Elucidation of Bacterial Peptidoglycan Signaling and Recognition in the Human Commensal Candida. albicans through the LRR domain of the peripheral Membrane Protein Cyr1p

The human commensal Candida. albicans (C. albicans) is the most common fungal resident of the human microbiome. In healthy individuals this fungus occupies a budding morphology, but during immune incompetence morphologically transitions to a pathogenic hyphal form. The long filamentous form of hyphae readily invades human epithelial cells and if not successfully treated, cause lethal systemic infections. One of the key molecular integrators in this morphological transition is the binding of bacterial peptidoglycan (PG) fragments to the Leucine Rich Repeat (LRR) domain of the adenyl cyclase Cyr1p. PG is a highly complex polymer consisting of repeating units of the disaccharide N-acetyl glucosamine-N acetyl muramic acid, which are crosslinked by pentapeptide side chains. Considerable diversity exists in this polymer within and among Gram (-) and Gram (+) species of bacteria, rendering a plethora of potential ligands for Cyr1p. Identification of specific PG fragments that are able to induce hyphal formation in C. albicans, and stabilize Cyr1p through the LRR domain can elude to small molecule inhibitors to combat these lethal fungal infections that are continuously becoming resistant to current antifungal therapies. To approach a congruency of PG fragments from both Gram (-) and Gram (+) bacterium that can serve as ligands for the LRR domain, we began by characterizing recombinant LRR and in doing so have determined this protein is a peripheral membrane protein that adopts an alpha helical conformation. Furthermore, to assess Cyr1p's ability to interact with a repertoire of PG fragments, we conducted Cellular Thermal Shift Assays (CETSA) with various PG fragments and have observed a temperature induced stabilization in comparison to apo-protein, suggesting that there is a positive interaction with the LRR domain and the fragments. We further investigated the ability of these PG fragments to induce hyphal formation in WT C. albicans cells via microscopy assays and evaluated upregulation of hyphae specific genes at various timepoints via RT-PCR. We observed differential hyphal gene regulation at various timepoints, suggesting that some fragments although able to initiate hyphal growth, failed to maintain growth and subsequent elongation. In addition, we observed that despite a positive association of the LRR with some fragments through CETSA assays, these fragments failed to illicit hyphal formation in WT cells. We infer that stabilization of the LRR is not linearly attributed to hyphal elongation, although hyphal initiation may be present. Collectively, these data help to identify new mechanisms to treat pathogenic fungal infections through the elucidation of promiscuity of the Cyr1p LRR domain, and steers us towards the identification of PG decoys and/or small molecule inhibitors for the treatment of these infections.