Research Progress on Commonly Used Antifungal Drugs and Their Resistance Mechanisms Against Candida Albicans Infections

Candida albicans is the primary cause of candidiasis, an acute, subacute, or chronic infection that poses a severe threat to human health due to its complex and varying clinical symptoms. Currently, the antifungal drugs commonly used in clinical practice are limited in variety, with narrow antibacterial spectrum and high drug resistance, which is one of the major reasons for the failure of clinical treatment and the high mortality rate of candidemia. The commonly used systemic antifungal drugs mainly include triazoles, polyenes, and echinocandins.

The Current Status of Commonly Used Anti-Candida albicans Drugs

1.Polyene Drugs

Polyene compounds are organic molecules belonging to the macrolide class. This class of drugs exhibits strong antifungal activity and a wide antifungal spectrum. Among them, nystatin and amphotericin B (AmB) are representative drugs commonly used in clinical practice. Nystatin has a significant killing effect on Candida albicans, but its poor absorption and significant adverse reactions limit its clinical use. AmB is the most effective drug with the broadest antifungal spectrum for deep fungal infections, but its nephrotoxicity and significant infusion-related adverse reactions restrict its clinical application. Additionally, the sensitivity of Candida albicans to AmB and nystatin significantly decreases after biofilm formation. In recent years, the development of AmB lipid-containing formulations has mitigated the nephrotoxicity of AmB. In China, the main lipid-containing formulation used is L-AmB, which has similar antibacterial spectrum, antibacterial activity, and clinical efficacy as AmB, but with lower nephrotoxicity. However, its high price means that L-AmB is currently only approved for patients who cannot tolerate the toxic reactions of AmB, experience severe toxicity related to intravenous administration, or do not respond to AmB treatment.

2.Triazole Drugs

Triazole antifungal drugs are currently the most widely used class of antifungal medications. Among them, fluconazole (FCA) is the preferred drug for early clinical empirical treatment of Candida infections, especially as a prophylactic agent for high-risk populations, making it remain at a sub-therapeutic level in the human body for a long time, leading to increased drug resistance. Voriconazole (VRC), as a new generation of triazole antifungal drugs, has the advantages of higher antibacterial activity, wider antibacterial spectrum, and higher bioavailability, and it has strong activity against even fluconazole-resistant strains. According to the CHIF-NET 2010-2014 multi-center study on invasive yeasts in China, 94% of Candida albicans strains were most sensitive to voriconazole, followed by fluconazole. With prolonged drug misuse and unnecessary drug exposure, the resistance of Candida albicans to azole drugs is increasing, limiting the treatment options for patients with azole-resistant Candida infections, posing an increasingly serious challenge for clinicians and patients worldwide.

3.Echinocandin Drugs

Currently, the echinocandin drugs available on the market include caspofungin, micafungin, and anidulafungin. Caspofungin has good activity against Candida albicans strains resistant to fluconazole and itraconazole. Micafungin has good inhibitory activity against Candida species, azole-resistant strains, and amphotericin B-resistant strains, and it has lower toxicity when treating severe Candida infections. Anidulafungin has a larger distribution volume and a wider antibacterial spectrum, with significantly higher antibacterial activity against Candida albicans than amphotericin B, itraconazole, and fluconazole. It also has a lower minimum inhibitory concentration against Candida species compared to fluconazole and caspofungin. With widespread clinical use, Candida albicans has developed varying degrees of resistance to these drugs. However, due to the lack of significant cross-resistance between echinocandins and azole drugs, echinocandins are often used clinically in combination with triazole or polyene drugs to treat invasive Candida infections.

Research Progress on Drug Resistance Mechanisms of Candida albicans

Currently, the drug resistance mechanisms identified in Candida albicans mainly include alterations in drug targets, overexpression of drug efflux pumps, changes in metabolic pathways, and the activation of adaptive stress responses.

