Cellular Pathology of fungal infection

Transcription

1 Cellular Pathology of fungal infection SCPA610 Cellular Pathology Somphong Narkpinit, M.D. Department of Pathobiology, Faculty of Science, Mahidol University somphong.nar@mahidol.ac.th

2 Overview of today s lecture Introduction to the fungal kingdom Role of morphology in fungal disease Candida albicans Blastomyces dermatitidis STM in Cryptococcus neoformans

3 Fungi are closely related to animal and plant hosts

4 Fungi: a diverse kingdom with great global impact 1.5 million species

5 Three types of fungal infection(mycoses) 1. Superficial and cutaneous mycoses: skin, hair, and nails 2. Subcutaneous mycoses: deeper layer of skin 3. Systemic or deep mycoses: internal organ involvement including opportunistic infection

6 Growth form of fungi Filamentous or hyphae Yeasts

7 Superficial and cutaneous mycoses Tinea (Ringworm) Ptyriasis versicolor

8 Subcutaneous mycoses Eumycotic mycetoma

9 Systemic or deep mycoses Mucormycosis or Zygomycosis

10 Systemic or deep mycoses Pulmonary aspergilllosis

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12 Virulence is a complex interrelationship between the infecting organism and the host. Pathogenesis involves interaction (and sometimes modification) of factors on both sides. This is particularly true of fungal pathogenesis. There is no single factor that causes or permits these organisms to be agents of diseases that range from superficial through invasive human infection

13 The complex interplay between fungal virulence factors and host defence factors will determine if a fungal infection will cause a disease. Infection depends on inoculum size and the general immunity of the host.

14 Primary Pathogens primary pathogens are those fungi that cause disease in non-compromised patients. This distinction is necessarily a gray one, because C. neoformans, a model opportunist, sometimes causes disease in healthy individuals, and the primary pathogens, such as C. immitis, are much more virulent in immunocompromised patients. Furthermore, infection by a primary pathogen often leads to subclinical disease

15 Opportunistic Pathogens Opportunistic pathogens incite disease in hosts whose local or systemic immune attributes have been impaired, damaged, or are innately dysfunctional. The pathogenesis of opportunistic infections involves production of virulence factors that allow individual organisms to be commensals during times when humans have normal immune systems.

16 POTENTIAL VIRULENCE FACTORS Because of the complex nature of the hostfungus interaction, there are few factors that are absolutely required for fungal virulence. because pathogenesis is complex, possession of a single putative virulence factor is not likely to render a fungus pathogenic; a complex mix of properties is usually required.

17 Fungal Pathogenicity (virulence factors) Ability to adhere to host cells by way of cell wall glycoproteins Production of capsules allowing them to resist phagocytosis Production of a cytokine called GM-CSF By Candida albicans that suppress the production of complement. Growth at Elevated Temperatures

18 Fungal Pathogenicity (virulence factors) Ability to acquire iron from red blood cells as in Candida albicans Ability to damage host by secreting enzymes such as keratinase, elastase, collagenase Ability to resist killing by phagocytes as in dimorphic fungi Ability to secrete mycotoxins

19 Fungal Pathogenicity (virulence factors) Having a unique enzymatic capacity Exhibiting thermal dimorphism Ability to block the cell-mediated immune defences of the host. Surface hydrophobicity

20 Growth at Elevated Temperatures The ability to grow at body temperature, 37 o C, and within the fever range, o C, of the human host is clearly an important requirement for systemic infection. This property is not a simple genetic one; experiments with Saccharomyces cerevisiae variants able to grow at physiological temperatures suggest that it is a multigenic trait.

21 Growth at Elevated Temperatures S. cerevisiae is normally not pathogenic, but Mc Cusker and co workers have found that strains, which are able to grow at 42 o C and form pseudohyphae, are able to infect and persist in mice. This property is highly correlated with the high-temperature growth phenotype.

