Genetic Screening of Candida albicans Inactivation Mutants Identifies New Genes Involved in Macrophage-Fungal Cell Interactions

Abstract

Candida albicans, an important fungal pathogen of humans, displays different morphologies, such as yeast, pseudo-hyphae and hyphae, which are recognized unequally by phagocytic cells of the innate immune response. Once C. albicans cells invade host tissues, immune cells such as macrophages are attracted to the site of infection and activated to recognize, engulf and kill the pathogen. We have investigated this fungal cell-macrophage interface by using high-throughput screening of the C. albicans GRACE library to identify genes that can influence this interaction and modify the kinetics of engulfment. Compared with the wild-type (WT) strain, we identified generally faster rates of engulfment for those fungal strains with constitutive pseudo-hyphal and hyphal phenotypes, whereas yeast-form-locked strains showed a reduced and delayed recognition and internalization by macrophages. We identified a number of GRACE strains that showed normal morphological development but exhibited different recognition and engulfment kinetics by cultured macrophages and characterized two mutants that modified interactions with the murine and human-derived macrophages. One mutant inactivated an uncharacterized C. albicans open reading frame that is the ortholog of S. cerevisiae OPY1, the other inactivated CaKRE1. The modified interaction was monitored during a 4 h co-culture. Early in the interaction, both opy1 and kre1 mutant strains showed reduced recognition and engulfment rates by macrophages when compared with WT cells. At fungal germ tube initiation, the engulfment kinetics increased for both mutants and WT cells, however the WT cells still showed a higher internalization by macrophages up to 2 h of interaction. Subsequently, between 2 and 4 h of the interaction, when most macrophages contain engulfed fungal cells, the engulfment kinetics increased for the opy1 mutant and further decreased for the kre1 mutant compared with Ca-WT. It appears that fungal morphology influences macrophage association with C. albicans cells and that both OPY1 and KRE1 play roles in the interaction of the fungal cells with phagocytes.

The nature of the fungal cargo induces significantly different temporal programmes of macrophage phagocytosis

“…This highlights the efficacy of our intact host defences in clearing challenges arising from regular encounters with environmental fungi and the ability of our immune system to establish a homeostatic relationship with our mycobiota ( Iliev, 2022 ). Phagocytosis by macrophages and neutrophils is a key element of the innate host defence response to invading fungal pathogens ( Lewis et al, 2012 , Rudkin et al, 2013 , Bain et al, 2021 , Godoy et al, 2022 ). The process of phagocytosis can be considered to comprise five progressive stages: migration and chemotaxis, target recognition, target internalization, phagosome maturation and pathogen degradation.…”

Going fishing: how to get what you want from a fungal genetic screen

“…Another major focus was on mutants that showed altered morphology, as multiple aggregating strains have been isolated from patients ( 3 – 5 ); screening for altered morphology was the focus of two recent insertional mutagenesis screens ( 6 , 7 ). In other fungi, the interesting biological phenomena and topics of screens range from identifying regulators of sex and meiosis ( 8 ), defining essential genes ( 9 – 11 ), identifying kinases that regulate morphogenesis ( 12 ), determining modulators of host immune responses ( 13 – 15 ), or identifying new potential avenues for antifungal therapies ( 16 – 19 ), to name a few. The critical task is to define what the question is and to spend the time to justify why this is the right question to ask using a screening approach.…”

“…The advent of high-throughput automated microscopy means that imaging is now a fantastic tool for screening, as long as the analysis can be equally high throughput and quantitative. Imaging-based screening also allows for analysis of more complicated phenotypes than growth-based screening, including assaying for morphological changes ( 17 ) or differential phagocytosis by host cells ( 15 ).…”

The advances in the regulation of immune microenvironment by Candida albicans and macrophage cross-talk

“…C. albicans, an important fungal pathogen in humans, exhibits different morphologies, such as yeast, pseudohyphae, and hyphae, which are recognized differently by the phagocytic cells of the innate immune response. Once C. albicans cells get into host tissues, immune cells like macrophages are drawn to the site of infection and activated to find, engulf, and kill the pathogen (Godoy et al, 2022). Understanding the virulence characteristics of C. albicans, the tissue-specific mechanisms of anti-Candida host defense, and its resistance mechanisms to existing antifungal agents should lead to better strategies to diagnose and treat affected individuals, which may help improve patient outcomes (Lopes and Lionakis, 2022).…”