Lecture 15: Saliva and Oral Yeast

mycology

study of fungi

yeast and mold

2 basic structural forms of fungi

dimorphic

exist in either yeast or mold form and can switch back and forth

eukarya

fungi domain

Yeast

unicellular with spherical or ovoid bodies (cells of different genera are almost indistinguishable under the microscope)

Molds

mulitcellular with specialized structures to preform specific functions (different shapes, colors, sizes, etc. between different genera)

Hyphae

structural unit of mold; multicellular with each cell separated by a septa that has pores

Mycelium

mass of hyphae that form the mold colony

c. albicans

both dimorphic (can switch between mold and yeast) and opportunistic

fuzzy mold colony

made from branching hyphae

smooth yeast colony

made from yeast cells

fungi reproduction

both sexual and asexual (most relevant clinically)

fungi classification

• yeast

• filamentous fungi

• dimorphic fungi

Yeast identification

biochemical reactions based on fermentation of and assimilation of carbohydrates and utilization of enzyme substrates and other metabolic activities

mold identification

identified based on color, texture, colony, and microscopic morphology (especially their specialized asexual structures)

fungi oxygen requirement

majority are aerobic, in rare cases anaerobic

fungi growth medium

• Sabouraud Dextrose Agar (SBD)

• Potato Dextrose Agar (PDA)

• Yeast Peptone Dextrose (YPD)

special features of fungal growth medium

• high sugar/carbohydrates

• acidic pH

• antibiotics to avoid contamination from bacteria

candida species

• most commonly found fungi in the oral cavity

• part of the normal oral flora

• opportunistic pathogen

Candidiasis

infection in which candida species are the primary infectious agents

localized candidiasis

oropharyngeal candidiasis (OPC): burning or pain, altered taste sensation, and difficulty swallowing liquids and solids (dysphangia); mucosal (skin, vaginal, and uro-genital) infections

Systemic candidiasis

deep-organ infection: 36-56% mortality, invasion of the GI mucosa: passage across the bowel wall, bloodstream (candidaemia, sepsis)

different OPC’s (oropharyngeal candidiasis)

• Pseudomembranous (white)

• Erythematous (red) Denture stomatitis: more inflammation of tissue as a response to the pathogen; less microbial load

c. albicans as an opportunistic infection

• c. albicans is a polymorphic fungus that normally colonizes at mucosal surfaces as harmless commensals

• part of the normal flora of the oral cavity, GI tract, genital tract, and skin)

• triggered by attentuation of normal host immunity or disturbance of normal microbial flora

• infection usually endogenous (inside the body) and even if exogenous, it is usually from mother to baby

populations susceptible to candida infections

• immune deficiency (neonates, elderly, AIDS, tumors)

• mechanical factors (burns, oral prosthesis, catheters, hemodialysis)

• pharmacology (antibiotics, corticosteroids (inhalers), antineoplasitc, psycho-active (opioid users))

commensal to pathogenic

host-fungal interactions determine the outcome of candida infection (host loses the fight, candida becomes opportunistic)

• rapid adaptation of C. albicans to the host environment mediated by signaling

• virulence genes are co-regulated with cell morphogenesis (yeast hyphae transition)

• escape from surveillance of immune system

morphological switching in response to environmental signaling

at different temperatures yeast (30°C) —> pseudohyphae —> hyphae (37°C)

• germ tube test: used to distinguish c. albicans and c. dubliniensis from other species

adhesins in candida invasion

sticky proteins on surface:

Als1p, Als5p, Hwp1p, Int1p

enzymes in candida invasion

break down proteins and tissues to allow candida to go deeper

• Saps, Plb1p

resorption in candida invasion

candida absorbed by cells

induced phagocytosis in candida invasion

induced vagination and go into the cell

sensing of host environment is critical in the transition from commensal to pathogenic

• protein picks up signal and sends it down to other proteins when virulence genes

• any change required by candida calls and signal to become opportunistic

relative cell surface hydrophobicity

non-specific adhearnce process

expression of cell surface adhesin molecules

facilitates specific adherence mechanisms

high frequency phenotypic switching

antigenic modification through frequent cell surface changes (morphs to different forms to evade immune cells)

hyphal development

• reduces likelihood of phagocytosis; allows phagocytosed yeast to escape phagocyte

• promotes invasion of oral epithelium

secreted aspartyl proteinase production

• secretory IgA destruction

• host cell and extracellular matrix damage

binding of complement molecules

antigenic masking

phospholipase production

damage to host cells

front line defense against c. albicans

rapidly acting (within minutes to hours), relatively non-specific

innate immunity against c. albicans

• b-glucans and mannans on fungal cell well interact with receptors on host cells

• these receptors are called pattern-recognition receptors (PRRs)

  • a major class of PRRs and toll like receptors (TLRs)

