All strains used in this paper were listed in Supplementary Table S1. The primer was listed in Supplementary Table S2. BWP17 was used as the wild-typle (WT) in the functional analysis and the parental strain for gene disruption. The MCP1 gene knockout is performed by homologous recombination (Liang et al., 2018). The MCP1:ARG4 fragment was transformed into the wild strain (WT). The heterozygous mutants (mcp1:ARG4/MCP1) were identified by PCR (MCP1-5det and MCP1-3det). After the MCP1:URA3 fragment was transformed into the heterozygous mutant (mcp1:ARG4/MCP1) homozygous mutant strains (mcp1:ARG4/mcp1:URA3) were identified by PCR. The URA3 was deleted in mcp1Δ/Δ (URA3) mutant strain to eliminate the interference of URA3 to the experiment, confirmed by PCR. For systemic infection assays, the WT, mcp1Δ/Δ and MCP1c were transformed with PstI/BglII-digested pLUBP (encoding URA3 and its adjacent gene, IRO1), obtaining the URA3-reconstituted strains WT^a, mcp1Δ/Δ^a, and MCP1c^a.

Cells were cultivated to log phase in YPD medium (2% glucose, 2% peptone, 0.5% yeast extract adding 80 μg/mL uridine). After collected, cells were transferred to SC-G medium (0.01% glucose, 0.67% yeast nitrogen base without amino acid, 0.2% amino acid drop-out mixture, 3% glycerin) and SD-N medium (1% glucose, 0.052% KCl, 0.152% KH₂PO₄, 0.052% MgSO₄$7H₂O supplemented with 0.1% traceelements, 0.1% vitamins) (Cui et al., 2019).

Fluorescence microscope was used to observe the location of proteins (Mcp1-GFP, Atg8-GFP, and Csp37-GFP). The strains were cultured in defined medium (SC-G and SD-N) at special time, and harvested. The green fluorescence was observed by fluorescence microscope (BX53, Olympus, Japan). After FM4-64 (dissolved in dimethyl sulfoxide (DMSO), final concentration 50 μg/ml, BBI) ((N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide)) staining at 30°C for 10 min (Fischer-Parton et al., 2000). The samples were observed by fluorescence microscope (BX53, Olympus, Japan). The morphology of mitochondria was observed after MitoTracker Red (dissolved in dimethyl sulfoxide (DMSO), final concentration 1 mmol/ml) staining at 37°C for 30 min (BX53, Olympus, Japan).

To investigate the sensitivity of mcp1Δ/Δ to non-fermentable carbon sources (Kundu, 2011), cells were cultured overnight, washed twice by non-fermentable carbon sources medium (SC-G), transferred to SC-G to OD6₀₀ of 0.1 and grown at 30°C with constant shaking at 160 rpm for 1–7 days. At the special time the same amount was taken on YPD solid plates and single colonies of C. albicans was counted. The data were shown from three replicates.

The cells were cultured in YPD medium for 4–6 h, transferred to SC-G medium for 1 day. After propidium iodide (PI) (final concentration 5μg/ml) staining at 30°C for 10 min (Ratnakumar and Tunnacliffe, 2006), the samples were observed by a microscope (BX53, Olympus, Japan).

The intracellular ATP concentration was measured using the ATP-based creatine kinase catalyzing adenosine triphosphate and creatine to produce creatine phosphate. In ATP assay, cell numbers are calculated by OD₆₀₀ where dead cells are excluded through PI-staining (Dong et al., 2015).

DCFH-DA (2′,7′-Dichlorodihydrofluorescein diacetate) was used to detect the total reactive oxygen species (ROS) concentration. The cells were cultured in SC-G at the special time, harvested and stained by DCFH-DA (final concentration 10 μmol/ml) at 37°C for 30 min. The samples were observed by a microscope (Ex = 480 nm, Em = 520 nm) (BX53, Olympus, Japan) (Yu et al., 2016).

The activity of SOD of cell was detected using a superoxide dismutase (SOD) assay kit (Hydroxylamine method) (Nanjing Jiancheng Bio., China). The samples were detected by a spectrophotometer (BIO-RED, United States) at OD₅₅₀ nm (Li et al., 2013).

The activity of CAT was detected using the UV absorption method. The reaction solutions include protein extract and 100 mmol/L H₂O₂ and were detected by a spectrophotometer (OD₂₄₀) (BIO-RED, United States).

The cells were washed twice by PBS, suspended in 2.5% glutaraldehyde fixative and fixed at 4°C for 12 h. Then cells were collected by centrifugation, 1% osmic acid was added, and the cells were suspended and fixed at room temperature for 1 h. Samples were fixed by gradient ethanol dehydration. The embedded samples were cut into 50–100 nm tissue sections using an ultra-microtome. Immunolabeling was performed using anti-GFP antibody (MBL598, 1:100) during 2 h at room temperature followed by Goat Anti-Rabbit IgG/Gold (10 nm) (Solarbio, 1:100 dilution) for 30 min at room temperature. The sample was observed by a transmission electron microscope (Tecnai G2 F-20, FEI, United States) (Elbaz-Alon et al., 2014).

Samples were cultured in defined medium at special time. The cells extracts were subjected to SDS-PAGE and western blotting. The specific antibody, anti-GFP (MBL598), anti-Tubulin (MBL PM054), anti-Rabbit IgG (PROMEGA W4018) was used to enhance the luminescence intensity of the substrate. The protein imprint was detected by Western Blotting exposure meter (Tanon, 5200, multi).

After cells were cultured in SC-G at the special time, harvested and stained by JC-1 (dissolved in DMSO, final concentration 1 μg/mL, Sigma, United States) at 37°C for 30 min (Ratnakumar and Tunnacliffe, 2006; Pina-Vaz and Rodrigues, 2010; Dong et al., 2015; Liang et al., 2018; Lee et al., 2019). Samples were measured by flow cytometry (FACS Calibar flow cytometry, BD, United States). Gated region R1 (Ex = 488 nm, Em = 525 ± 20 nm) includes cells with intact mitochondrial membranes and gated region R2 (Ex = 514 nm, Em = 585 ± 20 nm) depicts cells with decreased of mitochondrial membrane potential. All samples were obtained from three independent experiments.

Strains were cultured on the surface of solid hypha-inducing media (i.e., Spider and RPMI-1640 medium) at 30 and 37°C for 6 days. The strains were also culture in liquid hypha-inducing medium, i.e., the liquid RPMI-1640 medium, stained by Calcofluor White (CFW) (final concentration 10 mg/ml). The length of the hypha is measured by Image J.

All procedures were performed in accordance with the “Guide to the Care and Use of NIH Laboratory Animals” and approved by the Ethics Committee of Nankai University (20160004). All mice used in this study were placed at a constant temperature of 24 ± 2°C in room, a 12 h light/dark cycle, lighted at 7:00 in the morning, and fed at conditions of freedom in Nankai University Medical College. Every effort was made to minimize animal suffering and animal populations.

Thirty ICR female mice (4 weeks old) were divided into 3 groups, and kept in the breeding room for about 1 week. The WT^a, mcp1Δ/Δ^a, and MCP1c ^a strains were cultured in YPD medium to 12 h, washed with PBS, and mice were injected through the tail vein (the injection cells were 5 × 10⁶). The death of the mice was observed and recorded every day.

In order to determine the load of C. albicans in the kidneys, after injection on the 3rd day mice in each group were sacrificed. The kidneys were dissected and weighed. One kidney was made into tissue sections and observed by a light microscope (BX53, Olympus, Japan), another one was fully ground and spread on YPD solid medium to calculate the number of colonies per gram of sample.

The proteins of vCLAMP are located at the mitochondrion-vacuole connecting sites (John Peter et al., 2017). In the study, Mcp1 (orf 19.6550), a homologous protein of S. cerevisiae, was found through the online NCBI BLASTP software¹ in C. albicans. To prove whether Mcp1 is a core vCLAMP protein, the location of Mcp1 in C. albicans was investigated by fluorescence microscope. The results showed that Mcp1-GFP was located both on the mitochondria stained by MitoTracker Red, and on the vacuolar membrane stained by FM4-64 (Figure 1A). Strikingly, overexpression of Mcp1 caused a massive expansion of the contacts between vacuoles and mitochondria (Figure 1B). Moreover, immunoelectron microscopy (IEM) further confirmed that Mcp1 mainly accumulated at vCLAMP (Figure 1B). Therefore, Mcp1 may be a protein accumulated at vCLAMP.

To find whether Mcp1 participates in mitophagy, the strains were cultured in SC-glycerol (SC-G) medium (the medium containing the non-fermentable carbon source glycerol) for further investigation. The outcome shows that deletion of MCP1 gene impairs the growth on non-fermentable carbon sources (Figure 2A). Propidium iodide (PI) staining further showed that the mortality rate of mcp1Δ/Δ was significantly higher than the control strain (>40% vs. <1%) under SC-G medium (Figure 2B). Cell death is not only related to autophagy but also related to oxidative damage caused by reactive oxygen species (ROS) (Yu et al., 2016). To detect the effect of MCP1 deletion on ROS, the ROS levels were tested after DCFH-DA (2′, 7′-dichlorodihydrofluorescein diacetate) stained. The test shows that the ROS level of the mcp1Δ/Δ mutant was significantly higher than the control strains in the SC-G medium (Figure 2C). Moreover, the activity of superoxide dismutase (SOD) and catalase (CAT) was decreased remarkably in mcp1Δ/Δ mutant (Figures 2D,E). These results proved that deletion of MCP1 leads to cell death under the non-fermentable condition.

Mitochondria are sack-like structures surrounded by two bilayers, which is indispensable for oxidative phosphorylation and ATP production (Gomes et al., 2011). In the test, the ATP level was decreased in the mcp1Δ/Δ mutant after cultured in SC-G medium for 1 day (Figure 3A). qRT-PCR analysis demonstrated that the expression of mitochondrial DNA (mtDNA, e.g., COX2, NAD2, NAD5, and ATP6) dropped in the same condition (Figure 3B; Dong et al., 2015).

