a

number of them are symbiotic and may live in commensalism, mutualism or parasitism with other organisms

Of all these species, only around 600 species are human pathogens.

This fact that has led to several studies providing a better understanding of the relationship among parasite, host and virulence factors.

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Data from the late 1950s and early 1960s indicate that invasive fungal infections were extremely rare, even in immunocompromised cancer patients.

Now, fungal infections have dramatically increased in the past two decades as a result of improved diagnostics, high frequency of catheterization, instrumentation and an increasing number of immunosuppressed patients

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Virulence and Pathogenicity Pathogenesis is the ability of a

microorganism to infect the host and produce disease resulting from interaction of pathogen with host via expression of certain factors on both sides.

Virulence refers to quantitative ability for pathogenesis of a species.

Determinants of pathogenicity are called virulence factors.

The symbiotic-parasitic relationship produces an infectious process leading to lesions of the host tissues and establishment of disease due to a direct imbalance in parasite-host interaction.

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VIRULENCE FACTORS FUNCTIONS FUNGAL PATHOGENS

A. SURFACE COMPONENTS a. Cell wall glycoproteins

Adherence to epithelial surfaces

Candida species

b. Melanin pigment Shield against immunologically active cells, hydrolyses

Cr. NeoformansW. dermatitidis

c. Capsules, glucans

Anti - phagocytic Cr. neoformans

B. Thermotolerance Survive and replicate at 370C

Human pathogens

C. Resistance to microbiocidal products of neutorophils e.g. H2O2, by dimorphic primary pathogen

Evasion of host defence mechanisms by tissue phase (yeast, spherule) of virulent dimorphic fungi

Primary pathogensBlastomyces, Coccidiodes, Histoplasma, Paracoccidiodes, Sporothrix schenkii

D. Epithelial cell and monocyte cytocidal activity

Evasion of host defences Candida albicansCandida tropicalis

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VIRULENCE FACTORS

FUNCTIONS FUNGAL PATHOGENS

E. Exoenzymes

a. Elastase Degrades elastin, scleroproteins, enhances invasion of elastin containing tissue ( lung, skin, blood vessels)

Aspergillus flavus, A. FumigatusDermatophytes

b. Alkaline protease Degrades collagen, elastin, enhances invasion of lung tissue

Aspergillus flavus, A. FumigatusRhizopus spp.

c. Keratinase, collagenases

Degrades scleroproteins in skin Dermatophytes

d. Acid protease Cleavage of IgA2 Candida spp., A. Fumigatus

F. Toxins

a. Aflatoxin Hepatotoxicity Aspergillus flavus

b. Endotoxin Tissue necrosis A. Flavus, A. Fumigatus

G. Dimorphism Evasion of host defencesEnvironmental and tissue forms present different and surface features, requiring different host response of mechanisms to contain each form

True pathogensOpportunistic pathogens

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Cell wall components and capsule Capsule

› Produced by Cryptococcus

› Polysaccharide in nature

Cell wall› Chitin › Mannans› Glucans

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Capsule Cryptococcus

neoformans. viscous

polysaccharide capsule

composed of glucuronoxyomannan and other components.

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down regulate cytokine secretion, inhibit leukocyte accumulation, induce suppressive T-cells, inhibit antigen presentation, and inhibit lymphoproliferations Hence, it serves as a barrier to host

defenses in a variety of way.

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Virulence factors associated with cell wall components

Component Fungus Activity

α – (1,3) – glucan

B. dermatitidis Antigenic masking of WR – 1 adhesin

P. brasiliensis Resistance to digestion by phagocytosis

H. Capsulatum Destruction of macrophage in vitro

Glucuronoxylomannan C. neoformans Resistance to phagocytosis

Melanin C. neoformans Interference with oxidative metabolism of phagocytes

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MORPHOLOGY MORPHOLOGICAL VERSATILITY  Almost all pathogenic fungi can grow

in more than one form.  The major exception is C.

neoformans , which apparently exists only in the yeast form in vivo. In vitro it also grows mostly as a yeast; however, it does form filaments during the mating process.

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Transition from yeast form to hyphal form is facilitated by › nutrients, › near-neutral pH, › CO2 concentration about 5.5%, › presence of N-acetyl-D-glucosamine,

serum, some amino acids, and biotin. Reverse transition from hyphal to yeast

form is provoked by › acidic pH, › absence of serum, and› higher concentration of glucose

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Many molds form conidia , or vegetative spores; scattered by wind or water, these small, resistant cells serve as a mode of dissemination. In the case of aspergillosis, conidia serve as

the propagule that infects debilitated patients. Hydrophobicity is thought to contribute to

the efficacy of   Aspergillus conidia, already an ideal size for deposition into alveoli, to disperse in air.

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PHENOTYPIC SWITCHING Ability of organisms of single strain to

switch reversibly at high frequencies among different colony types.

 Observed in C.albicans, allows Candida to adapt to a different

host environment during infection. Colonies of C. albicans show

morphological variation, including smooth, rough, star, stippled, hat, wrinkle, and fuzzy at high frequency.

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At present, of all the phenotypes described, the white-opaque system in strain WO-1 is the most studied.

Opaque cells switch to white cells within one generation at 37oC.

OPAQUE PHENOTYPE WHITE PHENOTYPEFlat,Grey, opaque colonies Smooth, white colonies

elongated or bean shaped round ovoid

more virulent in a systemic mouse model of infection.

Less virulent in a systemic mouse model of infection.

