Adaptations of Cryptococcus to the host extracellular niche

Cryptococcus is an opportunistic human fungal pathogen with the potential to cause life- threatening infections of the central nervous system. Ubiquitous in the environment, Cryptococcus switches from a saprotrophic lifestyle in the environment to a pathogenic lifestyle in humans by altering key cellular functions required for adaptation and invasion of the host. Once inhaled, the host extracellular niche (particularly the lung mucosa) serves as a modulator for cryptococcal adaptive phenotypes which are critical for survival and proliferation. However, the cellular changes exhibited by Cryptococcus as it responds to the pulmonary environment is poorly understood. The surface of the lung mucosa is heavily loaded with secretions from host lung cells, such as Type I and II epithelial cells as well as alveolar macrophages. Upon inhalation, Cryptococcus comes into close contact with these secretions. The first part of this thesis probed and described the potency of mammalian cells secreted factors (including lung-associated cells) to stimulate phenotypic responses that are associated with Cryptococcus adaptive mechanisms. While C. neoformans responded to these secreted factors with rapid replication, its sister species C. gattii instead demonstrated a high capacity to form enormously enlarged titan-like cells. Within the lungs, Cryptococcus undergoes morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. In the second part of this thesis, a new in vitro titan-induction approach is introduced. Using this in vitro approach, I revealed a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterised strain-specific differences within the clade. In addition, this approach demonstrates for the first time that the cell-cycle-regulated phenotypes: cell size changes, DNA replication and budding, are not always synchronous during titanisation. i Titan cell formation is triggered by host-specific environmental conditions [such as physiological temperature (37°C) and CO2 level (5%) coupled with hypoxia, and nutrient limitation] and modulated by genetic regulators including those associated with cell cycle progression. The last part of this thesis established a strong correlation between progression of the cell cycle phenotypes (cell size, DNA replication and budding) and expression of cell cycle genes and identifies the role of cryptococcal quorum sensing peptide Qsp1 peptide and exogenous p-Aminobenzoic acid as a key inducer of titanisation in C. gattii

The Cryptococcus gattii species complex:Unique pathogenic yeasts with understudied virulence mechanisms

Members of Cryptococcus gattii/neoformans species complex are the etiological agents of the potentially fatal human fungal infection cryptococcosis. C. gattii and its sister species cause disease in both immunocompetent and immunocompromised hosts, while the closely related species C. neoformans and C. deneoformans predominantly infect immunocompromised hosts. To date, most studies have focused on similarities in pathogenesis between these two groups, but over recent years, important differences have become apparent. In this review paper, we highlight some of the major phenotypic differences between the C. gattii and neoformans species complexes and justify the need to study the virulence and pathogenicity of the C. gattii species complex as a distinct cryptococcal group.</p

Micrograph showing the budding nature of <i>C</i>. <i>deuterogattii</i> yeast (left panel) vs. titan (right panel) cells [44].

Scale bar = 5 μm.</p

Schematic diagram illustrating CGSC distinct phenotypic virulence traits as compared to <i>C</i>. <i>neoformans</i>.

Upon inhalation from the environment, CGSC yeast cells/spores responds to the lung extracellular niche by exhibiting phenotypic traits including larger capsule (with less immunogenic properties), larger cell body (with higher degree of homogeneity), and thinner but more compacted cell wall with higher chitosan content than C. neoformans. The manner in which CGSC strains exhibit these host-adaptive traits is perhaps responsible for its low affinity to dissemination from the lungs to the brain. Within the host macrophage, the intracellular phenotypes (mitochondrial fusion and “division of labour” proliferation mechanism mediated by extracellular vesicles) exhibited by C. deuterogattii (which drives the fatal Pacific Northwest outbreak) are absent in C. neoformans.</p

The role of Dectin-3 in host response to <i>C</i>. <i>deuterogattii</i> infection <i>in vitro</i> and <i>in vivo</i>.

(A) The C-type lectin receptor, Dectin-3, recognises C. deuterogattii (Cd) capsular glucuronoxylomannan (GXM) [64]. The recognition of GXM leads to the activation of NF-κB and ERK signalling pathways to drive proinflammatory cytokine production. (B) Dectin-3 deficient mice showed increased susceptibility to C. deuterogattii infection [64]. Figure created with BioRender.com.</p

List of virulence-related phenotypic traits whose underlying molecular, genetic, and metabolic mechanism has been studied in <i>C</i>. <i>neoformans</i> but not <i>C</i>. <i>gattii</i>.

List of virulence-related phenotypic traits whose underlying molecular, genetic, and metabolic mechanism has been studied in C. neoformans but not C. gattii.</p

Comparison of morphological attributes between 70 clinical isolates of cryptococci from HIV/AIDS patients in Botswana-Africa (adapted from [37] and summarized).

Comparison of morphological attributes between 70 clinical isolates of cryptococci from HIV/AIDS patients in Botswana-Africa (adapted from [37] and summarized).</p

Development of nanoantibiotic delivery system using cockle shell-derived aragonite nanoparticles for treatment of osteomyelitis

A local antibiotic delivery system (LADS) with biodegradable drug vehicles is recognized as the most effective therapeutic approach for the treatment of osteomyelitis. However, the design of a biodegradable LADS with high therapeutic efficacy is too costly and demanding. In this research, a low-cost, facile method was used to design vancomycin-loaded aragonite nanoparticles (VANPs) with the aim of understanding its potency in developing a nanoantibiotic bone implant for the treatment of osteomyelitis. The aragonite nanoparticles (ANPs) were synthesized from cockle shells by a hydrothermal approach using a zwitterionic surfactant. VANPs were prepared using antibiotic ratios of several nanoparticles, and the formulation (1:4) with the highest drug-loading efficiency (54.05%) was used for physicochemical, in vitro drug release, and biological evaluation. Physiochemical characterization of VANP was performed by using transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray powder diffraction, and Zetasizer. No significant differences were observed between VANP and ANP in terms of size and morphology as both samples were cubic shaped with sizes of approximately 35 nm. The Fourier transform infrared spectroscopy of VANP indicated a weak noncovalent interaction between ANP and vancomycin, while the zeta potential values were slightly increased from -19.4±3.3 to -21.2±5.7 mV after vancomycin loading. VANP displayed 120 hours (5 days) release profile of vancomycin that exhibited high antibacterial effect against methicillin-resistant Staphylococcus aureus ATCC 29213. The cell proliferation assay showed 80% cell viability of human fetal osteoblast cell line 1.19 treated with the highest concentration of VANP (250 µg/mL), indicating good biocompatibility of VANP. In summary, VANP is a potential formulation for the development of an LADS against osteomyelitis with optimal antibacterial efficacy, good bone resorbability, and biocompatibility

An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus