Cx43-mediated hyphal folding counteracts phagosome integrity loss during fungal infection

Phagolysosomes are crucial organelles during the elimination of pathogens by host cells. The maintenance of their membrane integrity is vital during stressful conditions, such as during Candida albicans infection. As the fungal hyphae grow, the phagolysosome membrane expands to ensure that the growing fungus remains entrapped. Additionally, actin structures surrounding the hyphae-containing phagosome were recently described to damage and constrain these pathogens inside the host vacuoles by inducing their folding. However, the molecular mechanism involved in the phagosome membrane adaptation during this extreme expansion process is still unclear. The main goal of this study was to unveil the interplay between phagosomal membrane integrity and folding capacity of C. albicans-infected macrophages. We show that components of the repair machinery are gradually recruited to the expanding phagolysosomal membrane and that their inhibition diminishes macrophage folding capacity. Through an analysis of an RNAseq data set of C. albicans-infected macrophages, we identified Cx43, a gap junction protein, as a putative player involved in the interplay between lysosomal homeostasis and actin-related processes. Our findings further reveal that Cx43 is recruited to expand phagosomes and potentiates the hyphal folding capacity of macrophages, promoting their survival. Additionally, we reveal that Cx43 can act as an anchor for complexes involved in Arp2-mediated actin nucleation during the assembly of actin rings around hyphae-containing phagosomes. Overall, this work brings new insights on the mechanisms by which macrophages cope with C. albicans infection ascribing to Cx43 a new noncanonical regulatory role in phagosome dynamics during pathogen phagocytosis. IMPORTANCE Invasive candidiasis is a life-threatening fungal infection that can become increasingly resistant to treatment. Thus, strategies to improve immune system efficiency, such as the macrophage response during the clearance of the fungal infection, are crucial to ameliorate the current therapies. Engulfed Candida albicans, one of the most common Candida species, is able to quickly transit from yeast-to-hypha form, which can elicit a phagosomal membrane injury and ultimately lead to macrophage death. Here, we extend the understanding of phagosome membrane homeostasis during the hypha expansion and folding process. We found that loss of phagosomal membrane integrity decreases the capacity of macrophages to fold the hyphae. Furthermore, through a bioinformatic analysis, we reveal a new window of opportunities to disclose the mechanisms underlying the hyphal constraining process. We identified Cx43 as a new weapon in the armamentarium to tackle infection by potentiating hyphal folding and promoting macrophage survival.