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FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS

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.

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FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS

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  1. 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

  2. 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

  3. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS FUNGUS • ATTACHMENT • & ENTRY INVASION MORPHOLOGICAL CHANGES TOXIGENICITY Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  4. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Adhesins Catalase Proteases Toxins SOD Mannitol Phospholipase Capsule Pigments Cell wall Urease Estrogen-binding proteins Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  5. 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 Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  6. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Huffnagle GB et al. Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  7. 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 Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  8. 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 Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  9. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Jackson BE et al. MicrobPathog 2007; 42: 88-93 Evaluated role of fungal hyphal extension in experimental C. albicanskeratitis Used geneticallyaltered 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

  10. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Jackson BE et al. MicrobPathog 2007; 42: 88-93 Wild-type human C. albicansisolate had a high degree of virulence in the murine cornea 4 C. albicansstrains deficient in genes regulating adherence or encoding membrane proteins did not significantly differ from the parental strain. 5 C. albicansstrains deficient in genes involved in filamentationcaused 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 filamentationin vitro. Phenotypic switching from yeasts to filamentous forms facilitates establishment & progression of experimental C. albicanscorneal disease Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  11. FusariumPathogenomics FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Ma L-J et al. Annu Rev Microbiol2013; 67: 399-416 Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  12. Pathogenesis of Fusarium keratitis FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS 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) Fusariumsolani implicated : 3/4 patients ((Jones, 1975) 3/3 patients (Kuriakose and Thomas, 1991) Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  13. Morphogenesis in Lasiodiplodia& Fusarium keratitis FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Thomas et al. (1991) -- TEM studies of keratoplasty samples from 3 patients with Lasiodiplodiatheobromae keratitis (failed treatment with saperconazole)--- intrahyphal hyphae Kiryuet al.(1991)-- TEM studies of samples from dexamethasone-treated ,Fusariumsolani-infected rabbit corneas -- double and triple cell walls, hypha-in-hypha structures Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  14. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS MORPHOLOGICAL CHANGES OF FUNGI EXPOSED TO ANTIFUNGALS Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  15. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Hua X et al. , Cornea 2010 ; 29 : 1440-4. Used an ex-vivo corneal model & 3 strains of Fusariumoxysporum: -- a wild-type isolate; -- apacC loss-of-function mutant(constructed from the wild-type); -- a pacC dominant-activating mutant constructed from the wild-type 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 stromacompared histopathologically in corneal sections. Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  16. 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 pacCloss-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

  17. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Fungal morphogenesis Phenotypic switching EVASION OF HOST DEFENCES Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  18. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  19. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Aspergillusfumigatusconidia human fibrinogen circulating or basement membrane-associated host proteins : laminin, complement, fibronectin, albumin, collagen, surfactant proteins Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  20. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  21. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Wu J , et al. MolImmunol. 2015; 64:235-43. A. fumigatusconidia in immortalized human corneal epithelial cells (HCECs) induced: -- increased expression of the pattern recognition receptors (PRRs) TLR-2 & nucleotide- binding oligomerizationdomain (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

  22. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS FUNGUS • ATTACHMENT • & ENTRY INVASION MORPHOLOGICAL CHANGES TOXIGENICITY Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  23. 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 (Fusariumequiseti)-- corneal and conjunctival lesions Dudley and Chick (1964)-- extract of Fusariummoniliforme 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

  24. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Jones et al. (1969) -- Fusariumsolani 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 LMM-N+collagen (Cohen, 1973)--inoculation into goat corneas--corneal liquefaction Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  25. KERATOCYTES IN GOAT CORNEAS INOCULATED WITH CULTURE FILTRATE OF Aspergillusflavus TEST CONTROL Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  26. COLLAGEN BUNDLES IN GOAT CORNEAS INOCULATED WITH CULTURE FILTRATE OF Aspergillusflavus TEST CONTROL Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  27. 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

  28. 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

  29. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS TOXIGENICITY IN LIFE AT DEATH EXOTOXINSPROTEASES(Zhu et al., 1990 Gopinathan et al.,2001) RELEASE OF ENDOTOXINS BY Aspergillus fumigatus Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  30. 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

  31. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Leema G, et al. MolVis2011;17:2889-97 Looked for transcriptional activation (expression) of key aflatoxinbiosynthetic pathway genes in 4 corneal isolates & 4 environmental isolates of Aspergillusflavus., namely: -- regulatory (aflatoxin regulatory [aflR] & aflatoxinJ [aflJ]) -- structural (polyketide synthase acetate [pksA] & norsolonicacid-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

  32. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Leema G, et al. MolVis2011;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 aflatoxigenicthan 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. flavusisolates used for comparison. Isolates of A. flavus from human keratitis patients shown to express regulatory and structural aflatoxinbiosynthetic pathway genes. Precise influence of the corneal microenvironment on expression of these genes &aflatoxinproduction by A. flavus infecting the cornea. needed Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  33. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Leemaet al. , Cornea 2013 ; 29 : 1440-4. A possible association between aflatoxigenicity&oxidative stress in keratitis due to Aspergillusflavusin an experimental rodent model. Wistarrats were divided into three groups of six each. -- Group I served as mock-inoculated controls. -- In Group II, experimental fungal keratitis was induced using aflatoxigenicA. flavus -- In Group III rats, experimental fungal keratitis was induced using non- aflatoxigenicA. 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

  34. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS Leemaet 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-aflatoxigenicA. 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. Aflatoxigenicityis associated with more intense oxidative stress in experimental A. flavuskeratitis. Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  35. 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

  36. FUNGAL VIRULENCE FACTORS IN OCULAR INFECTIONS FUNGUS ATTACHMENT & ENTRY INVASION MORPHOLOGICAL CHANGES TOXIGENICITY Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli

  37. 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

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