22 Adherence For most fungal infections, the ability of the host to resist the physical clearing of the infectious agent is important. For example, the lungs have effective means of clearing foreign particles, but Aspergillus species, H.capsulatum, and C. neoformans all infect via the bronchial route and must avoid clearance. C. albicans also must adhere to various host surfaces both as a commensal to avoid being washed out of its various niches and as a pathogen during the onset of hematogenous infections.

23 Adherence Adhesion in C. albicans has been the focus of much investigation, and several gene products have been implicated. Three of the most intensely studied adhesion mechanisms involve the HWP1 gene product, the ALS gene family, and the INT1 gene product.

24 Penetration and Dissemination Factors The first step in fungal infection is introduction of the agent to the host. Infections may be limited to portal of entry or they may become systemic, disseminating either via hematogenous or contiguous routes. Movement from the infecting surface into the blood stream requires tissue damage.

25 Penetration and Dissemination Factors This damage can be pre existing or can occur either by mechanical penetration or new tissue necrosis. Hence, the ability of fungi to penetrate host cells is crucial for progression of infection in the setting of intact skin or gut barriers. For Candida, it is the ability of hyphae to grow through host cell walls that is proposed to account for the importance of polymorphism in virulence. A. Fumigatus and other true molds are able to penetrate blood vessels and grow along the vessel lumen as they invade tissue.

26 Penetration and Dissemination Factors Hyphae respond thigmotropically (movement toward or away from a touch stimulus) and morphologically to cues such as the presence of a surface, pores, grooves, and ridges. Hyphae follow grooves and ridges of inert substrates and penetrate pores of filtration membranes. Thus, thigmotropism may enhance the ability of a hypha to invade epithelia of a host at sites of weakened integrity or to follow vasculature

27 Penetration and Dissemination Factors Fungi may also spread from the site of infection throughout the host by such mechanisms as host phagocytosis. C. albicans invades endothelial cells through being phagocytosed. H. capsulatum is phagocytosed by macrophages but does not seem to be killed and multiplies within the phagosome.

28 Necrotic Factors Necrotic factors are vehicles of virulence because they allow the fungus to overcome structural barriers that the human host uses to prevent invasive infection. Most necrotic factors are enzymes. Because the majority of fungal pathogens are opportunists, these enzymes may have evolved for saprophytic purposes and might be considered nutritional factors, but it seems more likely that their major role in infection is degradation of host tissue.

29 Necrotic Factors Among the factors that are thought to contribute to this damage are extracellular degradative enzymes, such as proteinases, phosphatases, and DNAses. The earliest identification of a potential necrotic factor was the extracellular proteinase of C. albicans, several different names were originally given to the gene for this enzyme, such as Opa1 and EPR but all correspond to members of the secreted aspartyl proteinase(sap) gene family.

30 Necrotic Factors A. fumigatus secretes at least two proteinases, both of which are active on elastin, a protein that constitutes about 30% of lung tissue C. albicans is known to secrete phospholipases and to possess a phospholipase gene family. Extracellular phospholipases have a role in the pathogenicity of C. albicans, as blood isolates produce significantly more extracellular phospholipase activity than do commensal strains

31 MORPHOLOGY MORPHOLOGICAL VERSATILITY Almost all pathogenic fungi can grow in more than one form. Aspergillus species, which are classical filamentous molds, form that are the infectious agent. 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.

32 SPECIFIC PARASITIC CELL FORMS H. capsulatum, Blastomyces dermatitidis, and several (but not all) species of Candida can grow both as yeasts and as hyphae. In C. albicans, both the yeast and hyphal forms are found at the site of infection, in Histoplasma and Blastomyces, the yeast form seems to be the major, if not the exclusive, parasitic form.

33 The transition to the parasitic form In H. capsulatum leads to a specific pattern of gene expression; this pattern facilitates many of the steps important in infection, including blocking acidification of the phagolysosome and synthesis of the calcium-binding protein, Cbp1p. In contrast to C. albicans, the H. capsulatum hypha seems to play no role in vivo

34 CRYPTOCOCCUS

35 MULTIPLE PARASITIC CELL FORMS Dimorphic fungi regulate their cellular morphology in response to environmental conditions. For example, ellipsoidal single cells of C.albicans (blastospores) predominate in rich media, whereas filaments composed of elongated cells attached end-to-end form in response to starvation, serum, and other conditions.

