RESUMEN
In the last decades, 3-bromopyruvate (3BP) has been intensively studied as a promising anticancer and antimicrobial agent. The transport of this drug inside the cell is a critical step for its toxicity in cancer and microorganisms. The Cryptococcus neoformans is the most sensitive species of microorganisms toward 3BP. Its cells exhibit the highest uptake rate of 3BP among all tested fungal strains. In Saccharomyces cerevisiae cells, the Jen1 transporter was found to be responsible for 3BP sensitivity. The deletion of Jen1 resulted in the abolishment of 3BP mediated transport. We functionally characterized the Jen4 protein, a Jen1 homologue of C. neoformans, and its role in the phenotypic 3BP sensitivity. The deletion of the CNAG_04704 gene, which encodes Jen4, was found to impair the mediated transport of 3BP and decrease 3BP sensitivity. Further heterologous expression of Jen4 in the S. cerevisiae jen1Δ ady2Δ strain restored the mediated transport of 3BP. The application of a green fluorescent protein fusion tag with the CNAG_04704, revealed the Jen4 labeled on the plasma membrane. The identification of 3BP transporters in pathogen cells is of great importance for understanding the mechanisms of 3BP action and to anticipate the application of this compound as an antimicrobial drug.
Asunto(s)
Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Piruvatos/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Proteínas Fluorescentes Verdes , Transportadores de Ácidos Monocarboxílicos/genética , Saccharomyces cerevisiae/genéticaRESUMEN
We have functionally characterized the four Saccharomyces cerevisiae (Sc) Jen1 homologues of Debaryomyces hansenii (Dh) by heterologous expression in S. cerevisiae. Debaryomyces hansenii cells display mediated transport for the uptake of lactate, acetate, succinate and malate. DHJEN genes expression was detected by RT-PCR in all carbon sources assayed, namely lactate, succinate, citrate, glycerol and glucose. The heterologous expression in the S. cerevisiae W303-1A jen1Δ ady2Δ strain demonstrated that the D. hansenii JEN genes encode four carboxylate transporters. DH27 gene encodes an acetate transporter (Km 0.94 ± 0.17 mM; Vmax 0.43 ± 0.03 nmol s(-1) mg(-1)), DH17 encodes a malate transporter (Km 0.27 ± 0.04 mM; Vmax 0.11 ± 0.01 nmol s(-1) mg(-1)) and both DH18 and DH24 encode succinate transporters with the following kinetic parameters, respectively, Km 0.31 ± 0.06 mM; Vmax 0.83 ± 0.04 nmol s(-1) mg(-1)and Km 0.16 ± 0.02 mM; Vmax 0.19 ± 0.02 nmol s(-1) mg(-1). Surprisingly, no lactate transporter was found, although D. hansenii presents a mediated transport for this acid. This work advanced the current knowledge on yeast carboxylate transporters by characterizing four new plasma membrane transporters in D. hansenii.