Chenpeng Xiao

Candida albicans autophagy, no longer a bystander: Its role in tolerance to ER stress-related antifungal drugs.

Autophagy is a degradation process involved in pathogenicity of many pathogenic fungi. However, its roles in Candida albicans, the leading fungal pathogen in human beings, remain to be detailed. Most recently, we found that endoplasmic reticulum (ER) stress-inducing conditions led to transcriptional up-regulation of C. albicans autophagy-related (ATG) genes, implying a possible link between autophagy and ER stress response in this pathogen. Using a series of C. albicans ATG mutants and autophagy reporting systems, we found that both treatment of ER stress-related drugs and loss of the ER calcium pump Spf1 promoted autophagic flux of Atg8 and Lap41 (a homologue of Saccharomyces cerevisiae Ape1), indicating that these conditions induce autophagy. Moreover, deletion of ATG genes in the spf1Δ/Δ mutant rendered cells hypersensitive to these drugs and caused activation of UPR, revealing a role of autophagy in alleviating ER stress. In addition, only treatment of tunicamycin and loss of Spf1 in combination increased autophagic flux of the ER component Sec63, suggesting that most of the ER stress-related conditions cause non-selective autophagy rather than selective ER phagy. This study uncovers the important role of C. albicans autophagy in ER stress response and tolerance to antifungal drugs.

The Calcium Channel Blocker Verapamil Inhibits Oxidative Stress Response in Candida albicans

Candida albicans is a common opportunistic fungal pathogen, causing both superficial candidiasis and life-threatening systemic infections in immune-compromised individuals. Calcium signaling is responsible for this pathogen in responding to several stresses, such as antifungal drugs, alkaline pH and membrane-perturbing agents. Our recent study revealed that it is also involved in oxidative stress response. In this study, we investigated the effect of verapamil, an L-type voltage-gated calcium channel blocker, on oxidative stress response in this fungus. The addition of verapamil resulted in increased sensitivity to the oxidative agent H2O2, which is associated with a decrease of calcium fluctuation under the stress. Moreover, this agent caused enhanced oxidative stress, with increased levels of ROS and enhanced dysfunction of the mitochondria under the oxidative stress. Further investigations in SOD activity, GSH contents and expression of oxidative stress response-related genes indicated that the effect of verapamil is related to the repression of oxidative stress response. Our findings demonstrated that verapamil has an inhibitory effect on oxidative stress response, confirming the relationship between calcium signaling and oxidative stress in C. albicans. Therefore, calcium channels may be potential targets for therapy to enhance the efficacy of oxidative stress against C. albicans-related infections.

Tfp1 is required for ion homeostasis, fluconazole resistance and N-Acetylglucosamine utilization in Candida albicans.

The vacuolar-type H+-ATPase (V-ATPase) is crucial for the maintenance of ion homeostasis. Dysregulation of ion homeostasis affects various aspects of cellular processes. However, the importance of V-ATPase in Candida albicans is not totally clear. In this study, we demonstrated the essential roles of V-ATPase through Tfp1, a putative V-ATPase subunit. Deletion of TFP1 led to generation of an iron starvation signal and reduced total iron content, which was associated with mislocalization of Fet34p that was finally due to disorders in copper homeostasis. Furthermore, the tfp1∆/∆ mutant exhibited weaker growth and lower aconitase activity on nonfermentable carbon sources, and iron or copper addition partially rescued the growth defect. In addition, the tfp1∆/∆ mutant also showed elevated cytosolic calcium levels in normal or low calcium medium that were relevant to calcium release from vacuole. Kinetics of cytosolic calcium response to an alkaline pulse and VCX1 (VCX1 encodes a putative vacuolar Ca2+/H+ exchanger) overexpression assays indicated that the cytosolic calcium status was in relation to Vcx1 activity. Spot assay and concentration-kill curve demonstrated that the tfp1∆/∆ mutant was hypersensitive to fluconazole, which was attributed to reduced ergosterol biosynthesis and CDR1 efflux pump activity, and iron/calcium dysregulation. Interestingly, carbon source utilization tests found the tfp1∆/∆ mutant was defective for growth on N-Acetylglucosamine (GlcNAc) plate, which was associated with ATP depletion due to the decreased ability to catabolize GlcNAc. Taken together, our study gives new insights into functions of Tfp1, and provides the potential to better exploit V-ATPase as an antifungal target.

The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans.