Mitophagy maintains the function and number of mitochondria through removal of damaged or superfluous mitochondria (Kanki et al., 2011; Böckler and Westermann, 2014). In the experiment, the mitochondrial membrane potential (MMP) of mcp1Δ/Δ was decreased significantly on non-fermentable carbon sources (Figure 3C). The results suggest that Mcp1 plays an important role on maintaining the membrane potential. The findings suggest that deletion of MCP1 severely impaired mitochondrial functions under the non-fermentable condition.

Mitophagy can be induced by non-fermentable carbon sources (Kundu, 2011). Csp37-GFP, a fusion protein localized on the outer membrane of mitochondria in C. albicans (Supplementary Figure S1), could be used as a mitophagy marker (Dong et al., 2015). During cultivation in the SC-G medium, Csp37-GFP was located inside the vacuoles in the WT (Figure 4A, up). However, Csp37-GFP was gathered around the vacuoles in mcp1Δ/Δ mutant (Figure 4A, bottom). Moreover, Western blotting revealed that the GFP alone was not detected in mcp1Δ/Δ mutant (Figure 4B and Supplementary Figures S2, S3).

When mitophagy is activated, the double-membrane autophagosomes form and are transported from the cytoplasm to vacuole (Böckler and Westermann, 2014). As indicated by transmission electron microscopy (TEM), the WT cells formed normal autophagosomes in the cytoplasm under the non-fermentable condition (Figure 5, up). In contrast, the mcp1Δ/Δ mutant did not form any autophagosomes, and showed abnormally enlarged mitochondria (Figure 5, bottom). These results revealed that deletion of MCP1 severely impaired the mitophagy pathway of the fungus under the non-fermentable condition.

As a protein of autophagosomes, Atg8 protein was transported into vacuoles and degraded in the vacuolar compartment during the process of autophagy (Maeda et al., 2017). Moreover, Lap41 is another marker protein in the process of autophagy. During the process of autophagy, Lap41 is transported to the vacuole through the Cvt pathway, forming mLap41 (Yu et al., 2015). In S. cerevisiae, nitrogen starvation culture can activate macroautophagy (Kohda et al., 2007). To explore the role of Mcp1 in the autophagic processes of C. albicans, Atg8 and Lap41 were tagged with GFP, respectively, in WT and mcp1Δ/Δ mutant (Johansen and Lamark, 2011; Cui et al., 2019). The results confirm that Atg8-GFP is located in the vacuole in SD-N medium (Figure 6A). Furthermore, the degradation of Atg8-GFP and Lap41-GFP was found to be normal in mcp1Δ/Δ mutant by Western blotting (Figures 6B–D and Supplementary Figures S4–S9). All the results indicate that macroautophagy and Cvt pathway are independent of Mcp1.

Maintenance of the vacuolar functions is essential for the autophagy process (Klionsky and Ohsumi, 1999). Quinacrine staining revealed that the vacuolar pH is stable in mcp1Δ/Δ mutant (Figure 7A). However, the expression of V-ATPase related gene (e.g., VAM2, VAM3, VPH1 and VPH2) (Jia et al., 2018) and vacuolar hydrolase related gene (CPY2 and CPY3) was significantly up-regulated in mcp1Δ/Δ mutant (Figure 7B; Parzych et al., 2018). This study indicates that deletion of MCP1 affected the activity of V-ATPase.

Some reports indicate that the stability of mitochondria is essential for hypha formation (Potapova et al., 2013). By culturing strains in solid hypha-inducing media (i.e., the Spider and RPMI-1640 medium), it can be seen that deletion of MCP1 affected the mycelial development of C. albicans (Figure 8A; Jia et al., 2018). To further investigate the role of Mcp1 in hyphal development, the strains were cultured in liquid hypha-inducing medium (i.e., the liquid RPMI-1640 medium), and then stained by Calcofluor White (CFW). Also, the growth rate of hyphae of mcp1Δ/Δ mutant was significantly lower than WT and MCP1c strain (Figures 8B,C). These results revealed that Mcp1 is important for hyphal development in C. albicans.

The above studies revealed that deletion of MCP1 can lead to dysfunction of mitophagy and impair mitochondrial function of C. albicans, which are closely related to virulence of C. albicans. Hence, it can be speculated that deletion of MCP1 may reduce the virulence of C. albicans. In a mouse model of systemic infection, while all mice were killed by the control strains in 11 days, about all of the mice survived in these days, and 20% mice remained alive even after 30 days (Figure 9A). Furthermore, deletion of MCP1 led to a significant decrease in kidney fungal burden (Figure 9B), with 10 times lower of kidney CFUs in the mutant-infected mice than that in the control strain-infected mice (Figure 9C). These results indicate that deletion of MCP1 significantly impaired the virulence of C. albicans.