Roles of Nucleolar Factor RCL1 in Itraconazole Resistance of Clinical Candida albicans Under Different Stress Conditions

Introduction

Invasive Candidiasis (IC) is a prevalent fungal infection associated with a high mortality rate of 70%. The manifestations of IC range from candidemia to fulminant sepsis, exhibiting varying degrees of severity.^1,2 Several factors, such as prolonged non-standard and high-dose application of immunosuppressants and glucocorticoids, an increase in acquired human immunodeficiency virus (HIV) infections, and the use of various catheters, stents, and other medical instruments, contribute to the annual rise in IC incidence.³ IC typically involves the invasion of the bloodstream by candida fungi, including species such as Candida albicans, Candida parapsilosis, and Candida tropicalis. C. albicans remains the primary pathogen causing IC.^1,2

C. albicans, a conditionally pathogenic fungus, can co-exist in healthy individuals as a part of the normal microbiota without causing disease.⁴ It commonly resides on mucosal surfaces, like the oral, vaginal, and gastrointestinal mucosa.⁵ However, under certain conditions, C. albicans can cause a spectrum of diseases, ranging from superficial mucous membranes infections to deep-seated systemic invasions, potentially leading to life-threatening deep fungal infections upon entering the bloodstream.⁶ Previous studies have indicated that a variety of factors, such as the transformation of yeast to mycelial morphology, phenotypic transformation, biofilm formation, and the expression of cell surface adhesins, enhance the pathogenicity of C. albicans.^7–9 Antifungal drugs, including itraconazole (ITR), fluconazole (FCA), and voriconazole (VRC), are effective against C. albicans. However, the abuse and prolonged use of these drugs have led to the emergence of drug-resistant strains, posing a significant challenge to the treatment of C. albicans infections.¹⁰ Therefore, it is imperative to elucidate the mechanisms of resistance in C. albicans to improve the prognosis of patients.

Ribosomes are arguably the most critical macromolecular machines, tasked with carrying out protein synthesis in cells. Ribosome biogenesis stands out as one of the most crucial and energy-consuming processes within cells.¹¹ Mature ribosomes consist of chemically modified components, including methylation of the ribose sugars at the C2-position, isomerization of uridines to pseudouridines, and various base modifications in both ribosomal RNA (rRNA) and ribosomal protein (RP).¹¹ In eukaryotes, ribosomes are composed of a small subunit (40Ss) and a large subunit (60S). The process of ribosome biogenesis is complex and subject to strict regulation to ensure the correct production of ribosomes under various environmental and metabolic changes.¹² Ribosome biogenesis begins with the transcription of rRNA in the nucleolus, and then the transcribed rRNA undergoes complicated folding, modification, processing, and binding to RP. During this process, precursor rRNA (pre-rRNA) is exported from the nucleus to the cytoplasm, eventually maturing into ribosomes with translational activity.^13,14 Over 200 ribosome assembly factors (AFs) and 80 small nucleolar RNAs (snoRNAs) are associated with ribosome biogenesis. Among them, GTPase BMS1 (BMS1) is the sole known GTPase required for the biosynthesis of the 40S ribosome subunit, interacting with RNA terminal phosphate cyclase like 1 (RCL1). RCL1, an RNA 3’-terminal phosphate cyclase, plays a crucial role in the biogenesis of 18S rRNA and pre-rRNA in eukaryotes.^15–17 Identified as a stress gene, RCL1 responds more rapidly to drugs compared to other ribosomal genes.¹⁸ In our previous studies, we uncovered that RCL1 is overexpressed in C. albicans during the immune response to drug treatment.¹⁷ Differential expression analysis and protein–protein interaction (PPI) network analysis of the CAF-21 expression data file (GSE65396) in C. albicans before and after treatment with the macrocyclic compound (RF59) in the GEO database revealed a notable increase in RCL1 and other ribosome-related genes following drug administration.¹⁷ However, the physiological significance of RCL1 in Candidal cells, particularly in drug-resistant strains and under different stress conditions, as well as how changes in RCL1 expression may impact the organism and/or the host in an infective situation remain unknown.

The conditions typically employed for the cultivation of C. albicans involve a temperature of 37°C, a CO₂ concentration of 0.03% CO₂ in the air, and a pH level of 4. It is noteworthy that the female vagina is characterized by weak acidity, with a pH range of 3.8–4.4 under normal circumstances. These specified conditions are considered standard and are henceforth referred to as common or normal conditions in this study. The primary objective of this investigation is to assess the impact of RCL1 on drug resistance in C. albicans through an examination of gene expression patterns. Concurrently, C. albicans was subjected to cultivation under diverse conditions to evaluate RCL1 expression and elucidate the role of RCL1 in the face of varying stressors.

