Design of high affinity ligands for C. albicans Als adhesins: inhibitors of biofilm formation

The purpose of this research project is to understand how Candida albicans, an 'opportunistic pathogen', forms aggregates known as biofilms and how small, designed molecules can inhibit this process.

Many microorganisms are allowed to live in our bodies, mainly in our skin and the digestive and urinary systems. Access to other body parts, as the blood and internal organs, is restricted by physical barriers and also by the immune system. In exchange for food and a niche to grow, these organisms break down some of the food we cannot digest and synthesise essential compounds we do not produce, such as vitamins. They also compete with pathogens, as they outgrow them and train our immune system against them. However, we know that conditions affecting our immune system break the natural balance of the human flora. In such conditions, some of these microorganisms can cause disease and hence we call them opportunistic pathogens. In this process, they use specific 'virulence determinants' to overcome its host.

Among these determinants there is a subset of proteins in their surface that specifically bind other molecules in our bodies. Pathogens have evolved a large array of these so-called 'adhesins' that are able to recognise the surface host cells. These interactions are essential to start the process of colonisation, cell invasion and then disease. A few fungal species can also colonise our body and live as commensals or opportunistic pathogens. As a nifty trick, the surface of Candida albicans contains adhesins that are able grasp unstructured or floppy 'tails' of proteins (peptides) on the host cell surface. The same molecule also facilitates the aggregation of Candida cells, which trigger the formation of biofilms, a large cellular mesh observed in advanced forms of this infection and resistant to clinical treatment.

We want to test if this 'promiscuous' mechanism can be inhibited at early stages by the use of molecules that replace the natural ligands found by Candida in our bodies. We will use techniques that show us the molecular structure of these adhesins and specifically, the pockets that bind these small compounds. These studies will facilitate the design drugs to inhibit the adhesin function and ultimately, improve our options in the treatment of Candida infections.

Candida spp. are ubiquitous commensals in the human flora. They are also the main source of opportunistic mycoses worldwide. Among them, C. albicans is the most commonly isolated species. C. albicans is a highly adhesive organism and can colonise almost any host surface, an unusual ability among microbial pathogens. Candida adhesion manifests in different forms, such as attachment to various cell types, recognition of specific proteins in the host cell surface and extracellular matrix, autoaggregation and the development of biofilms.

The Als surface glycoproteins are fundamental to the adhesive properties of C. albicans.

This work builds on progress from a previous funding period that solved the NT-Als domain structure and revealed a binding mechanism that recognizes up to seven C-terminal residues of peptides in an extended conformation. Our goal is to use cellular, biochemical and structural analyses to understand the aggregative properties of Als adhesins, and to place these results into the context of C. albicans ability to form biofilms. Specifically, it is suggested that a conserved amyloid-forming region can act as a switch between the ligand-bound form of Als adhesins or a free form that promotes cell aggregation.

This proposal also explores the use of Als adhesins as templates for the development of antifungal drugs. Blocking the activity of NT-Als domains is predicted to interfere with fungal adhesion and virulence. The peptide-binding site of NT-Als domains contains conserved features amenable for identification of antifungal compounds, e.g. using fragment-based screening by biophysical techniques including crystallography, NMR and thermofluorometry.

Fungal infections is a matter of concern for individuals of different age groups/medical conditions. Candida bloodstream infections carry ~50% attributable mortality and are associated with increased morbidity.

These issues underline the need to identify templates for the design of novel antifungals.

The most common antifungal drugs in the market target the fungal cell membrane (e.g. the synthesis of ergosterol) and enzymes involved in the formation of the cell wall. It is clear that new targets need to be identified for antifungal therapies. Adhesins and other cell wall proteins are readily available on the fungal cell surface and have not been fully explored as targets for antifungal therapy. Our new structural, cellular and biochemical information aims to describe how Als proteins, a common virulence factor in Candida albicans, can be targeted by small chemical compounds for inhibition of fungal growth and pathogenesis. These compounds could also help to reduce the burden of milder forms of candidiasis, sometimes recurrent as in oral thrush and vaginitis.

Candida Als proteins have also been associated with other (presumably non-adhesive) functions. Deletion of different Als genes leads to changes in morphology and the rate of cell growth in C. albicans. This is a relatively unexplored area that will greatly benefit from the discovery of a novel peptide binding mechanism in the N-terminal domain of these proteins, leading to new hypotheses on their interaction with other cell wall proteins and their repercussions on the physiology of C. albicans.

Interestingly, the agglutinin-like sequences (Als) are named after the alpha-agglutinin, a prototypical cell surface protein of S. cerevisiae. This protein specifically recognises the C-terminal end of the a-agglutinin. The new structural data will also promote research on the mechanism that allows this protein to recognise a specific sequence, in contrast to the broad ligand specificity observed in the Als family.