• TLR2/4 are responsible for recognizing fungal pathogens

• Leads to the activation of intracellular pathways leading to cytokine production, activation of the innate immune response, and release of AMPs

phagocytic clearance of candida

via PMNs (neutrophils) and monocytes (macrophages)

• stimulation of macrophages initiated by interaction of dectin recpetors (non TLR) on these with fungal cell wall components like b-glucans

cationic microbial peptide

antifungal peptides like histatins

c. albicans cell wall

• mannoproteins: outer sticky layer, since there are lots of sugars on the outside

• b 1,3-glucans and b 1,6-glucans: middle layer

• Chitin: inner layer

environmental changes can change c. albicans cell wall architecture and antifungal resistance

iron levels can change levels of cell wall components and the immune response will change

role of exposed b 1,3-glucan in host response

• changing the environment is a signal change that changes the fungal cell wall

• b 1,3-glucan masking: increases mannan structures

• b 1,3 glucan unmasking: decreases mannans to expose glucan

iron mediated cell wall remodeling alters c.albicans phagocytic response

iron deprivation enhances b 1,3 glucan masking and reduces phagocytosis

oral mucosa innate peptides

keratinocytes: physical barrier and secretion of antimicrobial peptides, such as b-defensins and calprotectin

saliva innate peptides

flow dislodges yeasts and bacteria from oral cavity AND saliva contains secreted fungicidal proteins, such as histatins, lysozyme, and lactoferrin

C. albicans growth

salivary proteins (proline rich proteins (PRPs), statherins, mucins) and oral epithelial transglutaminase (Tgase) can aid c. albicans growth by promoting adherance to oral tissues

C. albicans growth inhibition

• Inhibit growth through immune exclusion by binding and aggregating fungal cells (mucins and secretory IgA [sIgA]) to faciliate their clearance by swallowing

• Antifungal proteins, including salivary histatin 5 (Hst 5) or calprotectin and b-defensins (secreted by oral tissues) prevent c. albicans

cationic peptide properties

• 12-50 amino acids with a net positive charge of +2 to +7 due to large amount of basic amino acids

• greater than 50% of amino acids are hydrophobic

• these properties + the anionic microbial cell membrane and large transmembrane electrical potential make the peptide more accessible to pathogens

cationic peptide class I

linear, a-helical peptides without cysteines

cationic peptide class II

peptides with cysteines linked by a disulfide bridge

• disulfide bridge makes it close upon itself

cationic peptides class III

Unusual high proportion of specific amino acids

• histatin gives positive charge and attacks negatively charged membranes, fungi, bacteria, etc.

saliva secreted by salivary glands

contains antimicrobial proteins (AMPs) including Histatin 5 (Hst 5)

• Hst 5 added to whole saliva exhibits only 10-15% of its in vitro fungicidal activity

histatin degradation

histatin can get degraded by all the other stuff in our saliva

• degradation by proteases

• “masking” of activity by binding to:

  • salts

  • sugars

  • metal

  • host proteins

  • proteins secreted by C. albicans

effect of Saps of C. albicans on Hst 5 activity

Saps degrades histatins, Hst 5 activity is reduced by proteases secreted by C. albicans

effect of c. albicans and host mucins in Hst 5 activity

Hst 5 activity is reduced by C. albicans Msb2 and host mucins in saliva

mechanisms negitavely affecting Hst activity

proteins in saliva can reduce Hst 5 activity by degradation as well as sequestration

effects on mucins on C. albicans

reduction in Hst 5 activity by mucins can also be an indirect result of their effect on c. albicans cell surface properties

adaptive immunity to candida

• slow growing B and T cells require up to 7 days to mature

• hallmarks are specificity, inducibilty, and discrimination of self vs, non-self (dendritic cell-mediated T cell responses)

Th1 cell response

IL12, IFN-y: protective

activation of antimicrobial functions, respiratory burst, degranulation, isotype switching of Ab

Th2 cell response

IL4, 5, 10: non-protective

eosinophilia, hyper-IgE, hyper-IgG4 production which exacerbate the symptoms

Th17 cell response

IL-6, TGFb and IL-23: mew area

oral candidiasis and HIV conneciton

T helper cells (CD4+) counts go down due to HIV infection, then there is not enough signalling to fight the candidiasis infection

fungal cell wall

targets echniocandins

fungal cell membrane

targets polyene (cell membrane) (frequently used in dental practice for oral infections)

sterol synthesis

targets azoles (frequently used in dental practice for oral infections)

DNA and RNA synthesis

targets flucytosine

protein synthesis

targets sordarins

microtubule assembly

targets griseofulvin

Polyene drug

• binds to ergosterol and disrupts membrane by making it porous

• Amphotericin B, Nystatin (mycostatin)

• side effect: nephrotoxicity

azole drug

• intracellular targeting inhibits biosynthesis of ergosterol in fungal membranes

• clotrimazole (mycelex), etoconazole (nizoral), fluconazole (difulcan)

• There are azole resistant species