The Ccz1 mediates the autophagic clearance of damaged mitochondria in response to oxidative stress in Candida albicans

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Abstract

Autophagy plays a critical role in response to numerous cellular stresses, such as nutrient deprivation, hypoxia, starvation and organelle damage. The disruption of autophagy pathway affects multiple aspects of cellular stress response. Here we for the first time identified Ccz1 as an essential component for autophagy in Candida albicans. Our experiments demonstrated that loss of CCZ1 gene led to vacuolar fragmentation and disruption of the autophagy pathway. Our results also suggested that Ccz1 functioned in oxidative stress. In the ccz1Δ/Δ mutant, the levels of reactive oxidative species (ROS) sharply increased under H2O2 treatment. Further studies demonstrated that breakdown of the autophagic clearance pathway led to the accumulation of oxidative stress-damaged mitochondria, and consequently elevated cellular ROS levels in the ccz1Δ/Δ mutant. Furthermore, deletion of CCZ1 led to a significant defect in filamentous development at both 30 °C and 37 °C. The disruption of CCZ1 gene led to decreased capacity of macrophage killing and increased sensitivity to the macrophages. In addition, the ccz1Δ/Δ mutant exhibited attenuated virulence and decreased fungal burdens in the mouse systemic infection model, indicating that CCZ1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of autophagy-related gene in C. albicans.

Introduction

The fungus Candida albicans, a common opportunistic human pathogen, that colonizes mucocutaneous surfaces of the oral cavity, gastrointestinal tract, and vagina of many mammals, can cause both epithelial infections and life-threatening disorders in immunocompromised patients (Sudbery, 2011, Mayer et al., 2013). Candida infections are thought to be caused by endogenous C. albicans escaping the professional phagocytosis such as macrophages and polymorphonuclear cells (Rudkin et al., 2013). Therefore, a deeper understanding of the complex mechanisms underlying effective defense strategies against oxidative stress will provide valuable-insights into the treatment of C. albicans infection and drug development.

During invading the host cells, C. albicans has to be confronted with the various stresses, especially oxidative agents generated by host immune cells and nutrient deficiency (Almeida et al., 2009). Autophagy, which refers to any process that involves the delivery of cytoplasmic cargo to the lysosome/vacuole, is essential for survival, differentiation, development, and homeostasis. It allows the cell to respond to different types of stress by mediating the hydrolytic degradation of damaged proteins and organelles (Muller and Reichert, 2011, Chen and Chan, 2009). The complete autophagy pathway is essential for cell survival, especially in response to various stresses. To date, almost 40 autophagy-related genes (ATG genes) have identified by genetic screens for autophagy-defective mutants in yeas (Feng et al., 2015). In C. albicans, autophagy-related gene ATG1, ATG8, ATG9 and VMA2 have been identified (Palmer et al., 2007, Yu et al., 2015, Rane et al., 2014). However, the role of other autophagy-related genes is still little known in C. albicans. In Saccharomyces cerevisiae, Ccz1p is a membranous protein and belongs to the VPS (Vacuolar protein sorting) family. Deletion of ScCCZ1 leads to increasing the sensitivity to caffeine and divalent ions, such as Ca2+ and Zn2+ (Kucharczyk et al., 2000, Kucharczyk et al., 2009). Ccz1p, in a complex with Mon1p, is necessary for vacuolar protein trafficking and biogenesis, which is involved in the last step of fusion of multiple transport intermediates with the vacuole and in homotypic vacuole fusion (Piekarska et al., 2010, Cabrera and Ungermann, 2013, Kucharczyk et al., 2001). The disruption of ScCCZ1 blocks the process of vacuolar fusion of autophagosome (Meiling-Wesse et al., 2002). However, the function of Ccz1 is still little known in C. albicans. Recently, two marker proteins served as indicator during the process of autophagy had been identified (Hutchins and Klionsky, 2001, Yu et al., 2015). In C. albicans, Lap41, a homologue of S. cerevisiae Lap4, is expressed in the cytosol as high-weight precursor, and then is delivered from cytosol into vacuole through the Cvt pathway and autophagy for maturation (Palmer et al., 2007). In addition, Atg8, an autophagosome protein, is also delivered into vacuole accompanied with fusion of autophagosome and vacuole. Using the GFP-tagged Atg8 or Lap41, they are translocated into vacuole for degradation during autophagy activation. Because the GFP is stable in vacuole, the accumulation of GFP in the vacuole demonstrates activation of autophagy (Kanki et al., 2009a, Yu et al., 2015).