36 Candida albicans

37 Candida albicans

38 A variety of environmental changes, including a shift from an aerobic to a fermentative metabolism or growth on particular compounds such as N-acetylglucosamine, cause C. albicans to switch from yeast to filamentous growth. This change is accompanied by changes in carbohydrate metabolism and an interruption of electron transfer within the cell. Both temperature (a shift to 37 o C) and ph can regulate C. albicans dimorphism

39 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.

40 Aspergillus

41 PHENOTYPIC SWITCHING The capacity of fungi to undergo an epigenetic change (regulation of expression of gene activity without alteration of genetic structure) in colony morphology has come to be called phenotypic switching. This phenomenon was often observed in C.albicans Phenotypic switching is characterized by reversible change, usually occurring between10-3 and 10-5per cell division,

42 PHENOTYPIC SWITCHING A particular strain,wo-1, that has two major colony phenotypes, white and opaque. White colonies almost exclusively contain the classical yeast cells. Opaque colonies contain cells that are bean shaped and differ from white cells in a variety of ways, including surface properties, gene expression, and temperature sensitivity.

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44 Adaptations of Specific Organisms GROWTH AT DIFFERENT phs C. albicans can grow both at acid and at basic phs, a reflection of its ability to colonize several niches, ranging from the acid vagina to the neutral or opharyngealtract.

45 Adaptations of Specific Organisms MELANIN Melanins are scavengers of reactive oxygen intermediaries, making organisms relatively resistant to leukocyte attack. IRON AND CALCIUM Iron is an essential element for the growth and metabolism of fungi. C. neoformans capsular polysaccharide synthesis is increased by limitation of ferric iron.

46 Adaptations of Specific Organisms Most pathogenic microbes elaborate siderophores (molecules that can bind iron) to mobilize iron from ferric ligands. Hereditary or acquired hemochromatosis, the human disease with progressive iron overload leading to fibrosis and organ failure, poses an increased risk for the development of fungal infection.

47 SURFACE PROPERTIES The best known of the fungal immuno-evasion systems is the capsule of C.Neoformans. A viscous polysaccharide capsule composed of glucuronoxyomannan and other components, the capsule is believed to present a surface not recognized by phagocytes.

48 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.

49 Mycotoxins Many fungi produce biologically active compounds, several of which are toxic to animals or plants and are therefore called mycotoxins. Of particular relevance to humans are mycotoxins produced by molds causing food spoilage, and poisonous mushrooms. Particularly infamous are the lethal amatoxins in some Amanita mushrooms, and ergot alkaloids,which have a long history of causing seriouse pidemics of ergotism.

50 Toxic mushrooms

51 Mycotoxins Other notable mycotoxins include the aflatoxins, which are insidious liver toxins and highly carcinogenic metabolites produced by certain Aspergillus species often growing in or on grains and nuts consumed by humans.

52 Mycotoxins Mycotoxins are secondary metabolites (or natural products). Mycotoxins may provide fitness benefits in terms of physiological adaptation, competition with other microbes and fungi, and protection from consumption (fungivory)

53 Host defence factors: Physical barriers, such as skin and mucus membranes The fatty acid content of the skin The ph of the skin, mucosal surfaces and body fluids Epithelial cell turnover Normal flora

54 Host defence factors: Chemical barriers, such as secretions, serum factors Most fungi are mesophilic and cannot grow at 37 o C. Natural Effector Cells (polymorphonuclear leucocytes) and the Professional Phagocytes (monocytes and macrophages)

55 Factors predisposing to fungal infections: Prolonged antibiotic therapy Underlying disease (HIV infection, cancer, diabetes, etc.) Age Surgical procedures Immunosuppressive drugs Irradiation therapy Indwelling catheters Obesity Drug addiction Transplants Occupation

56 Immunity to fungal infections: Mechanism of immunity to fungal infections can be innate or acquired. Then on-specific immunity includes the physical barriers offered by skin and mucus membranes along with their secretions and normal flora. The ph, body temperature and serum factors along with phagocytic cells play an important part in providing non-specific immunity. Even though body mounts both humoral and cellmediated immunity, it is the latter that is the main stay of host defence.