Materials and Methods

Experimental Strains

The 15 strains of clinical ITR-resistant C. albicans and 1 strain of standard C. albicans were utilized for the Sanger sequencing. The quantitative real-time polymerase chain reaction (qRT-PCR) assays were conducted on 14 of the aforementioned 15 strains of clinical ITR-resistant C. albicans (the same strains used in sequencing), 14 strains of clinical C. albicans sensitive to ITR, FCA, and VRC (sensitive C. albicans), and 1 strain of standard C. albicans (the same strains used in sequencing). The standard strain (ATCC11006) was procured from the Center for Mycology and Mycosis Research, Peking University Health Science Center. All the clinical strains were isolated, identified, and preserved at the Fungal Laboratory, Department of Dermatology and Venereology, Second Hospital of Shanxi Medical University, China. This study received approval from the Ethics Committee of the Second Hospital of Shanxi Medical University.

DNA Extraction, PCR Amplification, and RCL1 Sequencing

The strains of C. albicans were cultured in Yeast Extract Peptone Dextrose (YPD) Liquid Medium (AOBOX BIOTECHNOLOGY, Beijing, China) at 37°C with a rotational speed of 200 rpm/min. After 24 hours of culture, total DNA from C. albicans was extracted using a Yeast DNA Extraction kit (Shanghai Sangong Biotechnology Co., LTD, Shanghai, China) following the manufacturer’s protocols. The concentration (OD260) and purity (OD260/280) of the isolated DNA were determined using a microplate reader (BK-9622, BIOBASE, Shandong, China). To obtain the full-length sequence of the RCL1 gene, the upstream and downstream primers for RCL1 (RCL1-Forward-5’-TGGCATCACCTTAGTCGCTG-3’, RCL1-Reverse-5’-GTAAACTCCCAGCCGAACCA-3’) were designed and synthesized by Shanghai Sangong Biotechnology Co., LTD. (Shanghai, China). Subsequently, the RCL1 sequence was amplified on a PCR system (ProFlex, Applied Biosystems, California, USA) using 2×Hieff^® Ultra-Rapid HotStart PCR Master Mix (with Dye) (10157ES03, YESEN, Shanghai, China) following the manufacturer’s instructions. The PCR products, with a length of 77 bp, were purified by 1% agarose gel electrophoresis, and sent to Shanghai Sangong Biotechnology Co., Ltd. for sequencing. Mutation sites in the RCL1 sequencing were observed using SnapGene 4.1.8 software (Insightful Science, California, USA).

Culture of C. albicans Under Diverse Stress Conditions

Culture of C. albicans Under Different Temperatures

Sterile inoculation rings were used to inoculate 14 strains of clinical ITR-resistant C. albicans and 14 strains of clinical sensitive C. albicans onto YPD solid medium (pH = 4, YPDA Medium, PM2011, Coolaber, Beijing, China). The strains were then incubated in either a 25°C or 37°C incubator (SPX-250B-Z, Shanghai Boxun Industrial Co., LTD, Shanghai, China) with 0.03% CO₂ for 48 hours. Subsequently, a single colony was selected and transferred into YPD liquid medium (pH = 4), shaken, and cultured in a 25°C or 37°C atmosphere with 0.03% CO₂ for an additional 24 hours.

Culture of C. albicans Under Different CO₂ Concentrations

The sterile inoculation rings were employed to introduce 14 strains of clinical ITR-resistant C. albicans and 14 strains of clinical sensitive C. albicans onto YPD solid medium (adjusted to pH = 4). Subsequently, the strains were incubated in a 37°C incubator, with 5% or 0.03% CO_2, for a period of 48 hour. Following incubation, a single colony was selected and transferred into YPD liquid medium (pH = 4), agitated, and cultured in a 37°C environment, under 5% or 0.03% CO₂ for an additional 24 hours.

Culture of C. albicans Under Different pH Value

Sterile inoculation rings were employed to inoculate 14 strains each of clinical ITR-resistant C. albicans and clinical sensitive C. albicans onto YPD solid medium at pH = 7 or pH = 4. Subsequently, the strains were incubated in a 37°C incubator with 0.03% CO₂ for 48 hours. Following this, a single colony was selected and transferred to YPD liquid medium (at pH = 7 or pH = 4), where it was shaken and cultured under a 37°C atmosphere with 0.03% CO₂ for an additional 24 hours.

