INSTITUTE OF PHYSIOLOGY CAS - Transporters of pathogenic yeasts as targets for new antifungal drugs

Some of the transporters existing in the membranes of pathogenic Candida species differ by its structure and activity from those of the host organism. These transporters thus may serve as targets for the development of new antifungal drugs that will affect yeast cells but not cells of the host.

Among more than 1400 known human pathogens, fungi and yeasts represent the second largest group - 22 % (after bacteria 38 %). Candidiasis is the most frequent fungal infection. Candida species are part of the normal human microflora, however these human commensals cause life-threatening infections in immunocompromised patients (due to AIDS, organ transplantations or cancer treatment) and can even kill human hosts in the case of treatment failure.

In 1929, Sir Alexander Fleming revealed to mankind a way of fighting against bacterial infections by the discovery of penicillin. Since that time, a wide collection of various antibiotics killing bacteria, including broad-spectrum ones, has been developed. The development of antifungal drugs has to overcome the obstacles of close evolutionary relationship between fungal and human cells. The animal (human) and yeast cells are eukaryotic, very similar to each other and simultaneously different from prokaryotic bacteria. Because of this evolutionary relationship, it is not easy to create drugs that would not damage the human host cells together with the fugal pathogen. In addition, in the course of evolution, the fungal cells have acquired a number of ways that enabled them to survive in adverse conditions and that help them now to gain resistance to antifungal agents.

Distribution of isolated Candida species from patients with candidemia (Pfaller et al., 2012):

C. albicans	42.1 %
C. glabrata	26.7 %
C. parapsilosis	15.9 %
C. tropicalis	8.7 %
C. krusei	3.4 %
C. lusitaniae	1.1 %
C. dubliniensis	0.9 %
C. guilliermondii	0.4 %
Other	0.8 %

Candida albicans is able to undergo reversible morphological transitions between yeast (left) and filamentous forms (right) in response to external stimuli – temperature, pH, presence of nutrients or human hormones etc. Although yeast cells are disseminated more effectively, filamentous forms are better adapted to penetrate and damage host tissue.

Two directions are followed in our search for new antifungal drugs:

We study the potassium transporters in Candida species, which differ from their human counterparts, and therefore represent a potential target of new antifungal drugs.

Potassium cations are crucial for many physiological processes (e.g. for negative charges compensations in macromolecules, for the regulation of intracellular pH, membrane potential or cell volume). Pathogenic yeast cells compete for K⁺ with the cells of their host; therefore Candida evolved efficient potassium transporters which belong to three groups: TRK, HAK and ACU. Whereas C. albicans has all three types of K⁺uptake systems (Husekova et al., 2016; Elicharova et al., 2016), C. glabrata genome contains only one gene encoding a TRK-type transporter (Llopis-Torregrosa et al., 2016). To characterize the transport properties of these systems, we use a combination of two approaches:

Deletion of genes encoding potassium transporters in Candida species
Heterologous expression of Candida K⁺ transporters in a S. cerevisiae strain lacking its own K⁺ transporters (BYT12, trk1∆ trk2∆)

Presence of transporters CaTrk1 from C. albicans and CgTrk1 from C. glabrata improves the cell growth of S. cerevisiae BYT12 cells in the presence of low concentrations of K⁺.

We would like to contribute to solving the problem of drug resistance, which has become an important medical issue not only for many infectious diseases of immunocompromised patients, but it complicates also the cancer treatment. One of the reasons of multidrug resistance is an active expulsion of drugs from the cell by membrane transporters – MDR pumps. Also the cells of pathogenic yeasts are able to effectively expel drugs that could damage them. Development of new drugs able to inhibit activity of these MDR transporters would strengthen the effect of conventional drugs. Such inhibitors when administered together with conventional drugs will help to combat bacterial and fungal infections or malignancies. Ultimately, the combination therapies may lead to the usage of drugs at lower concentrations that limit their negative side effects.

Multidrug resistance mediated by membrane transporters is the main defense system of pathogenic microorganisms and cancer cells. Transporters actively remove antimicrobial or chemotherapeutic agents from the cells and thus prevent their intracellular accumulation in toxic levels. According to a source of energy, which is used to transport the substrate, the MDR transporters are divided into two main categories:

Primary active transporters, so-called pumps, that get their energy from hydrolysis of ATP and belong to the family of ABC proteins that contain the ATP-binding cassette;
Secondary active transporters that utilize the electrochemical gradient of protons or sodium ions across the membrane. These proteins belong to the PMF (proton motive force) family and transport drug molecules out of cells and simultaneously H⁺ (Na⁺) into cells.

Candida species have both types of transporters involved in multidrug resistance. Their substrate specificity often overlaps, e.g. ABC proteins Cdr1 and Cdr2, as well as PMF transporter Mdr1, provide fluconazole resistance to C. albicans.

Kvasinky rodu Candida mají ve svých membránách oba druhy transportérů zodpovědné za mnohačetnou lékovou rezistenci. Jejich substrátová specifita se často překrývá, např. ABC proteiny Cdr1 a Cdr2 poskytují C. albicans odolnost k flukonazolu stejně jako PMF transportér Mdr1.

C. glabrata resistance to fluconazole is caused by MDR pumps Cdr1 and Cdr2. The absence of gene for Cdr1 pump (cdr1∆) in C. glabrata inhibits the cell growth in the presence of higher concentrations of fluconazole (left). Insertion of the CgCDR1 gene into S. cerevisiae AD1-8 strain, that lacks its own MDR pumps, confers the resistance to fluconazole (right).