Sequencing and heterologous expression in Saccharomyces cerevisiae of a Cryptococcus neoformans cDNA encoding a plasma membrane H+-ATPase

A cDNA containing an open reading frame encoding a putative plasma membrane H+-ATPase in the human pathogenic basidiomycetous yeast Cryptococcus neoformans was cloned and sequenced by means of PCR and cDNA library hybridization. The cloned cDNA is 3475 bp in length, containing a 2994 bp open reading frame encoding a polypeptide of 997 amino acids. As in the case of another basidiomycetous fungus (Uromyces fabae), the deduced amino acid sequence of CnPMA1 was found to be more homologous to those of P-type H+-ATPases from higher plants than to those from ascomycetous fungi. In order to prove the sequenced cDNA corresponds to a H+-ATPase, it was expressed in Saccharomyces cerevisiae and found to functionally replace its own H+-ATPase. Kinetic studies of CnPMA1 compared to ScPMA1 show differences in V(max) values and H+-pumping in reconstituted vesicles. The pH optimum and K(m) values are similar in both enzymes. (C) 2000 Elsevier Science B.V.This work was supported by the Spanish D.G.I.C.Y.T. (Grant PB97-0054 to F.P.) and F.I.S. (Grant 98/0008-02 to J.V.M.-S.). B.G. is a Fellow of Spanish Ministry of Education (Grant PN95-08930868).Peer Reviewe

Mefloquine and New Related Compounds Target the F(0) Complex of the F(0)F(1) H(+)-ATPase of Streptococcus pneumoniae

The activities of mefloquine (MFL) and related compounds against previously characterized Streptococcus pneumoniae strains carrying defined amino acid substitutions in the c subunit of the F(0)F(1) H(+)-ATPase were studied. In addition, a series of MFL-resistant (Mfl(r)) strains were isolated and characterized. A good correlation was observed between inhibition of growth and inhibition of the membrane-associated F(0)F(1) H(+)-ATPase activity. MFL was about 10-fold more active than optochin and about 200-fold more active than quinine in inhibiting both the growth and the ATPase activities of laboratory pneumococcal strain R6. Mutant strains were inhibited by the different compounds to different degrees, depending on their specific mutations in the c subunit. The resistant strains studied had point mutations that changed amino acid residues in either the c subunit or the a subunit of the F(0) complex. Changes in the c subunit were located in one of the two transmembrane α helices: residues M13, G14, G20, M23, and N24 of helix 1 and residues M44, G47, V48, A49, and V57 of helix 2. Changes in the a subunit were also found in either of the transmembrane α helices, helix 5 or 6: residue L186 of helix 5 and residues W206, F209, and S214 of helix 6. These results suggest that the transmembrane helices of the c and a subunits interact and that the mutated residues are important for the structure of the F(0) complex and proton translocation

Influence of shaking on antifungal susceptibility testing of Cryptococcus neoformans: a comparison of the NCCLS standard M27A medium, buffered yeast nitrogen base, and RPMI-2% glucose

Fil: Rodríguez-Tudela, Juan L. Instituto de Salud Carlos III. Servicio de Micología; España.Fil: Martín-Díez, Francisco. Instituto de Salud Carlos III. Servicio de Micología; España.Fil: Cuenca-Estrella, Manuel. Instituto de Salud Carlos III. Servicio de Micología; España.Fil: Rodero, Laura. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Carpintero, Yolanda. Instituto de Salud Carlos III. Servicio de Micología; España.Fil: Gorgojo, Begoña. Instituto de Salud Carlos III. Servicio de Micología; España.Cryptococcus neoformans is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640-2% glucose, and buffered yeast nitrogen base ¿BYNB) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35 degrees C for 48 h were determined. The results indicated that shaking and an inoculum size of 10(5) CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of C. neoformans and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI-2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the C. neoformans strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for C. neoformans

Influence of shaking on antifungal susceptibility testing of Cryptococcus neoformans: a comparison of the NCCLS standard M27A medium, buffered yeast nitrogen base, and RPMI-2% glucose

Fil: Rodríguez-Tudela, Juan L. Instituto de Salud Carlos III. Servicio de Micología; España.Fil: Martín-Díez, Francisco. Instituto de Salud Carlos III. Servicio de Micología; España.Fil: Cuenca-Estrella, Manuel. Instituto de Salud Carlos III. Servicio de Micología; España.Fil: Rodero, Laura. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Carpintero, Yolanda. Instituto de Salud Carlos III. Servicio de Micología; España.Fil: Gorgojo, Begoña. Instituto de Salud Carlos III. Servicio de Micología; España.Cryptococcus neoformans is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640-2% glucose, and buffered yeast nitrogen base ¿BYNB) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35 degrees C for 48 h were determined. The results indicated that shaking and an inoculum size of 10(5) CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of C. neoformans and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI-2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the C. neoformans strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for C. neoformans

Influence of Shaking on Antifungal Susceptibility Testing of Cryptococcus neoformans: a Comparison of the NCCLS Standard M27A Medium, Buffered Yeast Nitrogen Base, and RPMI–2% Glucose

Cryptococcus neoformans is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640–2% glucose, and buffered yeast nitrogen base [BYNB]) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35°C for 48 h were determined. The results indicated that shaking and an inoculum size of 10(5) CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of C. neoformans and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI–2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the C. neoformans strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for C. neoformans