Cryptococcosis generally occurs as an opportunistic infection in immunocompromised hosts. Patients with acquired immune deficiency syndrome (AIDS) or other immune deficiencies are especially at risk (Pappas et al., 2001; Singh and Forrest, 2009; DiNardo et al., 2015; Henao-Martínez and Beckham, 2015). Cryptococcus, as the causative agent of cryptococcosis, is found in various environmental sources, including contaminated milk, birds droppings, and soil. Pneumonia, meningitis, skin, soft tissue, bone, and joint infections (Cho et al., 2021) are typical cryptococcal symptoms; however, the infection may spread to other organs via the lymphatic system or the circulatory system.

Globally, cryptococcosis is one of the deadliest invasive mycoses due to its high morbidity and death rate (Perfect et al., 2010). About 200,000 individuals a year are killed by pathogenic species of Cryptococcus (Fonseca et al., 2019). Standard therapy is a typically an aggressive intravenous injection of an antifungal drug, followed by suppressive treatment taken orally for a period that varies depending on the patient’s condition (Cho et al., 2021). Amphotericin B (AmB) (typically lipid formulations) plus 5-fluorocytosine (5FC) for induction treatment for 2 weeks, followed by fluconazole as suppressive therapy, is the recommended treatment (Perfect et al., 2010; Cho et al., 2021). However, the high dosages needed for these infections, the severe toxicities of AmB have been a limiting factor in its use (Thakur and Revankar, 2011). Furthermore, in regions with higher disease load and death rates, the availability of 5-FC is limited (Maziarz and Perfect, 2016). Cryptococcosis remains a challenging management issue. Combination therapy with drug repositioning has been seen as a potential option due to the scarcity of novel antifungal medicines.

Everolimus (EVL), an analog of the naturally occurring macrolide rapamycin, is an orally bioactive inhibitor of the mammalian target of rapamycin (mTOR) serine/threonine kinase signal transduction pathway, which controls proliferation, cell growth, and survival. Its ability to directly inhibit tumor proliferation and cell growth and indirectly impede angiogenesis has garnered much interest as an anticancer drug (Hasskarl, 2018). TOR was first discovered in the yeast Saccharomyces cerevisiae, and subsequent research has shown that it is present in many other eukaryotic organisms, including plants, worms, flies, fungi, humans, and mammals (Crespo and Hall, 2002). Immunosuppressive medicines increase the likelihood of developing invasive fungal infections, although they also have antifungal activity. Rapamycin has shown intrinsic antifungal activity against Candida albicans, Microsporum gypseum, Trichophyton granulosum (Vézina et al., 1975), Aspergillus spp.(High and Washburn, 1997), Fusarium fujikuroi(Teichert et al., 2006)and cryptococcus neoformans(Cruz et al., 1999). Moreover, Manish Thakur et al. demonstrated a synergistic effect of caspofungin with rapamycin against Glomeromycetes(Thakur and Revankar, 2011). In addtion, Patrick Schwarz et al. observed that rapamycin may improve the efficacy of isavuconazole against Aspergillus species in vitro (Schwarz and Dannaoui, 2020). Despite this, EVL’s effectiveness against Cryptococcus has only been partially studied by itself or in conjunction with other treatments.

The rapalog effect of EVL against clinical Cryptococcus isolates was validated in vitro using AmB and azoles. To further investigate the potential treatment-related changes in larval survival, we extended these experiments to evaluate the effects of combination EVL+AmB or azoles therapy on Galleria mellonella larvae infected with Cryptococcus spp.

Since its discovery, the TOR signaling pathway—of which TOR kinase is the primary component—has been the subject of many studies and has come to be regarded as a key regulator of cell proliferation in eukaryotes (Crespo and Hall, 2002). Rapamycin, a classical allosteric TOR inhibitor, was first found in screening for new antifungal drugs and has since shown great medicinal promise. Researchers have shown that rapamycin has strong antifungal effects against various fungi, including Penicillium spp., Fusarium spp., Aspergillus spp., Cryptococcus spp., Candida spp., Dermatophytes spp.(Rohde and Cardenas, 2004).However, rapamycin’s much more potent immunosuppressive properties prevented it from being employed as an antifungal agent (Gao et al., 2016).