57 Cell mediated immunity: Immunity is provided non-specifically by effector cells (polymorphonuclear leucocytes) and professionalphagocytes (monocytes and macrophages) and specifically by T lymphocytes. The phagocytes are very important in defence against Candia, Aspergillus and Zygomycetes as is evidenced by their severity ingranulomatous diseases, myeloperoxidase deficiency and cytotoxicchemotherapy.

58 The risk of infection in patients is directly related to the quantity and quality of circulating neutrophils. For aspergillosis in particular, the risk of infection increases if the duration of neutropenia (neutrophil count below 0.5x10 9 cells/liter) is greater than 21 days

59 Neutrophils then migrate through the endothelium (diapedesis) into surrounding tissues, where the processes of chemotaxis and phagocytosis allow destruction of ingested fungi. The elastolytic proteinase of A. fumigatus appears to act as an inhibitory agent in vitro to the respiratory burst and chemotaxis activities in a concentration-dependent manner in the human neutrophil.

60 Aspergillus conidia are effective inducers of host chemokine responses that play a central role in the recruitment of neutrophils into the lung.

61 Expression of T-cell-mediated immunity to fungi includes: delayed-type hypersensitivity contact allergy chronic granulomatous reactions

62 Humoral immunity: Even though antibodies are produced against many fungi, their role in protection is not very clear. However, antibodies help in clearing fungal pathogens through opsonisation,which is important against Candida and Cryptococcus. Another component of humoral immunity is the complement, which can act as opsonins and may even cause damage to their cells through complement activation. Antibodies are important to fungal serodiagnosis.

63 Hypersensitivity: As a result of dermatophyte infection some fungus-free skin lesions of variable morphology occur elsewhere on the body, which are thought to result from hypersensitivity to the fungus. The sereactions are called "id reaction". These reactions are also seen n Candida infections. An inflamed boggy lesion of the scalp called the kerion may result from a strong immune reaction to the dermatophyte.

64 Id s reaction

65 Kerion

66 Granulomas due to intracellular fungi represent delayed hypersensivities. Many fungi are significant allergens to humans, the allergens being spores, conidia, hyphae and other fungal products. On inhalation they may produce allergic pulmonary diseases such as allergic bronchopulmonary aspergillosis, farmer's lung, maple barkstripper's lung, bronchial asthma etc, which maybe Type I or III hypersensitivity.

67 ENDOGENOUS Infection Routes C. albicans is part of the normal humanbody flora, and it can become pathogenic if it moves to a compartment that reacts toits presence. Other Candida species may become the major endogenous gut yeast when immunocompromised individuals have received anti fungal therapy that wipes out C. albicans.

68 A breakdown of gut mucosa, from oncologic chemotherapy, radiation, trauma, concurrent viral ulcers, etc. allows a commensal Candida strain to relocate from the gut to the bloodstream

69 EXOGENOUS Fungi that are environmental saprobes can cause invasive disease in humans if they enter the human body. Usually such organisms are carried in the air, inhaled into the pulmonary tree, and begin a localized invasive infection that may or may not disseminate further in the body.

70 Examples C. immitis among immunocompetent or the opportunistic A. fumigatus or Pneumocystis carinii among immunocompromised people

71 Fungi are closely related to animal and plant hosts

72 Fungi: a diverse kingdom with great global impact 1.5 million species

73 Fungi are pathogens of plants and animals Annual loss of rice to fungal infection is enough to feed 600 million people Crop losses due to fungi: $200 billion/yr Deadly aflatoxin 4th most common isolate in blood cultures 1 in 25 deaths in European teaching hospitals

74 Fungi are a major source of pharmaceuticals Antibiotics Cholesterol-lowering drugs Cyclosporin

75 A fungus may be largest known organism Armillaria ostoyae ( root rot ) Dubbed humungous fungus by U.S. Forest Service One colony underlies 2,200 miles of land adjacent to Prarie City, Oregon (1600 football fields)

