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Role of Chitin in Fungal Pathogenesis

Structure and functions of cell wall. Chitin synthesis and its regulation.

Chitin and innate immune response.


Chitinases and its antifungal activity. Severe asthma with fungal sensitization (Chitin Connection) Chitin and echinocandin resistance a paradoxical effect. Summary and conclusion

Structure of fungal cell wall:composed of glycoproteins and polysaccharides mainly glucan and chitin.

Cell wall function:

Maintaining cell shape. Mediating adhesion for cell migration and fusion. Protecting the cell against foreign substances. Mediating the absorption of molecules, transmitting intracellular signals from external stimuli. Synthesizing and remodelling cell wall components.

Provides mechanical strength to withstand osmotic pressure from environment.

Plasticity of the cell wall helps in cell growth, cell division.

Serves as a signaling center to activate signal transduction pathways within the cell.

Harbors enzymes responsible for tissue invasion.

Glycoproteins:

Protein component is tightly interwoven within chitin and glucans.

Cell wall proteins are glycoproteins.


Extensively modified with N-linked and O-linked oligosaccharides.

Forms of glycosylated proteins are mannoproteins in C. albicans and galactomannan in A. fumigatus


Proteins receive glycosylphosphatidylinositol (GPI) anchor. GPI anchor serves to direct and localize these proteins to plasma membrane and cell wall.

Glucans:

Major structural polysaccharide. -1-3 glucan serves as main structural constituent. Chitin and glycoproteins are covalently attached. Synthesis of beta-1,3-glucan is required for proper cell wall formation and the normal development of fungi. Glucan synthase catalyzes the formation of glucans. Inhibition of 1-3 glucan synthesis disrupts cell wall formation and prevent fungal growth. Echinocandins, are non-competitive inhibitors of the 1-3 glucan synthase complex.

Chitin:

long linear homopolymer of 1-4 linked N-acetylglucosamine.

Nascent primary polysaccharide folds back to form anti-parallel chains.

Chitin microfibrils are formed from interchain hydrogen bonding between anti-parallel chains.

Crystalline polymers has enormous tensile strength and contribute to cell integrity.

Fungal chitin is synthesized and deacetylated to chitosan by chitin deacetylase. Inhibition of chitin synthesis makes the cell osmotically unstable.

Chitin synthesis

Chitin synthesis in pathogenic fungi:

Chitin is synthesized by large family of chitin synthase (CHS) enzymes.

There are seven classes of CHS enzyme in fungi.

Class I acts as a repair enzyme that replenishes chitin in birth/bud scar immediately after cytokinensis. Class II synthesize primary septum formation. Class III appears to function at polarised growth sites and in forming septa during hyphal growth and conidia development.

Class IV - involved in both cell wall and septum synthesis. Class V and VII are N-terminal myosin motor like domains and Cterminal CHS domains localise to the sites of polarised cell wall expansion in an actin dependent manner. Chitin VI forms septa during hyphal growth and conidia development. The multipilicity of CHS enzymes suggests their role in cell wall synthesis. Expression and activity of CHS is highly regulated throughout cell cycle and under condition of stress.

Different classes of CHS genes in fungi

Transcripitional regulation of chitin synthesis:

Chitin and cell wall synthesis occurs at polarised growth.

During early bud growth cell wall material is deposited at the bud tip.
During isotropic growth occurs in large budded cells where material is deposited over the entire bud surface. Following nuclear division, a repolarisation phase begins where material is directed towards the mother-bud neck to prepare for cytokinesis. In hyphal or filamentous forms,cell extension is a continuous and indefinite process of apical growth.

Post- Transcripitional regulation of chitin synthesis:

Fungal CHS enzymes that are responsible for synthesising cell wall and/or septal chitin tend to be localised to sites of polarised growth. Both the localisation and stability of CHS enzymes can be regulated by phosphorylation. Protein kinases and the establishment of cell polarity appear to be important for cell wall regulation.