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•Ability to survive and replicate at 37°C.• Cryptococcus neoformans• Histoplasma capsulatum • Sporothrix schenckii

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Most isolates of C. neoformans var gattii that do not grow efficiently at

37oC are not able to produce fatal infection in mice

whereas isolates of var neoformans germinate and grow at 37°C producing lethal infection

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Low-virulence strains of H. capsulatum require more

time for mycelium-to-yeast-phase transition at 37ºC

whereas the more virulent strains are capable of withstanding

drastic temperature changes and of transforming more quickly

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Isolates of S. schenckii from systemic lesions can grow at

35ºC and at 37ºC, but isolates from fixed cutaneous lesions can

only grow at 35ºC It is believed that even small differences in temperature tolerance can influence the pathogenic potential of a microorganism as well as the form of disease presented by the host

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Biofilm formation A biofilm is an

assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix.

Biofilm may form on a wide variety of surfaces, including living tissues, indwelling medical devices, industrial or potable water system piping.

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•Keratinases•Collagenases •Gelatinases•Phospholipases •Lipases•Acid proteinases•Acid phosphatases

nutrient uptake

tissue invasion

adherence Disseminatio

n inside the host.

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PHOSPHOLIPASES Phospholipases are enzymes that hydrolyze

ester linkages of glycophospholipids. Classified into phospholipases A,B,C and D. Produced by candida albicans, cryptococcus

neoformans and aspergillus fumigatus. Roles

› Adherence to host cell› Penetration› Host cell injury› Signal transduction› Stimulation of host cells to release cytokines.

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Extracellular proteinsases Proteolytic activities attributed to secretory

aspartic proteinases. Produced by candida spp.(C. Albicans, C.

Tropicalis, C. Parapsilosis), Aspergillus spp., Coccidiodes immitis.

Role in › adherence and survival of the pathogen on mucosal

surfaces ,› invasion of host tissues and › digestion of immunoglobulins.

C. immitis endospores produce proteinases with elastase and collagenase activity

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Mycotoxins (Myco = of fungal origin) Secondary metabolites (chemicals) of a fungus that

produce toxic results in another organism. Unlike bacterial toxins, fungal toxins (mycotoxins) are

not proteins and therefore are not usually detectable by the immune systems of humans and animals

Lack of visible appearance of fungus does not negate presence of mycotoxins. Toxins can remain in the organism after fungus has been removed.

Cytotoxic: disrupt cell structures such as membranes, and processes such as protein, DNA, and RNA synthesis.

Can be heat stable, not destroyed by canning or other processes.

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How many mycotoxins are there? Today 300 - 400 mycotoxins are known Mycotoxins of human concern based on

toxicity: Aflatoxin Deoxyniva-lenol (DON) or Vomitoxin Zearalenone Fumonisin T-2 toxin Ochratoxin A

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Common toxigenic fungi

Aspergillus

Penicillium

Stachybotrys

Fusarium

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MYCOTOXINS

FUNGAL SPECIES SOURCE CLINICAL CONDITIONS

Aflatoxins A. Flavus,A. Parasiticus, A. Nominus, Penicillium puberulum

Nuts, maize Aflatoxicosis, Reye’s syndrome, hepatitis, hepatoma,

Fumonisins Fusarium moniliforme Maize Equine leukoencephalomalacia(ELEM), Porcine pulmonary edema(PPE)

Trichothecenes

Fusarium graminearum, F. Sporotrichoides

Maize, sorghum Human toxicosis, Alimentary toxic aleukia, Biological warfare

Ochratoxins Aspergillus ochraceus, A, niger, Penicillium verrucosum

Cereals, coffee beans, bread

Nephropathies i.e Balkan endemic nephropathy and Mycotoxic porcine nephropathy

Cyclopiazonic acid

A. Flavus, A. Versicolor, A. Oryzae, Penicillium cyclopium

Ground nut, corn, meat

Co – contaminant, kodua poisoning

Zearalenones Fusarium graminearum Wheat, maize, barley, sorghum

Genital disorders in animals i.e pigs

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When facing agressive conditions some fungi are able to use various and complex strategies involving

mechanisms such as

suppression of cytokine production

reduction of the fungicidal activity of macrophagesUtilization of the alternative complement pathway

Mechanisms of Evasion from Host Defenses

These mechanisms lead to immunoregulatory disturbances and

impairment of the host defenses

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Mechanisms FungusActivation of complement system C. Neoformans

Intracellular surviving and multiplication H. CapsulatumP. brasiliensis

Down regulation of antigen presentation by macrophages

C. Neoformans

Immunosuppresive effect of fungal antigen on the cytokine production by mononuclear phagocytes

C. Neoformans

Immunosuppression induced by antigenemia C. ImmitisH. CapsulatumP. brasiliensis

Stimulation of suppressor cells C. NeoformansP. brasiliensis

Interefence with fungicidal activity of phagocytes

H. Capsulatum

Escape mechanisms of host defences

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Conclusions: Understanding the pathogenicity

mechanisms that fungi use during infection is crucial for the development of new antifungal therapies and diagnostics.

Classically, antifungal drugs were designed to exert fungicidal activities.

Recently however, specifically targeting virulence factors has been proposed as a new and promising antifungal strategy.

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Several virulence factors, such as dimorphism, the secretion of proteases and the expression of adhesins and invasins, have been suggested as attractive targets.

As our detailed understanding of fungal pathogenicity mechanisms improves, the potential for developing novel therapeutic and diagnostic strategies expands.

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References Chander, J. Textbook of Medical Mycology, 3rd ed. Mehta

Publication: New Delhi, 2009. Hogan et al. Virulence factors of medically important fungi,

Clinical Microbiology Reviews, Oct. 1996, p. 469–488 Cilmery S K, Maria F S, Maria T S. Virulence factors in fungi

of systemic mycoses, Rev. Inst. Med. trop. S. Paulo vol. 40 n. 3 São Paulo May/June 1998

Ananthnarayan & Paniker. Textbook of Microbiology, 9th ed. Universities press: Hyderabad, 2014

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THANK YOU