Transcript 1000 tlr

FUNGAL VIRULENCE
FACTORS IN OCULAR
INFECTIONS
Philip A. Thomas
MD, PhD, MAMS, FABMS, FIMSA
Dept. of Ocular Microbiology
Institute of Ophthalmology
Joseph Eye Hospital,
Tiruchirapalli 620001, INDIA
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
AGENT FACTORS
• Adherence to ocular tissue
• Invasion of ocular tissue
• Liberation of enzymes and toxins
• Morphogenesis and phenotypic switching
• HOST FACTORS
• Tissue -derived enzymes (MMP 2 and MMP-9)
• Polymorphonuclear(PMN)-derived enzymes
• Plasminogen-activating system
• Immunological mechanisms
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN OCULAR
INFECTIONS
FUNGUS
ATTACHMENT
& ENTRY
INVASION
TOXIGENICITY
MORPHOLOGICAL
CHANGES
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Adhesins
Proteases
SOD
Phospholipase
Pigments
Urease
Catalase
Toxins
Mannitol
Capsule
Cell wall
Estrogen-binding
proteins
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Fungal virulence factors (including secreted/shed
fungal products) can facilitate evasion of host
defense mechanisms by:
• Modulating afferent phase cytokine signals by
-- down-regulation of pro-inflammatory cytokines
-- upregulation of anti-inflammatory cytokines
--promoting of switching of the immune response
• Altering antigen processing
• Blocking leucocyte recruitment to site of infection
• Inhibiting effector phase mechanisms
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Huffnagle GB et al.
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Adhesins of Candida albicans
•1,2 tetramannose epitope
•30 kDa antigen
•Fimbrial adhesin
•Als1-4/Ala 1p
•Hwp1
•Int1p
•Receptors for fibronectin, laminin and vitronectin
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
PHENOTYPIC SWITCHING
• changes antigenic surface molecules
• controls expression of capsule-- determines state of
commensalism or invasiveness
• allows host changes essential for life cycles
Phenotypic switching in Cryptococcus neoformans
• augments virulence in animal models
•  virulence associated with less organised inflammatory
response
• colony phenotypes associated with different antibody
responses
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Jackson BE et al. Microb Pathog 2007; 42: 88-93
• Evaluated role of fungal hyphal extension in experimental C. albicans
keratitis
• Used genetically altered yeast strains.
• Scarified corneas of adult BALB/c mice topically inoculated with
-- wildtype (SC5314)
-- 10 transposon-induced mutant strains of C. albicans
• Monitored for 4 days post -inoculation.
• In vitro growth kinetics and the yeast strains’ ability to bud into
pseudohyphae or hyphae also compared.
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Jackson BE et al. Microb Pathog 2007; 42: 88-93
• Wild-type human C. albicans isolate had a high degree of virulence in the
murine cornea
• 4 C. albicans strains deficient in genes regulating adherence or encoding
membrane proteins did not significantly differ from the parental strain.
• 5 C. albicans strains deficient in genes involved in filamentation caused fully or
partially attenuated keratomycosis (P < 0.0001).
• The overall growth kinetics of wild-type and mutant strains were similar in rich
media.
• Mutants with deficient morphogenesis had reduced filamentation in vitro.
• Phenotypic switching from yeasts to filamentous forms facilitates
establishment & progression of experimental C. albicans corneal disease
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Ma L-J et al. Annu Rev Microbiol 2013; 67: 399-416
Fusarium
Pathogenomics
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Pathogenesis of Fusarium keratitis
• Invasion of anterior chamber by fungi infecting corneal tissue
• Formation of lens-iris-fungal mass at pupillary area
• Interference with normal drainage of aqueous
`Fungal malignant glaucoma’(Jones, 1975)
`Keratomycotic malignant glaucoma’(Kuriakose
and Thomas, 1991)
• Fusarium solani implicated :
3/4 patients ((Jones, 1975)
3/3 patients (Kuriakose and Thomas, 1991)
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Morphogenesis in Lasiodiplodia & Fusarium keratitis
Thomas et al. (1991) -- TEM studies of keratoplasty samples from
3 patients with Lasiodiplodia theobromae keratitis (failed
treatment with saperconazole)--- intrahyphal hyphae
Kiryu et al.(1991)-- TEM studies of samples from
dexamethasone-treated ,Fusarium solani-infected rabbit corneas
-- double and triple cell walls, hypha-in-hypha structures
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FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS
MORPHOLOGICAL CHANGES OF FUNGI EXPOSED TO
ANTIFUNGALS
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Hua X et al. , Cornea 2010 ; 29 : 1440-4.