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77 Fungal cells have cell wall

78 Antifungals Amphotericin B Azoles Flucytosine Echinocandins

79 General characterisitics of opportunistic fungi Fail to induce disease in immunocompetent hosts Predominant in people with compromised cell-mediated immunity or neutropenia (BMT, cancer chemotherapy, HIV) 20 g mouse 20 kg immunocompromised person 10 8 Aspergillus conidia 100 Aspergillus conidia No disease Invasive aspergillosis

80 Fungi have distinct morphologic states Sudbery et al., 2004, 12(7):

81 Fungal morphologies associated with disease Gow et al., 2002, 5:

82 Host stimulates changes in morphology Host has signals that trigger a change in morphology (e.g. temperature, serum) How is morphology related to virulence? Shape could facilitate tissue penetration Hyphae and pseudohyphae invade agar Shape could facilitate dissemination Shape could facilitate intracellular colonization Shape could be linked with expression of virulence factors

83 Overview of Candida albicans, the most common human fungal pathogen Chemotherapy Prematurity Suzanne Noble Diabetes AIDS

84 C. albicans alternates between yeast and filamentous morphological forms Yeast Hyphae Differences between yeast and hyphae include: invasiveness of agar and host tissues antigenicity secreted factors

85 Candida albicans forms filaments and yeasts in tissues Mouse kidney 24 hr p.i. Inglis and Johnson, Mol Cell Biol, 2002, p. 8669

86 Yeast and hyphae interact differently with the host immune system Yeast -> elicit Th1 cytokine response (more effective) Hyphae -> elicit Th2 response (less effective) d Ostiani et al (2000) J Exp Med 191: Yeast expose beta glucan at bud scars and activate Dectin-1 --> phagocytosis, immune activation Calderone and Braun (1991) Microbiol Rev 55:1-20 Gantner et.al (2005) EMBO J 24:

87 Hyphae appear to invade epithelial cells Newborn mouse skin model Rat tongue explant T L Ray and C D Payne (1988) Infect Immun. 56: J Howlett and C A Squier (1980) Infect Immun. 29:

88 Hyphae may invade whereas yeast may disseminate Hyphal tip extension Generation of tip pressures for penetration Secretion of proteases, lipases from tip cell Newly formed filaments are more adherent than yeasts Yeast cells taken up by macrophages produce filaments and pierce the macrophage Hyphal growth by contact guidance Locate points of weakened surface integrity and grow along those planes Hyphal morphology may impede dissemination

89 Signals that trigger hyphal growth ph Serum Temperature (37 o C) Nutrient limitation

90 Signal transduction pathways involved in hyphal growth Saville et al., Eukaryotic Cell, 2(5), 2003.

91 tup1/tup1 and nrg1/nrg1 Mutants are Constitutively Filamentous TUP1/TUP1 TUP1/tup1 tup1/tup1 Braun and Johnson, Science, 1997, 277, p. 105

92 tup1/tup1 and nrg1/nrg1 Mutants Have a Virulence Defect Murad et al., EMBO, 20(17), 2001.

93 efg1/efg1 cph1/cph1 mutants show reduced filamentation Lo, et al., Cell, 1997, 90, p. 939

94 efg1/efg1 cph1/cph1 mutants have a virulence defect Lo, et al., Cell, 1997, 90, p. 939

95 Summary of Morphology Thus Far Mutants trapped in filamentous form are avirulent. Mutants trapped in yeast form are avirulent. Model: Need to switch between 2 forms in host: Perhaps yeast form disseminates Perhaps filamentous form invades tissues

96 Conditional NRG1 Expression is a Tool to Study Role of Filaments in vivo Nrg1p is a repressor of filamentous growth. Nrg1 ON: Cells grow only as yeast form Nrg1 OFF: Cells can grow as filaments Replace Nrg1 promoter with tetracycline-regulatable promoter. No doxycycline: Nrg1 constitutively expressed repress filament formation Add doxycycline: filaments are able to form Can add doxycycline to mouse drinking water and regulate filament production in vivo.