1. Resistance Mechanisms to Azole Drugs

① Alteration of Drug Targets. Mutations or overexpression of the azole target enzyme gene ERG11 in Candida albicans can maintain the activity of the target enzyme and produce resistance. Studies have shown that 140 missense mutations have been identified in the ERG11 gene, and multiple mutation sites such as Y123F, K143R, F449V, and G464S have been confirmed to be related to the resistance of Candida albicans to fluconazole. Point mutations in ERG11, especially those occurring in the three "hot spot" regions between amino acids 105-165, 266-287, and 405-488, can reduce the sensitivity of Candida to azole drugs. The overexpression of ERG11 in Candida albicans resistant strains is mainly caused by gain-of-function mutations in Upc2p, such as G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Research reports have indicated that ERG3, an upstream gene of ERG11, can lead to resistance to azole drugs in Candida albicans when its function is inactivated.

② Overexpression of Drug Efflux Pumps. Candida albicans possesses two types of drug efflux pumps: ABC transporters (CaCdr1 and CaCdr2) and MFS transporters (CaMdr1). Mutations in CaTAC1, which regulates the expression of CaCdr1 and CaCdr2, and CaMRR1, which regulates the expression of CaMDR1, lead to the overexpression of drug efflux pumps and, consequently, resistance to azole drugs in Candida albicans. ABC transporters utilize ATP binding to nucleotide-binding domains to acquire energy for drug efflux, with CaCdr1 being a major determinant of azole resistance. Deletion of Cdr1 in clinical isolates can reduce azole resistance by 4 to 8 times. Recent studies have found that the deletion of the transporter CaCdr6/Roa1 results in excessive activation of the TOR signaling pathway, inhibiting drug influx, and thus leading to resistance of Candida albicans to azole drugs. Additionally, the ABC transporter CaMlt1 regulates the resistance of Candida albicans to azole drugs through a vacuolar uptake mechanism. Furthermore, the mediator complex plays a crucial role in Tac1-mediated azole resistance, and the deletion of the mediator tail module in Tac1 gain-of-function mutants reduces CDR1 transcription, thereby increasing sensitivity to fluconazole. MFS transporters rely on electrochemical gradient diffusion for drug efflux, and among the 95 MFS transporters in Candida albicans, only Mdr1 participates in resistance to azole drugs. The Swi/Snf chromatin remodeling complex in Candida albicans is considered a major co-activator of Mrr1, and in functional strains with acquired Mrr1, deletion of the catalytic subunit SNF2 of the Swi/Snf complex leads to a sharp reduction in Mdr1 activation and resistance to fluconazole.

③ Regulation of Stress Responses. Heat shock proteins (Hsps) can regulate the resistance of Candida albicans to azole drugs through the calcium-calcineurin, MAPK, and Ras1-Camp-PKA signaling pathways. Studies have found that Hsp90 substrate proteins and Hsp90 post-translational modifications can act as upstream regulators to alleviate drug resistance. Sterol C-22 desaturase EGR5 and phosphatidylinositol-4 kinase (PI4K) STT4 are involved in regulating Hsp90. Additionally, metal ion stress can affect the sensitivity of Candida albicans to azole drugs. Research has reported that iron deficiency can lead to downregulation of CaERG11 expression and upregulation of ERG3, thus increasing the sensitivity of strains to azole drugs. Iron transporter proteins Ftr1, Ftr2, and Ftr11 participate in iron-mediated drug sensitivity. Furthermore, CaUpc2 and heat shock factor 1 (Hsf1) are also involved in iron-mediated drug sensitivity. Resistance to azole drugs in Candida albicans is also related to the formation of biofilms. Other studies have shown that during the production of Candida albicans biofilms, the expression levels of CaERG25 and CaERG11 increase, leading to resistance to azole drugs.