Chitosan

Nascent chitin is converted into chitosan (deacetylated form) by enzyme chitin deacetylase (CDA). First discovered in Mucor rouxii. Chitin deacetylases has been divided into 2 subgroups: 1. intracellular chitin deacetylases. - secretes into the periplasmic space. - contributes to the formation of chitosan in the cell wall. 2. extracellular chitin deacetylases. - secretes in the extracellular environment. - contributes for hyphal penetration into the tissues.

Chitin is an essential component of vegetative growth, but chitosan is not. During spore wall formation both chitin and chitosan are essential.

Chitosan based spore structure is important for cell to retain structural rigidity and resistance to various stresses.
In Cryptococcus neoformans, - chitosan is necessary for maintains cell integrity and aids in bud separation. - maintains normal capsule width.

CDA plays important role in protecting pathogenic fungal hyphae being lysed from host cell chitinases.

Chitin regulation of innate immune response

Large chitin polymers are shown to be biologically inert.


Chitin fragments are known to regulate innate immune response.

Chitin as immunomodulator
Chitin modulates adaptive type 2 immune response:

Chitin can affect allergen induce adaptive type II response. Type I cytokines produced by innate immune cells down regulate type 2 immune response. Chitin-induced alternatively activated macrophages play a central role in the recruitment of eosinophils and other immune cells by augumenting Th2 cytokine production and type 2 inflammatory response.

Macrophage activation by chitin:

The stimulation of macrophages with chitin or chitin derivatives shows significant increase in type 1 proinflammatory cytokines such as TNF-, IL-12 and IFN-. Reese et al demonstrated that chitin alternatively activated macrophages that were chitin- induced inflammatory response. Chitin directly stimulates macrophages to express TNF- and IL-17 via TLR-2 and MyD-88.

Chitin can interact with diverse cell surface receptors such as macrophage mannose receptor and TLR-2 response.

Chitin effects on other immune cells:

Epithelial cells endogenously express chitinases and chitin binding proteins or their expression is significantly induced by fungal cell wall chitin stimulation. As epithelial and dendritic cells are key players in pathogen recognition and processing. The response of these cells to chitin to understand the immune regulatory function of chitin in vivo. Airway epithelial cells are exposed to allergen pro- and antiinflammatory cytokines are produced from epithelial cells. The innate immune cells produces chitinases to cleave the fungal chitin.

Chitin stimulation of innate immune cells

Yonsei Med J;2009, 50(1):22-30

Antifungal activity of chitinase:

Chitinases belongs to the glycosyl hydrolase 18 family.

Mammalian cells produce several forms of chitinases with chitinolytic activity, including chitotriosidase(CHIT1), acidic mammalian chitinase (AMCase), and other chitinases that apparently lack activity, like YKL-40.
Chitinases with chitinolytic activity are thought to play a role in the innate immune response to fungal infections. CHIT1 is produced by activated macrophages, and elevated levels of CHIT1 in serum are present in humans during infection.

AMCase was first noted to be induced during Th2 inflammation through an IL-13-dependent mechanism. It was also shown to play an important role in the pathogenesis of Th2 inflammations and IL-13 effector pathway activation. Chitinases play a major role as mediators of allergic inflammation. Early studies in mice demonstrated that Ym1 and Ym2 (murinespecific chitinases) and AMCase are induced in an experimental model of asthma. AMCase, by virtue of its chitinolytic activity, has been reported to reduce allergic inflammation induced by chitin.

The nonchitinolytic chitinase YKL-40 has also been linked to allergic inflammation. YKL-40 promotes inflammation by preventing the death of inflammatory cells (including eosinophils) and promoting alternative activation of macrophages. Chitinases appear to play a protective role in the innate response to fungal infection but also mediate adaptive TH2 inflammation.

Chitinase activity in airway epithelium


Fungal

Chitinases in allergic inflammation

Fungal infections in asthma

Emerging concepts in severe Ashtma, fungal infection & Chitinases

Fungal antigens are an important cause of allergeninduced asthma. Sensitization to fungal allergens is thought to occur as a result of transient, repeated exposures without invasion or colonization of host tissue. Fungi can also elicit asthma symptoms in association with persistent colonization or superficial invasion of host tissue.

