Introduction to the Project: The Role of Endocytosis in Regulating Cell Wall Composition in Candida Albicans

Background

Candida Albicans (C. albicans)

C. albicans is the most common human fungal pathogen, which causes various types of Candidiasis depending on the anatomical site of disease manifestation. It is perhaps most readily associated with thrush, which occurs in the throat and vagina. Both in vitro and in vivo, Candida are capable of growing in a variety of morphogenic forms, namely yeast, pseudohyphae and true hyphae. These forms can be selectively induced by particular experimental conditions; for example, growing C. albicans at 28^oC and 37^oC induces yeast and true hyphal forms, respectively.

Pseudohyphal, Yeast, and True Hyphal Forms of Candida albicans. Source: Sudbery, 2011 

Endocytosis, AP-2 and Polarised Growth

Yeast budding and hyphal growth are highly polarised events, that are co-ordinated in-part by the regulation of cell surface composition via endocytosis (membrane internalisation) and exocytosis (secretion). Both processes are highly complex and involve a plethora of proteins with both overlapping and distinct function. Most pertinant to this project is the endocytic adaptor protein, AP-2. 

Exocytic and Endocytic Events at the Growing Hyphal Tip. Cargo destined for the plasma membrane is transported to the hyphal tip on microtubules (grey). At the tip they can either be sent back towards the base of the tip or to the Spitzenkörper (S), where they are then trafficked to the plasma membrane via actin filaments. Vesicles then fuse with the membrane (exocytosis), resulting in hyphal growth. Membrane components are endocytosed at the collar of the hyphal tip to ensure polarised growth. Source: Schultzhaus & Shaw, 2015

AP-2 is a heterotetrameric protein complex, comprised of α, β, mu2 and σ2 subunits, the latter two of which are responsible for binding to various cargo proteins. Homozygous APM4 deletion mutants (apm4Δ/apm4Δ), which lack the gene encoding the mu2 subunit, display altered growth dynamics in that: (i) on solid media apm4Δ mutants can penetrate media but cannot spread; and (ii) in liquid culture, apm4Δ mutants form wider, shorter and less straight filaments more akin to pseudohyphae. As the mutants are able to undergo morphological changes associated with hyphal growth, these results suggest that AP-2 may be involved in the maintenance, rather than the induction, of polarized growth. 

APM4 is Required for the Maintenance of Polarized Hyphal Growth. (A) Wild type and apm4Δ homozygous mutant Candida albicans grown on solid spider (A) and liquid (B) media. Source: Chapa-y-Lazo et al, 2013.

Candida Colonisation and Invasion

At any given time, Candida colonises the skin and mucosal surfaces of 30-70% of individuals without  causing noticeable disease. However, when immune recognition and control of Candida replication fails, individuals develop Candidiasis. The extent to which the morphological forms of C. albicans contribute to colonisation and disease-causing invasion is still a subject of debate; although mutant strains which are locked into yeast or hyphal forms are avirulent and thus the ability to switch between the two is integral to pathogenesis. 

Steps in Invasion by Candida albicans. Candida first adhere to and colonise epithelial surfaces. Growing hyphae then penetrate and invade epithelial cells and subsequently blood vessels, which results in vascular dissemination of yeast cells, endothelial colonisation and penetration, and disseminated disease. Source: Gow et al, 2012.

Candida and the Innate Immune Response 
Innate immune cells employ non-specific pattern recognition receptors (PRRs), for example toll-like receptors (TLRs), to detect pathogen associated molecular patterns (PAMPs) that are common to multiple infectious agents. Many Candida PAMPs are located in the cell wall, which is comprised of chitin, glucan and mannans organised into two distinct layers. For example, TLR2 and TLR4 recognize phospholipomannan and O-linked mannosyl residues, respectively. 

The Cell Wall of Candida Albicans. Source: Gow et al, 2012 

What this Project will Entail
Due to the fact that: (i) endocytosis regulates cell wall composition; and (ii) apm4Δ mutants have altered hyphal formation, it is plausible that apm4Δ mutants may have aberrant immune interaction and invasive properties and thus, altered virulence. This project aims to investigate the effects of the apm4Δ mutation on Candida cell wall composition; and furthermore establish how any observed changes impact on the interaction between Candida and the innate immune system. 

References

• Centres for Disease Control and Prevention (CDC). Candidiasis
• Gow N.A.R. et al (2012). Candida albicans morphogenesis and host defence: discriminating invasion from colonization. Nature Reviews Microbiology. 10: 112-122
• Schultzhaus Z.S. & Shaw B.D. (2015). Endocytosis and exocytosis in hyphal growth. Fungal Biology Reviews. 29(2): 43-53
• Chapa-y-Lazo B. et al (2013) Yeast endocytic adaptor AP-2 binds the stress sensor Mid2 and functions in polarized cell responses. Traffic. 15(5): 546-557
• Mukaremera L. et al (2017) Candida albicans yeast, pseudohyphal, and hyphal morphogenesis differentially affects immune recognition. Frontiers in Immunology. 8: 629
• Zheng et al (2015) The role of pattern recognition receptors in the innate recognition of Candida albicans. Virulence. 6(4): 347-361