Real-Time Quantitative PCR (RT-qPCR)

The total RNA from C. albicans cultured under various conditions was extracted using the Spin Column Yeast Total RNA Purification Kit (B518657, Shanghai Sangong Biotechnology Co., LTD, Shanghai, China) following the manufacturer’s protocols. The MightyScript First-Strand cDNA Synthesis Master Mix (B639251, Shanghai Sangong Biotechnology Co., LTD, Shanghai, China) was employed for reverse transcription of the total RNA into complementary DNA (cDNA) in accordance with the instructions of the manufacturer. Subsequently, qRT-PCR analysis was conducted using 2×SYBR Green PCR Master Mix (SR1110, Solarbio, Beijing, China) as outlined previously.¹⁹ The transcription level of RCL1 was normalized to ACTB (β-actin), and the relative expression of RCL1 was calculated using the 2^−ΔΔCt method. The primers used in the qRT-PCR assay were as follows: RCL1-Forward-5’-GCCAACAGGATGCGAGGTTA-3’, RCL1-Reverse-5’- TCAGCGACTAAGGTGATGCC-3’; ACTB-Forward-5’-TGTTGACCGAAGCTCCAATGA-3’, ACTB-Reverse-5’-ACCGGTGGTTCTACCAGAAG-3’.

Statistical Analysis

All data were analyzed using SPSS software (version 26.0, SPSS, Inc., Chicago, IL, USA) and were presented as mean ± standard deviation (SD). The differences between the two groups were assessed using the Student’s t-test. *P < 0.05 indicated a statistical difference.

Results

RCL1 Sequencing Analysis

The RCL1 gene of 16 strains of C. albicans was subjected to bidirectional sequencing in this study. The forward sequencing revealed successful sequencing of six C. albicans strains, including the standard strain ATCC11006 and the ITR-resistant strains SY13, SY17, SY18, SY32, and SY35. RCL1 gene sequences of the five ITR-resistant strains, which were successfully sequenced in the forward direction, were compared to those of the standard strain ATCC11006. Table 1 illustrates that a total of three missense mutations were identified in ITR-resistant C. albicans, namely C6A, G10A, and A11T.

Table 1 Base Mutation Sites and Amino Acid Mutation for RCL1 in Candida albicans Strains Through Forward Sequencing

Results from reverse sequencing demonstrated the successful sequencing of all 16 strains of C. albicans. Comparative analysis with the RCL1 gene of the standard strain ATCC11006 revealed one synonymous mutation (C1T) and four missense mutations (C1T, A3T, A7G, and T8G) in the 15 ITR-resistant strains that were successfully sequenced in the reverse direction (Table 2).

Table 2 Base Mutation Sites and Amino Acid Mutation for RCL1 in Candida albicans Strains Through Reverse Sequencing

Expression of RCL1 in the Sensitive and ITR-Resistant C. albicans

Subsequently, we determined the expression levels of RCL1 in both susceptible and ITR-resistant strains of C. albicans. The data revealed a significant upregulation of RCL1 expression in ITR-resistant C. albicans compared to the susceptible strain under normal conditions (37°C, 0.03% CO₂, pH 4.0) (P < 0.05, Figure 1A). At a lower temperature (25°C, 0.03% CO₂, pH = 4.0), the expression levels of RCL1 in both sensitive and ITR-resistant C. albicans were comparable, with no statistically significant difference (P > 0.05, Figure 1B). Although the expression of RCL1 in ITR-resistant C. albicans was slightly elevated with increased CO₂ concentration (37°C, 5% CO₂, pH 4.0), the difference did not reach statistical significance (P > 0.05, Figure 1C). Intriguingly, the expression of RCL1 in ITR-resistant C. albicans was notably higher than in sensitive strains when the pH of the culture conditions was increased to 7.0 (37°C, 0.03% CO₂, pH 7.0) (P < 0.05, Figure 1D). These findings suggest that RCL1 expression varies between sensitive and resistant C. albicans strains under different culture conditions.

Figure 1 Expression of RCL1 in the sensitive and resistant C. albicans. (A) RCL1 expression in sensitive and ITR-resistant C. albicans in normal condition (37°C, 5% CO2, pH = 4.0). N = 14. (B) RCL1 expression in sensitive and ITR-resistant C. albicans at low temperature (25°C, 0.03% CO2, pH = 4.0). N = 14. (C) The level of RCL1 in sensitive and ITR-resistant C. albicans at high CO2 concentration (37°C, 5% CO2, pH = 4.0). N = 14. (D) The expression of RCL1 in sensitive and ITR-sensitive C. albicans in neutral conditions (37°C, 0.03% CO2, pH = 7.0). N = 14. *P < 005, compared with the sensitive strains.