• Used an ex-vivo corneal model & 3 strains of Fusarium
oxysporum :
-- a wild-type isolate;
-- a pacC loss-of-function mutant(constructed from the wild-type);
-- a pacC dominant-activating mutant constructed from the wildtype isolate
• 27 human donor corneas (maintained in tissue culture)
superficially scarified & topically inoculated with the strains (9
corneas in each group)
• Relative hyphal invasion into the stroma compared
histopathologically in corneal sections.
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Hua X et al. , Cornea 2010 ; 29 : 1440-4.
• All 3 strains showed comparable exponential growth rates in vitro.
• Wild-type F. oxysporum
-- invaded into the corneal tissue within 1 day
-- penetrated through anterior stroma during next 4 days.
• The pacC dominant-activating strain behaved similar to the wild-type strain.
• The pacC loss-of-function mutant invaded explanted corneas
significantly less than the wild-type strain on days 1 & 3 .
• The PacC pathway regulating the transcription of fungal genes
-- allows fungal adaptation to the ocular surface
-- enables invasion of the injured cornea by F. oxysporum.
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS
Fungal
morphogenesis
Phenotypic
switching
EVASION OF HOST DEFENCES
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FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Aspergillus fumigatus conidia
• human fibrinogen
• circulating or basement membrane-associated host
proteins :
laminin, complement, fibronectin, albumin,
collagen, surfactant proteins
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FUNGAL VIRULENCE FACTORS IN OCULAR INFECTION
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Wu J , et al. Mol Immunol. 2015; 64:235-43.
• A. fumigatus conidia in immortalized human corneal epithelial cells (HCECs)
induced:
-- increased expression of the pattern recognition receptors (PRRs) TLR-2 &
nucleotide- binding oligomerization domain (NOD)- 2 like receptor;
--release of multiple inflammatory cytokines incl. interleukin (IL)- 6 & IL-8 & TNF-α.
• NOD2 expression was up-regulated by its agonist muramyl dipeptide (MDP),
along with receptor interacting protein 2 (RIP2), NFκB-p65 & inhibitor of NFκB
(IκB)-α.
• Zymosan, a TLR2 agonist, promoted the expression of NOD2 and RIP2 in a
TLR2-dependent manner.
• The increased expression of NOD2 and RIP2 caused by A. fumigatus conidia
occurred in part through a TLR2-dependent pathway.
• These findings suggest the existence of complex interactions between TLR2
& NOD2 in the inflammatory response of HCECs against A. fumigatus infection.
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN OCULAR
INFECTIONS
FUNGUS
ATTACHMENT
& ENTRY
INVASION
TOXIGENICITY
MORPHOLOGICAL
CHANGES
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
CONTRIBUTION OF FUNGAL EXTRACELLULAR PRODUCTS TO
CORNEAL DAMAGE
• Burda and Fisher (1960) -- extract of Cephalosporium spp.  rapid
corneal opacification, ulceration and liquefaction
• Brian et al. (1961) --diacetoxyscirpenol (Fusarium equiseti)-corneal and conjunctival lesions
• Dudley and Chick (1964)-- extract of Fusarium moniliforme
collagenolytic activity in vitro
 corneal lesions after intracorneal inoculation
 corneal lesions due to extract eventually self-limiting;
corneal lesions due to living fungus were progressive
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
• Jones et al. (1969) -- Fusarium solani from human corneal
ulcers found to secrete elastase in vitro -- possible
contribution to fungal virulence
• Cuero (1980) -- purified extracts and culture filtrates of F.