Initially created to treat cancer, EVL was an orally active, potent TOR kinase inhibitor that directly inhibited tumor cell proliferation and growth and indirectly blocked angiogenesis (Hasskarl, 2018). We tested EVL’s antifungal potential against Cryptococcus spp. in vitro, alone and in combination with other antifungal drugs. The results discovered that EVL alone was active against all tested strains, which could be explained by the fact that FKBP protein (FK-506 Binding Proteins) and TOR protein are ubiquitous in eukaryotes, and rapalogs could bind to the FKBP12, and then, this complex inhibits mTOR to regulate the response of fungi to the external environment, and kills microorganism (Stan et al., 1994; Hasskarl, 2018). Furthermore, synergistic activities between EVL and AmB(88.9%), FLU(61.1%), ITR(55.6%), POS(50%), VOR(33.3%) were observed in Cryptococcus spp. The effective working ranges of EVL were 0.0625-0.5 μg/ml against Cryptococcus spp.; no antagonism was observed. The synergy between EVL and FLU was seen in as many as 61.1% of Cryptococcus strains, suggesting that EVL might increase the in vitro susceptibility of FLU-inactive Cryptococcus strains even when their MICs against FLU were high (0.5-32 μg/ml).

Rapamycin has been shown to have synergistic interactions with POS (40%), ITR (50%), and AmB (70%) against Mucorales (formerly called zygomycetes) by the broth microdilution checkerboard procedure (Dannaoui et al., 2009). Additionally, it has been reported that Mucorales exhibits antagonism of rapamycin/ITR, and no substantial agonism of rapamycin/POS (Dannaoui et al., 2009; Narreddy et al., 2010). In contrast, no antagonism was identified between EVL and POS or ITR in the current investigation, and EVL increased the antifungal activity of POS (50%) and ITR (55.6%). The different effect of EVL and rapamycin on TOR and antifungals, as well as the differential responsiveness of the tested species to these drugs, may account for these discrepancies.

Since G. mellonella larvae exhibit immunological responses comparable to those of mammals without the accompanying ethical concerns with advantages such as being easy to manipulate and inexpensive, these larvae have recently emerged as an appropriate in vivo model for the preclinical investigation of the antifungal effects of novel medicines (Vilcinskas, 2011; Favre-Godal et al., 2014; Liu et al., 2017). To further verify the interaction between EVL and azoles, EVL and AmB detected in our study, we tested the in vivo effect of this combination against one of the isolates of C.neoformans (G7) with the G. mellonella model. We found that EVL-POS, EVL-FLU, and ELV-ITR combination treatment were associated with significant improvements in larval survival compared to POS, FLU, or ITR treatment in isolation, further confirming the synergistic benefits of EVL-POS, ELV-FLU, and ELV-ITR combination treatment when used to treat infections caused by C.neoformans spp.

Ergosterol biosynthesis in fungal cell membranes was inhibited by azole antifungal compounds by inhibiting lanosterol 14α-demethylase activity (Keady and Thacker, 2005; Singal and Khanna, 2011; Jo Siu et al., 2013). Micropores in the cell membrane are formed, and the membrane’s permeability to monovalent and divalent cations is increased when ergosterol on the fungal cell membrane binds with AmB to form the sterol-polyene complex, killing fungus.

As an oral mTOR protein kinase inhibitor, EVL might control cell growth to kill fungi and potentiate the activities of azoles by inhibiting TOR signaling which could affect the amino acid permeases regulation, ribosome biogenesis, protein synthesis initiation, actin cytoskeleton organization, autophagy inhibition, control of phosphatases by TOR, and transcriptional control of nutrient metabolism(Crespo and Hall, 2002).

In conclusion, the current investigation expands prior results in the combination interactions between conventional antifungals and TOR inhibitors. Against Cryptococcus spp., EVL may augment the antifungal activity of AmB, POS, FLU, ITR, and VOR in vitro. In addition, for some individuals with clinical cancer, combining EVL with AmB or azoles may be a safe alternative for treating Cryptococcus infections. However, further research is required to clarify the underlying process and identify viable, safe therapeutic applications.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

PL conceived and designed the study. PL performed all the experiments, analyzed the data and wrote the manuscript. JS and QL provided general guidance and revised the manuscript. All authors contributed to the article and approved the submitted version.