Reference Laboratory and Research in Mycology

Reference Laboratory and Research in Mycology
National Center of Microbiology. Carlos III Health Institute

Óscar Zaragoza Hernández, Irene García Barbazán, Nuria Trevijano Contador, Ainhize Curiel Iglesias

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From left to right: Nuria Trevijano Contador, Ainhize Curiel Iglesias,
Irene Garcia Barbazán, Óscar Zaragoza Hernández

Yeasts are microscopic, single-celled fungi that divide by budding, fission, or septation. There are many species of yeast, some of them of great interest as a research model or in biotechnology. The clearest example is Saccharomyces cerevisiae, since due to its role in the production of bread and wine, it has become for decades one of the organisms most used in research to understand multiple biological processes, such as metabolism, cell cycle and response to stress. Another example is fission yeast. Schizosaccharomyces pombe, which due to its peculiar way of dividing, has been one of the models used to discover the molecular bases that regulate the cell cycle in eukaryotes. Other yeasts, such as Pichia pastoris O Yarrowia lypolytica they are considered small factories to produce heterologous proteins and are widely used in biotechnology.

But the importance of yeasts goes beyond biotechnology and food production, as some species can cause disease in humans. In particular, there are two genera of clinical interest, which are Candida and Cryptococcus. These two yeasts differ widely in evolution, being Candida ascomycete yeasts and Cryptococcus basidiomycetes. In addition, from a clinical point of view, they also differ very significantly in the mechanisms of virulence, the route of entry in humans, and the clinical picture they cause. However, both have in common that they are capable of causing serious and disseminated diseases in immunosuppressed patients, which is why they are considered opportunistic pathogenic yeasts.

The vast majority of yeasts of the genus Candida they are human commensals, being found in the microbiota of the intestine, mucous membranes and skin. The most important species is C. albicans, which can cause mucosal or disseminated disease. C. albicans It is one of the most frequent causative agents of vaginitis, which affects millions of women in the world. It also causes oropharyngeal infections in HIV+ patients. The most serious clinical picture are those diseases in which the yeasts spread throughout the body and invade different organs, the kidneys being one of the sites where they are found. C. albicans preferential accommodation. Disseminated diseases are closely associated with immunosuppressed patients, especially those who undergo intestinal surgery and require immunosuppressive treatments. In these cases, the most frequent origin of the infection is the yeasts that are part of the intestinal microbiota, and that are capable of passing through the tissues and spreading through the blood. These diseases have a high mortality rate and are a very common cause of nosocomial infection. Other species of the genus Candida that are disease-causing agents are C. glabrata, C. parapsilosis, y C. tropicalis. In addition, in recent years, there is another species, C. ear, which has emerged as a worrying pathogen, since it causes hospital outbreaks that are difficult to control and eliminate.

Diseases caused by Cryptococcus are different from those caused by Candida, and have a very characteristic clinical picture. The yeasts of the genus Cryptococcus they have a very typical phenotypic characteristic, which is a polysaccharide capsule that surrounds the cell and is not found in other yeasts. This capsule is easily visible after suspension of the yeasts in India ink, appearing as a white halo around the cell. The capsule is necessary for virulence, as it has been reported to have multiple deleterious effects on the immune response.

The main pathogenic species of this genus is Cryptococcus neoformans. This species is found in multiple niches in the environment, and it is believed that they are acquired by inhalation, the lung being the first organ they colonize. Although yeasts are normally controlled and eliminated by the immune system of the lung, there is evidence that C. neoformans it can also remain in this organ for a long time, causing asymptomatic chronic infections. The most serious clinical picture occurs when the yeasts are capable of spreading from the lung to other organs, the brain being the organ where they preferentially lodge, and which occurs in patients with a low CD4+ lymphocyte count, especially HIV+. Therefore, the most characteristic clinical picture is meningoencephalitis, which has a very high associated mortality (around 20%). diseases caused by C. neoformans They have a high incidence, especially in developing regions, such as Sub-Saharan Africa, where they cause more than one hundred thousand deaths annually.