2.Resistance Mechanisms to Polyene Drugs

Candida species develop resistance to polyene drugs through mechanisms that reduce the binding affinity of the drug to the membrane or alterations in enzymes that consume ergosterol from the membrane. Research has shown that mutations in Candida albicans' ERG2, ERG3, ERG5, or ERG11 genes can lead to strain resistance to Amphotericin B (AmB). The adaptability and survival rate of AmB-resistant Candida isolates heavily depend on the expression and function of Hsp90. Therefore, pharmacological inhibition of Hsp90 in Candida albicans resistant strains can eliminate AmB resistance.

Candida albicans exhibits a compensatory antioxidant response upon exposure to AmB. Studies on AmB-resistant Candida suggest a significant reduction in the accumulation of reactive oxygen species (ROS), decreased protein carbonylation, reduced mitochondrial basal respiration, and a significant increase in catalase production. Additionally, transcriptional regulators such as Rlm and Smil regulate the production of β-glucan, a major component of the biofilm matrix in Candida, leading to drug resistance.

3.Resistance Mechanisms to Echinocandin Drugs

Glucan synthase is both the target and the site of resistance for echinocandin drugs. Mutations in the 1,3-β-glucan synthase encoding gene FKS1 can lead to Candida albicans resistance to echinocandin drugs. The main mutations associated with this are S645P and S645F. In susceptible strains, mutations in this region increase the strain's tolerance, leading to a significant reduction in the antibacterial effect of echinocandin drugs. In addition to FKS1, Candida albicans expresses FKS2 and FKS3, which are related to echinocandin resistance. Recent studies have found that FKS2 and FKS3 act as negative regulators of FKS1 expression. When FKS2 and FKS3 are deleted in Candida albicans, FKS1 expression is significantly upregulated, affecting its sensitivity to echinocandin drugs.

At the same time, testing of clinically isolated Candida albicans resistant strains has found that mutations in Ser645 occur most frequently. Currently, three mutations have been discovered: S645P, S645F, and S645Y. When mutations occur in this region in susceptible strains, the strains will develop resistance. The Candida albicans transcription factor Cas5 is a cytokine that affects fungal tolerance to echinocandins. Homozygous deletion of Cas5 can reduce FKS1-mediated echinocandin resistance. Furthermore, magnesium ion deficiency can cause mutations in the Candida albicans histidine kinase gene NIK1, inhibiting Hog1 activation and increasing the strain's sensitivity to caspofungin.

4.The Resistance Mechanism to 5-Fluorocytosine

Genomic studies on Candida albicans have confirmed that the resistance to 5-fluorocytosine (5-FC) is determined by recessive genes in specific strains, with mutations in the FUR1 gene being the most common. The FUR1 gene, which encodes UPRTase, undergoes mutations at the 301 locus, reducing the ability of 5-FC to convert into cytotoxic metabolites. Additionally, mutations in the FCA1 gene, which encodes cytosine deaminase, may lead to reduced drug uptake, both of which can affect the metabolism and intracellular accumulation of toxic compounds, resulting in drug resistance. Furthermore, functional defects in cytosine permease, cytosine deaminase, or alterations in thymidylate synthase activity may also contribute to drug resistance.

References

[1] Zhang Qianqian, Luo Chuanyu, Chen Jiaqi, et al. Current Status of Candida albicans Infection and Research Progress in Antifungal Drugs [J]. Chinese Journal of Mycology, 2021, 16(05): 356-360.

[2] Zhang Qianqian, Feng Xiaochuan, Zhang Kaixuan, et al. Research Progress on Drug Resistance Mechanisms of Candida albicans to Commonly Used Clinical Antifungal Drugs [J]. Chinese Journal of Mycology, 2022, 17(03): 251-254.

About the Author

Xiaomichong is a researcher in drug quality who has been committed to drug quality research and validation of drug analysis methods for a long time. Currently, she works for a large domestic pharmaceutical research and development company, engaging in drug inspection analysis and validation of analytical methods.