solani (human corneal ulcers, vegetation ) provoked marked
ocular inflammation when dropped into rabbit eyes
• Thomas(1990)- Ocular isolate of A.flavus-- grown in LMMN+collagen (Cohen, 1973)--inoculation into goat corneas-corneal liquefaction
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KERATOCYTES IN GOAT CORNEAS INOCULATED
WITH CULTURE FILTRATE OF Aspergillus flavus
TEST
CONTROL
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COLLAGEN BUNDLES IN GOAT CORNEAS
INOCULATED WITH CULTURE FILTRATE OF
Aspergillus flavus
TEST
CONTROL
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Zhu et al.(1990)
• extracellular proteases of Aspergillus flavus
• growth on MM(milk protein as N2 source)-- mainly
metalloprotease
• growth on MM (insoluble collagen or elastin as N2
source )-- serine , cysteine and metalloproteases
• ? collagenase activity mediated severe corneal
destruction caused by the isolate
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Gopinathan et al. (2001)
extracellular proteases produced in vitro by A.flavus
and F. solani using collagen as sole nitrogen
source
gelatin zymography revealed : in A. flavus
multiple protease bands (MW 100 to 200 kDa),
in F. solani one single band (MW 200 kDa )
fungal cultures in vitro contained
predominantly serine protease activity
(metalloprotease activity to lesser extent)
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN OCULAR INFECTION
TOXIGENICITY
IN LIFE
EXOTOXINS
PROTEASES
(Zhu et al., 1990
Gopinathan et al.,2001)
AT DEATH
RELEASE OF ENDOTOXINS BY
Aspergillus fumigatus
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
• Fusaric acid (Fusarium species)--` vascular wilt’ syndrome’ in
plants
-- intracorneal injection of upto 1000 ug/ml - no effect (Thomas
, 1990)
• 18 isolates of Fusarium species from human keratitis-- in vitro
production of nivalenol, T-2 toxin, deoxynivalenol and
diacetoxyscirpenol-- no correlation between sterol
content/toxin production and severity or outcome of keratitis
(Raza et al.,1994)
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Leema G, et al. Mol Vis 2011;17:2889-97
• Looked for transcriptional activation (expression) of key
aflatoxin biosynthetic pathway genes in 4 corneal isolates
& 4 environmental isolates of Aspergillus flavus., namely:
-- regulatory (aflatoxin regulatory [aflR] & aflatoxin J [aflJ])
-- structural (polyketide synthase acetate [pksA] &
norsolonic acid-1 [nor-1]) genes by reverse transcription
PCR.
• The aflatoxin-producing potential of each strain also
detected by thin-layer chromatography & quantified by
spectrophotometry.
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Leema G, et al. Mol Vis 2011;17:2889-97
• All isolates expressed aflJ, nor-1, and pksA, while all but one expressed aflR.
• Overall, significantly higher mean expression levels occurred in aflatoxigenic
than in non-aflatoxigenic corneal isolates.
• A significant positive correlation noted between mean expression level of aflR
& the quantum of aflatoxin production by the corneal isolates.
• Essentially similar patterns of expression of these genes were noted in four
environmental A. flavus isolates used for comparison.
• Isolates of A. flavus from human keratitis patients shown to express regulatory
and structural aflatoxin biosynthetic pathway genes.
• Precise influence of the corneal microenvironment on expression of these genes
&aflatoxin production by A. flavus infecting the cornea. needed
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN OCULAR
INFECTIONS
Leema et al. , Cornea 2013 ; 29 : 1440-4.
• A possible association between aflatoxigenicity &oxidative stress in keratitis
due to Aspergillus flavus in an experimental rodent model.
• Wistar rats were divided into three groups of six each.
-- Group I served as mock-inoculated controls.
-- In Group II, experimental fungal keratitis was induced using aflatoxigenic A.
flavus
-- In Group III rats, experimental fungal keratitis was induced using nonaflatoxigenic A. flavus
• Clinical features were scored for 5 days post-inoculation.
• Test corneas excised and examined histologically.
• Expression of IL1β andTNFα genes was sought in excised corneas.
• Levels of malondialdehyde (MDA) & reduced glutathione (GSH) measured
• Activities of key antioxidant enzymes measured in excised corneas
& fungal mycelial homogenates.
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS
Leema et al. , Cornea 2013 ; 29 : 1440-4.
• In mycelial homogenates of aflatoxigenic A. flavus:
-- mean levels of MDA & GSH & mean activities of catalase, superoxide
dismutase & glutathione peroxidase were significantly (p<0.05) higher than
those in homogenates of the non-aflatoxigenic A. flavus.
-- Increased numbers of well-stained isoforms were detected.
• Significantly (p<0.05)higher expression profiles of IL1β and TNFα
genes, MDA & GSH levels & antioxidant enzyme activities were noted
in Group II than in Group III rat corneas.
• Clinical and histological scores suggested a more severe keratitis in
Group II than in Group I & Group III rat corneas.
• Aflatoxigenicity is associated with more intense oxidative stress in
experimental A. flavus keratitis.
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Conclusive proof of involvement of fungal
proteases/exotoxins in clinical keratitis requires:
. demonstration of proteases/exotoxins in corneal
tissue of patients with mycotic keratitis
.protease/exotoxin -deficient mutants should
produce less severe corneal disease than that
produced by protease/exotoxin-secreting parent
strain
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN OCULAR
INFECTIONS
FUNGUS
ATTACHMENT
& ENTRY
INVASION
TOXIGENICITY
MORPHOLOGICAL
CHANGES
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli
FUNGAL VIRULENCE FACTORS IN
OCULAR INFECTIONS
Relevance of studying fungal virulence factors to
treatment of ocular infections
•Compounds to prevent fungal adherence to &
invasion of ocular tissue
•Antibodies or synthetic molecules for neutralisation
of toxic extracellular fungal products
•Compounds to prevent morphogenesis and
phenotypic switching
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli