Molecular mechanisms that govern infection and antifungal resistance in Mucorales

Abstract

SUMMARYThe World Health Organization has established a fungal priority pathogens list that includes species critical or highly important to human health. Among them is the order Mucorales, a fungal group comprising at least 39 species responsible for the life-threatening infection known as mucormycosis. Despite the continuous rise in cases and the poor prognosis due to innate resistance to most antifungal drugs used in the clinic, Mucorales has received limited attention, partly because of the difficulties in performing genetic manipulations. The COVID-19 pandemic has further escalated cases, with some patients experiencing the COVID-19-associated mucormycosis, highlighting the urgent need to increase knowledge about these fungi. This review addresses significant challenges in treating the disease, including delayed and poor diagnosis, the lack of accurate global incidence estimation, and the limited treatment options. Furthermore, it focuses on the most recent discoveries regarding the mechanisms and genes involved in the development of the disease, antifungal resistance, and the host defense response. Substantial advancements have been made in identifying key fungal genes responsible for invasion and tissue damage, host receptors exploited by the fungus to invade tissues, and mechanisms of antifungal resistance. This knowledge is expected to pave the way for the development of new antifungals to combat mucormycosis. In addition, we anticipate significant progress in characterizing Mucorales biology, particularly the mechanisms involved in pathogenesis and antifungal resistance, with the possibilities offered by CRISPR-Cas9 technology for genetic manipulation of the previously intractable Mucorales species.

Keywords: CotH; Mucor; RNAi; Rhizopus; dimorphism; genome duplication; host responses; invasins; iron uptake; mucoricin.

Fig 1

Problems associated with the management of mucormycosis.

Fig 2

Manifestations of mucormycosis. Most remarkable features of the different types of infections caused by Mucorales are indicated.

Fig 3

Experimental models to study mucormycosis. The models are displayed in order of frequency of use, with the most used in the top left and the least used in the bottom right. Text above and below each image describes the advantages and limitations of each model, respectively. Mammals are shown in light blue boxes, other vertebrates in green boxes, and invertebrates in yellow boxes.

Fig 4

Fungal invasion and cellular response of the host. The most significant routes of infection are illustrated: rhino-orbital, pulmonary, and skin breaches. (A) Rhino-orbital infections primarily occur in individuals with DKA, likely because associated features induce the expression of both GRP78 and Rhizopus CotH3. Rhizopus CotH3 binds to GRP78, facilitating invasion of both respiratory epithelium and vascular tissue, respectively. Attachment to epithelial cells is possible only for germlings (enlarged for better visualization), but the entry of sporangiospores (brown spheres) after breaching epithelial integrity could also be a potential route. Epithelial damage is mediated by the secretion of mucoricin. Other secreted molecules, such as rhizoferrin, may also contribute to tissue damage. Hyphae cannot be phagocytosed by phagocytes, but they are sensitive to neutrophil cationic peptides. (B) Pulmonary infections are frequently associated with a deficient immune system. A similar host response to nasal infection occurs, except for the presence of alveolar macrophages, which play a primary role in sporangiospore phagocytosis. Despite a significant influx of neutrophils, inflammatory monocytes, and dendritic cells in the lungs of immunocompetent mice, most sporangiospores are phagocytosed by alveolar macrophages, with only a few by interstitial macrophages and neutrophils. Failure in the immune response reduces this reaction, potentially resulting in fungal invasion, mediated by fungal CotH7, which interacts with integrin β1 on alveolar epithelial cells, leading to the activation of epidermal growth factor receptor (EGFR). (C) Skin damage due to trauma or burns can result in mucormycosis in immunocompetent individuals. Breaches in the skin epithelium facilitate the entry of sporangiospores, which readily adhere to laminin or type IV collagen of the basement membrane. This entry triggers a robust immune response, recruiting various types of immune cells that either phagocytose the sporangiospores or restrict their dispersion and germination by forming early innate granulomas, in which the sporangiospores keep alive for long time. Similar structures have been observed in other types of mucormycosis in animal models and humans. Eventually, the fungus may reach the vascular system and disseminate.

Fig 5

Mechanisms involved in the fungal colonization of the host. (A) Phagocytosis represents one of the primary host responses to prevent fungal infection. Mucorales species have evolved mechanisms to hinder phagosome maturation, with melanin- and calcineurin-dependent mechanisms observed in Rhizopus and Mucor, respectively. They also employ strategies to evade phagocytosis, such as the endosymbiont-mediated secretion of antiphagocytic factors in certain R. microsporus isolates. Furthermore, phagocytosis triggers a significant remodeling of fungal gene expression in response to reactive oxygen species and nutritional immunity, including iron deficiency, which inhibits spore germination. In Mucor, the non-canonical RNAi pathway (NCRIP) plays a significant role in this transcriptomic response. This includes the involvement of transcription factors Atf1 and Atf2, which are essential for germination under acidic conditions. (B) Iron plays a crucial role in Mucorales infectivity, as an increase in free iron resulting from conditions like diabetic ketoacidosis, hyperglycemia, and other forms of acidosis can enhance their virulence. These fungi employ a high-affinity reductive system for iron uptake, comprising a ferric reductase (Fre), multicopper ferroxidase (Fet3), and high-affinity iron permease (Ftr1). Additionally, at least Rhizopus can acquire iron bound to xenosiderophores, such as deferoxamine (D), utilizing the Fob1 and Fob2 proteins. Trf refers to transferrin. (C) Mucor and other Mucorales species exhibit growth dimorphism. In Mucor, mycelial growth is associated with virulence. The transition from yeast to hyphae is regulated by oxygen levels, CO₂ levels, and the carbon source. The calcineurin and PKA pathways play opposing roles in dimorphism. G-proteins repress yeast growth under low oxygen levels by inhibiting adenylate cyclase (AC).

Fig 6

Mechanism of innate antifungal drugs resistance in Mucorales. Resistance of Mucorales to antifungals is linked to gene duplication of genes encoding antifungal efflux pumps (pdr), targets of echinocandins (fks), and proteins involved in biosynthesis of azoles (erg6 and erg11). In addition, the mutation Y129F (spiked red ball) in one of the Erg11 proteins (Cyp51F5) causes resistance to short-tailed azoles.