Treatment of invasive fungal diseases remains a challenge. There are three families of antifungals used clinically to treat these diseases: polyenes (amphotericin B), azoles (fluconazole, itraconazole, posaconazole, voriconazole, and isavuconazole), and candins (caspofungin, micafungin, anidulafungin, and rezafungin). These antifungals have several limitations. Many have side effects, such as amphotericin and azoles. In other cases, the spectrum of action is limited. The most active antifungal against most fungi is amphotericin, the rest being active against a limited number of yeasts. For example, azoles have reduced activity against C. glabrata and C. the ear. In the case of candins, they have limited activity against C. parapsilosis and they are not active against C. parapsilosis. Another problem associated with the administration of antifungals is the selection of resistance. These drugs are frequently administered as prophylactic or empiric treatment, that is, without being certain that a proven infection exists. Thus, the massive use of antifungals in recent decades has produced a selection of species that have reduced sensitivity or intrinsic resistance to certain antifungals, as is the case of C. glabrata, C. parapsilosis O C. ear. Finally, the price of most antifungals is high, which limits their administration. Therefore, the development of new therapeutic strategies is necessary to reduce the impact of these diseases in our society.

LINES OF INTEREST AND RESEARCH ACTVITY

The group is interested in elucidating the mechanisms involved in the development of pathogenic yeast diseases, mainly C. neoformans. In addition, in recent years it has started other lines whose objective is to identify new therapies based on the repositioning of drugs not subject to patents (off-patent drug repurposing).

Adaptation mechanisms of Cryptococcus neoformans to the host. Role of titan cells. Among all pathogenic fungi, C. neoformans offers a unique research model. In addition to the fact that it is a yeast that causes a large number of deaths per year, C. neoformans It has multiple mechanisms of adaptation to the host that make it able to evade the immune response very effectively. The polysaccharide capsule protects the yeasts against stress factors induced by the immune system, such as free radicals or antimicrobial peptides. But the polysaccharide in the capsule can also act as a virulence factor and interfere with the immune response. Another characteristic of this yeast is its ability to survive within phagocytic cells, thus behaving as a facultative intracellular pathogen.

Our group is interested in another of the mechanisms that allow C. neofomans survive in the host, which is the formation of so-called titan cells. These cells are formed by massive growth of both the capsule and the cell body (delimited by the wall). The normal size of yeast under laboratory conditions is 5-7 microns. However, in animal models, we have found that there is a population of cells that can reach more than 40 microns in diameter.

To understand the role of these cells in the virulence of C. neoformans, we are carrying out different approaches:

• in vivo studies. To find out the host factors that regulate the formation of titan cells, we are using different animal models that have a different polarization in the immune response (KO mice, administration of cytokines, blocking antibodies, etc). Our results indicate that under conditions in which the main immune response is of the Th2 type, there is a higher proportion of titan cells in the lungs. These studies can help understand the susceptibility factors of certain patients to develop diseases caused by C. neoformans, but also help predict the effectiveness of antifungal treatments.
• Molecular mechanisms that regulate the formation of titan cells. Recently, our group has been able to describe laboratory media (nutrient limitation with serum and CO2) in which C. neoformans increases in size significantly (diameter about 30 microns). Although these cells do not have the size observed in vivo, we have called them "titan-like" and we consider that they offer an optimal model to investigate the processes that trigger this morphological process. We have carried out several approaches, such as RNAseq, which have allowed us to identify new elements that regulate this process. Recently, using the drug repositioning strategy (see below), we have also identified drugs that inhibit cell growth in C. neoformans, which will allow us, on the one hand, to identify new regulatory elements after identifying the target of these drugs, and also to evaluate their therapeutic potential.

Mechanism of actionohn of antifúngicos. The group is also interested in improving antifungal treatments. Of the families described above, the antifungal that has the greatest fungicidal activity is Amphotericin B (AmB), although it is also one of those with the greatest associated toxicity. Classically, the mechanism of action of this antifungal has been based on its binding to ergosterol, the main sterol of the fungal membrane, favoring the formation of pores and cell death. Recent studies have shown that there are also other mechanisms by which AmB exerts its function, such as ergosterol sequestration and induction of oxidative stress. As for the first, AmB binds to ergosterol, separating it from the membrane and causing cell damage. Regarding oxidative stress, AmB favors the increase of reactive oxygen species, leading to alterations in the structure of proteins, lipids and nucleic acids, and causing cell damage. Therefore, AmB causes damage both at the membrane level and at the intracellular level, so it is necessary to continue investigating the role of free radicals in the action of this molecule in order to design more effective therapies with less toxicity.