Mitochondria are required for cellular energy production via oxidative phosphorylation and the conserved processes of iron metabolism, programmed cell death, and intermediary metabolism (Veatch et al., 2009). Meanwhile, mitochondria are the generator of reactive oxygen species (ROS), and the target of oxidative damage. In normal condition, the delicate overall balance is maintained between ROS production and elimination. Once, disruption of the balance might lead to accumulation of ROS in the mitochondria (Venditti et al., 2013). The accumulation of ROS activates the autophagy pathway. In turn, damaged mitochondria are removed by activated autophagy to protect cells against oxidative stress. However, little is known regarding the function of autophagy-related gene in oxidative stress response in C. albicans. In S. cerevisiae, Om45 (the homolog of Csp37 in C. albicans) is a mitochondrial outer membrane protein. The GFP-tagged Om45 protein also localizes on the mitochondrial outer membrane (Kanki et al., 2009b). When mitophagy is induced, the Om45-GFP into the vacuole is degraded. However, GFP is relatively stable within the vacuole and is often released as an intact protein. The Om45-GFP served as the marker protein and GFP can be detected by immunoblotting as semi-quantitative evidence for mitophagy (Kanki et al., 2009a).

In the present report, we identified C. albicans homologue of S. cerevisiae Ccz1 protein by BLAST search of the Candida genome database. And we constructed a CCZ1 null mutant and found that Ccz1 was required for maintenance of vacuolar morphology and ion homeostasis in C. albicans. Furthermore, we found that Ccz1p played an essential role in autophagy and oxidative stress response. The loss of CCZ1 led to accumulation of oxidative stress-damaged mitochondria. Detailed investigation further demonstrated that Ccz1 played an important role in filamentous development and virulence in C. albicans. Hence, this study uncovered a novel relationship between oxidative stress and autophagic clearance of damaged mitochondria in C. albicans.

Section snippets

Strains and growth conditions

The C. albicans strains used in this study are listed in Table 1. SN76 was used as the wild-type strain in the functional analysis and the parental strain for gene disruption. Cells were grown at 30 °C or 37 °C in YPD medium or, when indicated, in synthetic complete (SC) (adding 80 μg/ml uridine). The growth conditions of the C. albicans cells are presented in Supplemental Information.

Strains and plasmids construction

All deletion strains were obtained with the SN76 background (Noble and Johnson, 2005). The ccz1Δ/Δ was constructed

The vacuolar morphology and function in ccz1Δ/Δ mutant

In S. cerevisiae, the CCZ1 deletion shows increased sensitivity to caffeine, calcium and zinc and the CCZ1 encodes a member of class B VPS (Vacuolar Protein Sorting) protein (Kucharczyk et al., 1999). Here, we identified a homologue of S. cerevisiae Ccz1, termed CCZ1 (orf.19.5725) by performing BLAST searches in the Candida Genome Database (http://www.candidagenome.org/), which encodes a protein of 693 amino acids. We disrupted the CCZ1 gene by PCR-mediated recombination for deeply studying the

Discussion

In our study, we found that the loss of CCZ1 gene resulted in the vacuole fragmentation (Fig. 1A) and we speculated that loss of CCZ1 might impair the membrane fusion. Uptake of the endocytic markers FM4-64 demonstrated that Ccz1 functioned at the very last stage of delivery of the cargo to the vacuole (Kucharczyk et al., 2000). Here, we further explored the function of vacuole in ccz1Δ/Δ. Our data suggest that fragmented vacuoles have no obvious difference in vacuolar acidity and membrane

Acknowledgments

We are grateful to Dr. Suzanne M. Noble (University of California-San Francisco, USA), Dr. Gerald Fink (Whitehead Institute for Biomedical Research, MIT, USA), Dr. Julia R. Koehler (Boston Children's Hospital, USA) and Dr. P. Brown (University of Aberdeen, UK) for the generous gift of strains and plasmids. This study was supported by the National Natural Science Foundation of China (No. 81471923, No. 81171541, No. 31400132), the Tianjin Research Program of Application Foundation and Advanced

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