Severe ashtma with fungal sensitization (SAFS)


Fungal infection and ashtma are coincidental observations.

SAFS highlights the expanding awarness of fungi in asthma.


SAFS - characterized by Failure of step 4 asthma therapy,

Elevated serum IgE levels, and Sensitization to one of several environmental fungi may represent one point on a spectrum of fungus-associated asthma. Importantly, asthma control is improved in SAFS patients using prolonged itraconazole therapy.

Model of hypothesized role of chitinases in fungus associated ashtma

Genetic polymorphism of chitinase gene:

Fungus-associated asthma could represent an imbalance between chitinolytic and nonchitinolytic chitinases. Mutations resulting in decreased activity of chitotriosidase confer increased susceptibility to fungal infection. Contributing to increased asthma severity, possibly via enhanced induction of YKL-40.

Fungal cell wall chitin and echinocandin resistance a paradoxical effect

Fungal cell wall ideal target for theraputic treatment. Echinocandins are cyclic hexapeptides modified with lipid side chains for antifungal activity. Echinocandins binds to Fks1, a subunit of -1,3-glucan synthase, results in the depletion of -1,3-glucan polymers within the fungal cell wall. Paradoxical effect:- echinocandin can inhibit the -1,3-glucan synthesis but as a compensatory mechanism chitin content in the fungal cell wall is increased.

Mechanism behind the paradoxical attenuation of echinocandin activity

Possible mechanism are - Mutations within the gene encoding -1,3-glucan synthase. - Increase in the cell wall chitin content. - Up regulation of the cell wall integrity pathways.

Cell wall integrity pathways are:- Protein kinase C (PKC) signalling pathway (MAP kinase). - High osmolarity glycerol signalling pathway (MAP Kinase). - Ca2+ - Calcineurin pathways.
Recent studies suggest that cell wall integrity pathways play a major role in increasing the chitin cell wall content.

Protein kinase C signalling:

PKC singalling pathway is activated via Mitogen activated protein kinase. PKC signalling is essential for sensing cell wall integrity.

Studies demonstrated the coordination of the fungal response to cell wall damage by echinochandins.
Activation of the pathway by cell wall damaging agents resulted in activation of increased chitin synthase activity and elevated cell wall chitin content.

HOG pathway:

HOG singalling pathway is activated via Mitogen activated protein kinase. Major controller of governing cellular response to diverse external stimuli including osmotic shock, UV irradiation. Involved in growth, morphology, virulence and maintains cell wall integrity.

Ca2+ calcineurin pathway:

Ca2+ is a second messenger that mediates cellular response to changes in external stimuli. Activates calcineurin by Ca2+ calmodulin protein kinases and leads to the stimulation of products to withstand environmental stress and maintain calcium homeostasis. Cyclosporin A inhibits Ca2+ calcineurin pathway.

Fungal Genetics and biology;2010;47:117-126

Inhibitors of chitin synthesis

Inhibitors of Calcineurin pathway

Dephosphorylation of NF-AT is inhibited by cyclosporine A (CsA) and FK506.

Summary & conclusions

Chitin is the minor but essential component of the cell wall. Chitin synthesis are regulated by chitin synthase genes, mainly to maintain cell structure. Regulation of chitin synthesis can occur when the cell wall is under environmental stress.

Chitin can modulate immune response, but the mechanism is largely unknown.

Chitin is masked by the glucans in the cell wall, so the chitin may or may not stimulate the pattern recognition receptors.

Chitinase produced by the host innate immune cells play a major role in the antifungal immunity.
A newer concept of fungal chitin, chitinase plays a major role in asthma. SAFS is mainly due to the repeated exposure to the fungi where the respiratory epithelium produces host chitinases and leads to the severity of the disease.

Chitin plays a major role in the echinocandin resistance. A paradoixcal effect is seen in between echinocandin resistance and chitin synthesis. The combination therapy with the chitin synthase inhibitor or with calcineurin inhibitor along with echinocandins shows much better results in in-vivo models.

Role of chitin in the fungal diseases are yet to be studied in detail.

Thank you

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