Expression of RCL1 in C. albicans Under Diverse Temperature Conditions

The expression levels of RCL1 in C. albicans under various temperature conditions were determined using qRT-PCR. In the sensitive strains of C. albicans, RCL1 expression at 25°C was significantly higher than that at 37°C (P < 0.05, Figure 2A). In contrast, the level of RCL1 was remarkably lower at 25°C compared to 37°C in the ITR-resistant strains of C. albicans (P < 0.05, Figure 2B). These data indicate that temperature could influence the expression of RCL1 in both the sensitive and resistant strains of C. albicans.

Figure 2 Expression of RCL1 in C. albicans under diverse temperature conditions. (A) RCL1 expression in sensitive C. albicans at 37°C or 25°C. N = 14. (B) RCL1 expression in ITR-resistant C. albicans at 37°C or 25°C. N = 14. *P < 005, compared with the strains cultured at 37°C.

Expression of RCL1 in C. albicans Under Diverse CO₂ Conditions

The RCL1 expression in C. albicans was investigated under different CO₂ conditions. Figure 3A illustrates that an increased CO₂ concentration (5% CO₂) had minimal impact on RCL1 expression in the sensitive strains of C. albicans (P > 0.05). However, in ITR-resistant C. albicans, the RCL1 level significantly decreased under 5% CO₂ conditions compared to 0.03% CO₂ conditions (P < 0.05, Figure 3B). These results demonstrate that CO₂ concentration influences the expression of RCL1 in the resistant C. albicans but not in the sensitive C. albicans.

Figure 3 Expression of RCL1 in C. albicans under diverse CO2 condition. (A) The RCL1 level in sensitive C. albicans under 0.03% or 5% CO2 concentration. N = 14. (B) The RCL1 expression in ITR-resistant C. albicans under 0.03% or 5% CO2 concentration. N = 14. *P < 005, compared with the strains cultured with 0.03% CO2.

Expression of RCL1 in C. albicans Under Diverse pH Conditions

Further analyses revealed that an elevated pH value (pH = 7.0) significantly diminished the expression levels of RCL1 in both the sensitive and ITR-resistant strains of C. albicans (P < 0.05, Figure 4A and B). These findings suggest that distinct pH values modulate RCL1 expression in both the sensitive and resistant strains of C. albicans.

Figure 4 Expression of RCL1 in C. albicans under different pH condition. (A) The RCL1 expression in sensitive C. albicans under pH = 4 or pH = 7 condition. N = 14. (B) The RCL1 expression in ITR-resistant C. albicans under pH = 4 or pH = 7 condition. N = 14. *P < 005, compared with the strains cultured at pH = 4.

Discussion

C. albicans is a common human pathogen, and IC caused by this fungus poses a threat to the lives and health of patients.²⁰ RCL1 is a phosphate cyclase closely associated with the processing of rRNA.²¹ Growing evidence indicates that RCL1 can bind to BMS1, a typical GTPase involved in the synthesis of 18S rRNA.²² It has been reported that the RCL1–BMS1 complex plays a crucial role in processing yeast pre-rRNAs.²³ Moreover, our previous study demonstrated a significant increase in RCL1 expression in C. albicans upon treatment with RF59, a macrocyclic compound.¹⁷ These findings suggest that RCL1 contributes to the improved the physiological status of C. albicans. However, the expression 1 changes of RCL in C. albicans have not been thoroughly investigated. In this study, we conducted RCL1 sequencing on 16 C. albicans isolates, revealing mutation sites in the RCL1 gene in ITR-resistant C. albicans compared to the standard strain. Specifically, among the five successfully sequenced ITR-resistant strains, three missense mutations were identified in the RCL1 sequence: C6A, G10A, and A11T. Reverse sequencing of 15 successfully sequenced ITR-resistant strains unveiled one synonymous mutation (C1T) and four missense mutations (C1T, A3T, A7G, and T8G) in the RCL1 sequencing. These results suggest that reverse sequencing detected more mutations than forward sequencing, potentially due to base mutations.²⁴ The full length of the RCL1 gene in our study was 77 bp, which may not be sufficient, increasing the likelihood of base mutations.²⁵ Additionally, mutations were only identified in ITR-resistant C. albicans, indicating a potential association between RCL1 mutations and ITR resistance. However, further studies are needed to validate the correlation between RCL1 mutations and ITR resistance.

