Influence of cell wall modifications by chitin deacetylases in Cryptococcus neoformans on the human immune response

Hellmann, Margareta J.

Zusammenfassung

The fungus Cryptococcus neoformans was recently classified by the World Health Organization as the most critical fungal pathogen for human health, prevalently infecting immunocompromised patients. One suspected reason for the successful fungal immune evasion is the partial deacetylation of immunogenic cell wall chitin to chitosans, which is performed by the four fungal chitin deacetylases (CnCDAs 1-4). This thesis aims to investigate the characteristics of CnCDA-generated cryptococcal cell wall chitosans, including their fraction and pattern of acetylation (FA and PA, respectively), and how these may influence the host immune response. Given that the characterization of chitosans and related enzymes relies on powerful analytical methods, we extended the toolbox for chitosan analytics with two novel approaches. Size exclusion chromatography coupled to detection by refractive index and mass spectrometry offers a unique opportunity to monitor in vitro enzymatic reactions in their entirety, including both the polymer substrates and the emerging oligomer products. In addition, researchers in the field of linear binary copolymers, such as chitosans, can now utilize the LCP Simulator web tool to simulate cleavage products or pattern analyses in silico, which facilitates the prediction, interpretation, or contextualization of in vitro results. Both methods have contributed to the unexpected identification of a more regular PA in heterogeneously deacetylated chitosans, thereby resolving a decade-old debate and underscoring the impact of the chitosan production method. Equipped with various analytical tools, we first focused on the fungal pathogen and its CnCDAs. A comprehensive review of the enzyme class of CDAs summarizes the current state of research, provides guidance for the analysis of CDAs, and introduces a novel concept of three intertwining effects that guide the enzymes’ mode of action. This proved valuable during our characterization of CnCDA3, which is the second in vitro analysis of a cryptococcal CDA after CnCDA4. Because CnCDA3 was observed to favor partially deacetylated substrates, converting them into chitosans of lower FA and random PA, we hypothesized that its function in vivo may be to deacetylate products of other CnCDAs, thus further decreasing their immunostimulatory potential. Interestingly, the CDA-based immune evasion employed by C. neoformans represents a rather universal fungal stealth strategy, as emphasized in our review on the role of CDAs in plant pathogenic fungi. An extensive biochemical analysis of cell wall chitosans of the C. neoformans wildtype and CDA knockout mutants cultivated under various conditions highlights the importance of CnCDA1 and growth conditions for cell wall deacetylation. Moreover, it was possible to correlate the extent of the host immune response with both the cell wall FA and the accessibility of fungal chitin and chitosans. Subsequently, we turned to the human host and investigated the fate of chitosans within the human body by characterizing all three human chitinolytic enzymes – the GH18 chitinases chitotriosidase and acidic mammalian chitinase, as well as human lysozyme – with regards to their substrate and subsite preferences. Whereas the former two exhibited a behavior typical for GH18 endo-chitinases, the latter demonstrated an even greater specificity for highly acetylated substrates, strongly favoring acetylated units at three consecutive subsites. In a final study, we could show that chitotriosidase is capable of converting immunologically inert crystalline chitin into soluble mobile oligomers, which elicit immune responses via the human Toll-like receptor 2 (TLR2), thereby shedding light onto chitin-related immunity in humans. Overall, the findings of this thesis support the hypothesis that CnCDAs are important virulence factors of C. neoformans and provide insights into the interaction between the human host and the fungal pathogen with focus on cell wall chitin and chitosans. Our results form the basis for future studies on the structure-function relationships of cryptococcal chitosans and on the roles of the associated enzymes of host and fungus. Ultimately, this may lead to urgently needed improvements of therapies against cryptococcosis and to the development of effective cell-based vaccines against cryptococcal infections.