Candida albicans is a common pathogenic fungus and has aroused widespread attention recently. Actin cytoskeleton, an important player in polarized growth, protein secretion and organization of cell shape, displays irreplaceable role in hyphal development and cell integrity. In this study, we demonstrated a homologue of Saccharomyces cerevisiae Sac1, in C. albicans. It is a potential PIP phosphatase with Sac domain which is related to actin organization, hyphal development, biofilm formation and cell wall integrity. Deletion of SAC1 did not lead to insitiol-auxotroph phenotype in C. albicans, but this gene rescued the growth defect of S. cerevisiae sac1Δ in the insitiol-free medium. Hyphal induction further revealed the deficiency of sac1Δ/Δ in hyphal development and biofilm formation. Fluorescence observation and real time PCR (RT-PCR) analysis suggested both actin and the hyphal cell wall protein Hwp1 were overexpressed and mislocated in this mutant. Furthermore, cell wall integrity (CWI) was largely affected by deletion of SAC1, due to the hypersensitivity to cell wall stress, changed content and distribution of chitin in the mutant. As a result, the virulence of sac1Δ/Δ was seriously attenuated. Taken together, this study provides evidence that Sac1, as a potential PIP phosphatase, is essential for actin organization, hyphal development, CWI and pathogenicity in C. albicans.

Loss of Ssq1 leads to mitochondrial dysfunction, activation of autophagy and cell cycle arrest due to iron overload triggered by mitochondrial iron-sulfur cluster assembly defects in Candida albicans.

Iron-sulfur clusters perform essential functions in enzymatic catalysis and homeostatic regulation. Here we for the first time identified Ssq1 as an essential component for iron-sulfur cluster assembly in Candida albicans. Ssq1 played an important role in cell growth. Shutting off SSQ1 led to accumulation of intracellular iron, especially in mitochondria, and disorder of intracellular iron regulation. In tetO-SSQ1, iron overloading triggered the oxidative damage of mitochondrial function. Surprisingly, disruption of SSQ1 activated autophagic pathway. The mitochondrial dysfunction was further aggravated when CCZ1 (which is essential for autophagy) and SSQ1 was simultaneously deleted, suggesting that autophagy played a critical role in maintenance of mitochondrial function in tetO-SSQ1. In addition, double deletion of SSQ1 and CCZ1 further elevated cellular iron levels in comparison with tetO-SSQ1, indicating that autophagy participated in maintenance of iron homeostasis. Furthermore, we found that loss of SSQ1 led to increasing protein expression of Rnr1 and redistribution of Rnr2 from the nucleus to cytoplasm, and further resulted in cell cycle arrest. The results implied that cell cycle arrest was caused by activating the checkpoint pathway because of impairing the iron-sulfur cluster assembly in tetO-SSQ1. Shutting off SSQ1 led to a significant defect in filamentous development. Interestingly, the tetO-SSQ1ccz1Δ/Δ growth was inhibited on hyphae-inducing solid media. Both tetO-SSQ1 and tetO-SSQ1ccz1Δ/Δ exhibited extremely attenuated virulence, indicating that Ssq1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of iron-sulfur cluster assembly-related gene in C. albicans.

The Candida albicans fimbrin Sac6 regulates oxidative stress response (OSR) and morphogenesis at the transcriptional level

The actin cytoskeleton coordinates numerous fundamental cellular processes. Fimbrins are a class of evolutionally conserved ABPs that mediate actin bundling and regulate actin dynamics and functions. In this study, we identified the fimbrin Sac6 from the important fungal pathogen, Candida albicans. Interestingly, deletion of SAC6 led to increased tolerance to oxidative stress, while its overexpression caused hyper-susceptibility to this stress. Further investigations revealed that Sac6, by interaction with actin, negatively regulated the cytosol-to-nucleus transport of the key OSR (oxidative stress response) transcription factor Cap1 and consequent expression of OSR genes. Moreover, loss of Sac6 enhanced hyphal maintenance, and its overexpression caused a defect in hyphal development, which was attributed to abnormal expression of morphogenesis-related genes. In addition, Sac6 was involved in regulation of secretion of lytic enzymes and virulence of C. albicans. This study reveals a novel mechanism by which fimbrin transcriptionally regulates OSR and morphogenesis, and sheds a novel light on the functions of actin cytoskeleton.