Stress responses involve various mechanisms that enable microorganisms to survive in adverse and fluctuating environments.⁹ Microorganisms typically possess intricate “circuits” capable of responding to diverse stresses, exemplified by the response of Candida to antifungal drugs.²⁶ There is a growing body of evidence indicating that C. albicans exhibits different cell morphologies under varying conditions, including “white, opaque, gray”, “yeast state”, and “mycelium state”. These cell morphologies could be converted to each other, which endows C. albicans with diverse pathogenic abilities.²⁷ Taken together, we speculated that stress may exert an essential role in the drug resistance of C. albicans.

Additionally, stress often leads to the reorganization of the nuclear structure. The nucleolus serves as a stress sensor, coordinating the stress response.²⁸ Recently, accumulating evidence has demonstrated that damage to the integrity of the nucleolar system and disruption of ribosome generation can activate the nucleolar stress signaling pathway, ultimately compromising its role in cell growth, apoptosis, and DNA regulation.²⁹ The viral stress gene RCL1 encoding a nucleolar protein is essential for 18S rRNA in ribosome biosynthesis.^15,17 Moreover, Enjalbert et al verified that RCL1 is the main regulatory factor in C. albicans under various stress conditions, including oxidation and heavy metal stress.¹⁸ As C. albicans is well known for its characteristic dimorphism, existing as both a yeast and mycelium form, different environmental stimuli, such as a neutral pH value, 25°C and 5% CO₂ can induce changes in its dimorphism of C. albicans.^30,31 The transition from yeast to mycelia is a crucial virulence factor in C. albicans.³² However, the impact of different culture environments (temperature, CO₂ concentration, and pH value) on the expression of RCL1 in C. albicans remains unclear.

In this study, we investigated the expression of RCL1 in C. albicans under normal conditions (37°C, 0.03% CO₂, pH 4.0). We observed higher RCL1 expression in ITR-resistant C. albicans compared to sensitive C. albicans. The decrease in temperature to 25°C and the increase in CO₂ concentration to 5% CO₂ both led to similar RCL1 expression levels in both sensitive and resistant strains of C. albicans. Notably, an up-regulated pH value (pH = 7.0) resulted in increased RCL1 expression specifically in the ITR-resistant C. albicans compared to the sensitive C. albicans. These findings suggest that RCL1 expression is inherently higher in resistant C. albicans under normal conditions. Interestingly, alterations in temperature and CO₂ concentration effectively eliminated the differences in RCL1 expression levels between sensitive and resistant C. albicans.

Subsequent experiments revealed that, when compared to 37°C, a temperature of 25°C led to a significant increase in the RCL1 level in sensitive C. albicans, while concurrently decreasing it in ITR-resistant C. albicans. Conversely, exposure to 5% CO₂ showed no significant effect on RCL1 content in sensitive C. albicans, in contrast to the notable reduction observed in ITR-resistant strains, when compared to 0.03% CO₂. Furthermore, an increase in pH was found to up-regulate RCL1 expression in both sensitive and resistant C. albicans.

This study had certain limitations. A significant constraint is the absence of cross-condition testing, which is essential for establishing the effects of stressors (temperature, CO₂, and pH) on the expression of RCL1. The findings indicate a notable increase in RCL1 expression in ITR-resistant C. albicans was notably higher compared to sensitive strains, suggesting a potential involvement of RCL1 in ITR resistance. However, this conclusion warrants further exploration in future studies. Furthermore, additional experiments will be conducted to investigate whether various conditions (temperature, CO₂ concentration, and pH) regulate the morphological transformation and pathogenicity of C. albicans by influencing RCL1 expression.

Conclusion

In summary, this study unveils that ITR-resistant C. albicans harbors several mutation sites in the RCL1 gene. Forward sequencing identified three missense mutations (C6A, G10A, and A11T) in the five strains of ITR-resistant C. albicans, while reverse sequencing revealed one synonym mutation (C1T) and four missense mutations (C1T, A3T, A7G, and T8G) among the 15 strains of ITR-resistant C. albicans. Different stress conditions (25°C, 5% CO₂, pH = 7.0) exert distinct regulatory effects on RCL1 expression in both sensitive and resistant C. albicans strains. These findings suggest that C. albicans may activate its stress response through the RCL1 protein, providing a novel perspective for understanding the molecular targets of potential antifungal drugs for treating patients with invasive candidiasis.