Drug repositioning. In an attempt to solve the problems presented by the aforementioned drugs, in recent years work has been under way on strategies based on the repositioning of non-patent drugs (“off-patent drug repurposing”). This is intended to search for new activities of already marketed drugs that can improve current therapies and reduce the cost and toxicity of treatment. Drugs, such as the antibiotic erythromycin, have been found to increase the effectiveness of AmB, which opens the door to proposing new therapeutic strategies based on new combinations of drugs.

In parallel, we have used this strategy to find drugs that are active against emerging pathogenic fungi, as is the case with Candida auris, which has become a yeast of special concern for being the cause of multiple hospital outbreaks worldwide in recent years.

CONTRIBUTIONS AND SELECTED PUBLICATIONS

1. Rossi SA, de Oliveira HC, Agreda-Mellon D, Lucio J, Mendes-Giannini MJS, García-Cambero JP, Zaragoza O. Identification of Off-Patent Drugs That Show Synergism with Amphotericin B or That Present Antifungal Action against Cryptococcus neoformans and Candida spp. Antimicrob Agents Chemother. 2020; 64(4):e01921-19.
2. de Oliveira HC, Monteiro MC, Rossi SA, Pemán J, Ruiz-Gaitán A, Mendes-Giannini MJS, Mellado E, Zaragoza O. Identification of Off-Patent Compounds That Present Antifungal Activity Against the Emerging Fungal Pathogen Candida auris. Front Cell Infect Microbiol. 2019 2;9:83.
3. Trevijano-Contador N, de Oliveira HC, García- Rhodes R, Rossi SA, Llorente I, Zaballos Á, Janbon G, Ariño J, Zaragoza O. Cryptococcus neoformans can form titan-like cells in vitro in response to multiple signals. PLoS Pathog. 2018; 14(5):e1007007.
4. Rueda C, Puig-Asensio M, Guinea J, Almirante B, Cuenca-Estrella M, Zaragoza O; CANDIPOP Project from GEIH-GEMICOMED (SEIMC) and REIPI. Evaluation of the possible influence of trailing and paradoxical effects on the clinical outcome of patients with candidemia. Clin Microbiol Infect. 2017; 23(1):49.e1-49.e8.
5. Mesa-Arango AC, Rueda C, Román E, Quintin J, Terrohn MC, Luque D, Netea MG, Pla J, Zaragoza O. Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host. Antimicrob Agents Chemother. 2016; 60(4):2326-35.
6. García-Barbazán I, Trevijano-Contador N, Rueda C, by AndrIt'ss B, PIt'stav floorárez R, Herrero-Fernández I, Gaspar ML, Zaragoza O. The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2-type responses. Cell Microbiol. 2016; 18(1):111-24.
7. Trevijano-Contador N, Herrero-Fernández I, García- Barbazán I, Scorzoni L, Rueda C, Rossi SA, García-Rodas R, Zaragoza O. Cryptococcus neoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleria mellonella. Virulence. 2015;6(1):66-74.
8. Mesa-Arango AC, Trevijano-Contador N, Román E, Sánchez-Fresneda R, Casas C, Herrero E, Argüthem JC, Plan J, Cuenca-Estrella M, Zaragoza O. The production of reactive oxygen species is a universal action mechanism of Amphotericin B against pathogenic yeasts and contributes to the fungicidal effect of this drug. Antimicrob Agents Chemother. 2014; 58(11):6627-38.
9. García-Rhodes R , Lamb RJ , Trevijano-Counter N , Janbon G , Moyrand F , Casadevall A , Zaragoza O . Capsule growth in Cryptococcus neoformans is coordinated with cell cycle progression. mBio. 2014; 5(3):e00945-14.
10. Wheel C, Cuenca-Estrella M, Zaragoza O. Paradoxical growth of Candida albicans in the presence of caspofungin is associated with multiple cell wall rearrangements and decreased virulence. Antimicrob Agents Chemother. 2014;58(2):1071-83.
11. Zaragoza O, García-Rodas R, Nosanchuk JD, Cuenca-Estrella M, Rodríguez- Tudela JL, Casadevall A. Fungal cell gigantism during mammalian infection. PLoS Pathog. 2010. 6(6):e1000945.