Mucor circinelloides Thrives inside the Phagosome through an Atf-Mediated Germination Pathway

Abstract

Mucormycosis is an emerging fungal infection that is often lethal due to the ineffectiveness of current therapies. Here, we have studied the first stage of this infection—the germination of Mucor circinelloides spores inside phagocytic cells—from an integrated transcriptomic and functional perspective. A relevant fungal gene network is remodeled in response to phagocytosis, being enriched in crucial functions to survive and germinate inside the phagosome, such as nutritional adaptation and response to oxidative stress. Correspondingly, the phagocytic cells induced a specific proinflammatory and apoptotic response to the pathogenic strain. Deletion of fungal genes encoding putative transcription factors (atf1, atf2, and gcn4), extracellular proteins (chi1 and pps1), and an aquaporin (aqp1) revealed that these genes perform important roles in survival following phagocytosis, germination inside the phagosome, and virulence in mice. atf1 and atf2 play a major role in these pathogenic processes, since their mutants showed the strongest phenotypes and both genes control a complex gene network of secondarily regulated genes, including chi1 and aqp1. These new insights into the initial phase of mucormycosis define genetic regulators and molecular processes that could serve as pharmacological targets. IMPORTANCE Mucorales are a group of ancient saprophytic fungi that cause neglected infectious diseases collectively known as mucormycoses. The molecular processes underlying the establishment and progression of this disease are largely unknown. Our work presents a transcriptomic study to unveil the Mucor circinelloides genetic network triggered in fungal spores in response to phagocytosis by macrophages and the transcriptional response of the host cells. Functional characterization of differentially expressed fungal genes revealed three transcription factors and three extracellular proteins essential for the fungus to survive and germinate inside the phagosome and to cause disease in mice. Two of the transcription factors, highly similar to activating transcription factors (ATFs), coordinate a complex secondary gene response involved in pathogenesis. The significance of our research is in characterizing the initial stages that lead to evasion of the host innate immune response and, in consequence, the dissemination of the infection. This genetic study offers possible targets for novel antifungal drugs against these opportunistic human pathogens.

A non-canonical RNAi pathway controls virulence and genome stability in Mucorales

“…Our transcriptomic analysis found that all of these marker genes were also controlled by NCRIP during saprophytic growth (S1 Dataset), and thus, they were employed here to validate the transcriptional pre-activation observed in the mutants rdrp1 Δ and r3b2 Δ without macrophages (Fig 3C). We found that the four marker genes showed significant induction in the two mutants without macrophages, similar to the previously reported increased expression observed in the wildtype during phagocytosis [13], indicating that NCRIP controls the response to phagocytosis by repressing it during non-stressful conditions.…”

“…Here, we have directly analyzed the transcriptomic profiles in NCRIP key mutants, identifying a substantial number of DEGs in both rdrp1 and r3b2 mutants compared to the wild-type strain. However, a significantly lower number of genes were regulated in these mutants upon phagocytosis compared to the complex response observed in the wild-type strain [13]. The principal component analysis and the comparison of the four profiles among them further confirmed this strong bias among samples.…”

“…Previous studies revealed an intricate network of genes activated in response to phagocytosis, which is essential for the pathogenic potential of Mucorales [13]. Considering the large number of genes regulated by NCRIP, and the functional processes involved, we postulated that some of these genes might participate in the response to phagocytosis.…”

“…Spores were harvested and filtered using a Falcon® 70 µm cell strainer before confronting with macrophages or animal models. The host-pathogen interactions were performed confronting spores from R7B, MU419, and MU412 with mouse macrophages (cell line J774A.1; ATCC TIB-67) in a ratio 1.5:1 (spores:macrophages) following the protocol described in [13]. In summary, the interactions were maintained at 37ºC in L15 medium (Capricorn Scientific) supplemented with 20% of Fetal Bovine Serum (Capricorn Scientific) for 5 hours, ensuring all the spores were phagocytosed.…”

Early diverging fungus Mucor circinelloides lacks centromeric histone CENP-A and displays a mosaic of point and regional centromeres

“…Mucoralean species are the causative agents of mucormycosis, an emerging fungal infection with extremely poor clinical outcomes, probably as a consequence of their innate resistance to all current antifungal drugs 27,28 and their ability to evade host innate immunity 29,30 . In spite of their clinical relevance, limited information is available on essential biological processes in these fungal pathogens, especially nuclear division and cell cycle.…”