97 Forcing Nrg1 Expression Eliminates Virulence Saville et al., Eukaryotic Cell, 2(5), 2003.

98 Forcing Nrg1 Expression Doesn t Reduce Fungal Burden in Tissues Saville et al., Eukaryotic Cell, 2(5), 2003.

99 Conditional NRG1 Expression Reveals Filaments not Required for Invasion No doxycycline (repress filaments) Add doxycycline (can make filaments) Saville et al., Eukaryotic Cell, 2(5), 2003.

100 Turning off constitutive NRG1 during infection restores virulence Saville et al., Eukaryotic Cell, 2(5), 2003.

101 Is Filament Formation Required for Virulence? Filament formation is not necessary for tissue invasion. Accumulation of yeast-form cells in tissues is not sufficient to kill mice, suggesting that hyphae play some role in virulence (other than invasion). BUT These regulatory genes (NRG1, CPH1, EFG1, etc.) affect expression of other virulence genes (adhesins,saps, iron assimilatory functions) Further characterization of mutants with a more specific defect is necessary before establishing a true causeand-effect relationship between morphology and virulence

102 The systemic dimorphic fungi Histoplasma capsulatum Coccidioides immitis Blastomyces dermatitidis Paracoccidioides brasiliensis Saprophytic fungi, primary pathogenshyphal (mycelial) form in environment Yeast form in host Histoplasma capsulatum Images from Anita Sil and Medical Mycology, Kwon-Chung and Bennett

103 Host bias for the dimorphics Coccidioides: Dissemination is more common in African Americans, Filipinos, Hispanics Paracoccidioides: Male to female ratio of 12:1 Blastomyces: Male to female ratio of 9:1 Histoplasma: No ethnic or gender bias

104 Distribution of Coccidioides species Hector and Laniado- Laborin (2005) PLoS Medicine

105 Histoplasma Epidemiology 500,000 new infections every year Most common cause of fungal respiratory infections

106 Blastomyces dermatitidis (Blastomycosis) Hyphal form (soil) Yeast form (host) Big, blue, broad-based, budding yeast--blasto Endemic in Mississippi, Ohio, Missouri River Valleys Causes pneumonia Primary cutaneous infection Dissemination to skin, bone, other sites

107 Contrasting incidence of systemic dimorphs Chu et al., CID, (# hospitalized patients/106 people)

108 Temperature regulates morphology 25oC 37oC soil host Conversion inside a host cell is faster than in culture

109 Switch to the yeast form might be essential for virulence + PCMS-treated + No evidence of disease

110 Genetic screen to identify regulators of yeast-specific genes in Blastomyces Nemecek et al., 2006 Science 312: BAD1: B.d. yeast-specific gene Genetic screen using PBAD1-lacZ reporter wild-type drk1 mutant 25 oc 37 oc

111 DRK1 is required for yeast-form growth at 37 o C DRK1 encodes a histidine kinase Histidine kinases classically respond to environmental signals and regulate developmental processes

112 DRK1 is required for yeast-form growth at 37 o C in H. capsulatum

113 DRK1 is required for virulence of Blastomyces and Histoplasma Dramatic decrease in lung CFUs from DRK1-RNAi strains

114 Is cell shape linked to virulence? 37 oc 37 oc DRK1 and virulence genes virulence genes

115 Systematic Genetic Analysis of Virulence in Cryptococcus neoformans Infection: different Cryptococcus strains Found in environment (bird droppings, eucalyptus trees) Infection route: inhalation Disseminates to CNS (meningitis) Most common life-threatening fungal disease in AIDS patients

116 STM screen: Cryptococcus neoformans Liu et al., 2008, Cell, 135: p

117 Survival curves of mice infected with individual strains

118 Distribution of phenotypes

119 GAT201 inhibits phagocytosis STM hypovirulent mutants that exhibit growth at 37ºC are particularly interesting Gat201 is a transcription factor that is required to inhibit phagocytosis

120 Thank you