Novel mechanisms of surfactants against Candida albicans growth and morphogenesis

Candida albicans is a common opportunistic fungal pathogen, causing not only superficial mucosal infections but also life-threatening systemic candidiasis in immune-compromised individuals. Surfactants are a kind of amphiphilic compounds implemented in a wide range of applications. Although their antimicrobial activity has been characterized, their effect on C. albicans physiology remains to be elucidated. In this study, we investigated the inhibitory effect of two representative surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), on C. albicans growth and morphogenesis. Both surfactants exhibited inhibitory effect on C. albicans growth. This effect was not attributed to plasma membrane (PM) damage, but was associated with mitochondrial dysfunction. Excitingly, the surfactants, especially CTAB, showed strong inhibitory effect on hyphal development (IC50=0.183 ppm for CTAB and 6.312 ppm for SDS) and biofilms (0.888 ppm for CTAB and 76.092 ppm for SDS). Actin staining and Hwp1-GFP localization further revealed that this inhibition is related to abnormal organization of actin skeleton and subsequent defect in polarized transport of hyphae-related factors. This study sheds a novel light on the antimicrobial mechanisms of surfactants, and suggests these agents as potential drugs against C. albicans hyphae-related infections in clinical practice.

Function of glutaredoxin 3 (Grx3) in oxidative stress response caused by iron homeostasis disorder in Candida albicans

AIM Glutaredoxin is a conserved oxidoreductase in eukaryotes and prokaryotes. This study aimed to determine the role of Grx3 in cell survival, iron homeostasis and the oxidative stress response in Candida albicans. MATERIALS & METHODS A grx3Δ/Δ mutant was obtained using PCR-mediated homologs recombination. The function of Grx3 was investigated by a series of biochemical methods. RESULTS Deletion of GRX3 impaired growth and cell cycle, disturbance of iron homeostasis and activated the oxidative stress response. Furthermore, disruption of GRX3 caused oxidative damage and growth defects of C. albicans. CONCLUSION Our findings provide new insights into the role of GRX3 in C. albicans.

Arf1 regulates the ER–mitochondria encounter structure (ERMES) in a reactive oxygen species‐dependent manner

The Arf family of small GTP‐binding and ‐hydrolyzing proteins are some of the most important intracellular regulators of membrane dynamics. In this study, we identified the Golgi‐localized Arf family G protein Arf1 in Candida albicans and confirmed its conserved function in regulating the secretory pathway. Interestingly, deletion of ARF1 resulted in intracellular reactive oxygen species (ROS) accumulation, and induced formation of the endoplasmic reticulum (ER)–mitochondria encounter structure (ERMES). Moreover, N‐acetylcysteine‐mediated ROS scavenging in the arf1Δ/Δ strain attenuated ERMES formation, indicating that intracellular ROS accumulation resulting from ARF1 deletion facilitated ERMES formation. In addition, Arf1 regulated many key physiological processes in C. albicans, including cell cycle progression, morphogenesis and virulence. This study uncovers a role for Arf family G proteins in regulating ERMES formation and sheds new light on the crucial contribution of ROS to membrane dynamics.

The malfunction of peroxisome has an impact on the oxidative stress sensitivity in Candida albicans.

The peroxisome plays an essential role in eukaryotic cellular metabolism, including β-oxidation of fatty acids and detoxification of hydrogen peroxide. However, its functions in the important fungal pathogen, C. albicans, remain to be investigated. In this study, we identified a homologue of Saccharomyces cerevisiae peroxisomal protein Pex1 in this pathogen, and explored its functions in stress tolerance. Fluorescence observation revealed that C. albicans Pex1 was localized in the peroxisomes, and its loss led to the defect in peroxisome formation. Interestingly, the pex1Δ/Δ mutant had increased tolerance to oxidative stress, which was neither associated with the Cap1 pathway, nor related to the altered distribution of catalase. However, under oxidative stress, the pex1Δ/Δ mutant showed increased expression of autophagy-related genes, with enhanced cytoplasm-to-vacuole transport and degradation of the autophagy markers Atg8 and Lap41. Moreover, the double mutants pex1Δ/Δatg8Δ/Δ and pex1Δ/Δatg1Δ/Δ, both of which were defective in autophagy and peroxisome formation, showed remarkable attenuated tolerance to oxidative stress. These results indicated that autophagy is involved in resistance to oxidative stress in pex1Δ/Δ mutant. Taken together, this study provides evidence that the peroxisomal protein Pex1 regulates oxidative stress tolerance in an autophagy-dependent manner in C. albicans.