RESUMO
The resistance of Candida albicans biofilms to a broad spectrum of antimicrobial agents has been well documented. Biofilms are known to be heterogeneous, consisting of microenvironments that may induce formation of resistant subpopulations. In this study we characterized one such subpopulation. C. albicans biofilms were cultured in a tubular flow cell (TF) for 36 h. The relatively large shear forces imposed by draining the TF removed most of the biofilm, which consisted of a tangled mass of filamentous forms with associated clusters of yeast forms. This portion of the biofilm exhibited the classic architecture and morphological heterogeneity of a C. albicans biofilm and was only slightly more resistant than either exponential- or stationary-phase planktonic cells. A submonolayer fraction of blastospores that remained on the substratum was resistant to 10 times the amphotericin B dose that eliminated the activity of the planktonic populations. A comparison between planktonic and biofilm populations of transcript abundance for genes coding for enzymes in the ergosterol (ERG1, -3, -5, -6, -9, -11, and -25) and beta-1,6-glucan (SKN and KRE1, -5, -6, and -9) pathways was performed by quantitative RT-PCR. The results indicate a possible association between the high level of resistance exhibited by the blastospore subpopulation and differential regulation of ERG1, ERG25, SKN1, and KRE1. We hypothesize that the resistance originates from a synergistic effect involving changes in both the cell membrane and the cell wall.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Ergosterol/genética , Ergosterol/metabolismo , Esporos Fúngicos/efeitos dos fármacos , beta-Glucanas/metabolismo , Candida albicans/genética , Candida albicans/ultraestrutura , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , DNA Fúngico/biossíntese , DNA Fúngico/genética , Farmacorresistência Fúngica/fisiologia , Genes Fúngicos/genética , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , RNA Fúngico/biossíntese , RNA Fúngico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esporos Fúngicos/genética , Esporos Fúngicos/ultraestrutura , Transcrição GênicaRESUMO
Activation of P2Y2 receptor (P2Y2-R) in inner medullary collecting duct (IMCD) of rat decreases AVP-induced water flow and releases PGE(2). We observed that dehydration of rats decreases the expression of P2Y2 receptor in inner medulla (IM) and P2Y2-R-mediated PGE(2) release by IMCD. Because circulating vasopressin (AVP) levels are increased in dehydrated condition, we examined whether chronic infusion of desmopressin (dDAVP) has a similar effect on the expression and activity of P2Y2-R. Groups of rats were infused with saline or dDAVP (5 or 20 ng/h sc, 5 or 6 days) via osmotic minipumps and euthanized. Urine volume, osmolality, and PGE(2) metabolite content were determined. AQP2- and P2Y2- and V2-R mRNA and/or protein in IM were quantified by real-time RT-PCR and immunoblotting, respectively. P2Y2-R-mediated PGE(2) release by freshly prepared IMCD was assayed using ATPgammaS as a ligand. Chronic dDAVP infusion resulted in low-output of concentrated urine and significantly increased the AQP2 protein abundance in IM. On the contrary, dDAVP infusion at 5 or 20 ng/h significantly decreased P2Y2-R protein abundance (approximately 40% of saline-treated group). In parallel, the relative expression of P2Y2-R vs. AQP2- or V2-R mRNA was significantly decreased. Furthermore, the P2Y2-R-mediated PGE(2) release by IMCD was significantly decreased in rats infused 20 ng/h but not 5 ng/h of dDAVP. Urinary PGE(2) metabolite excretion, however, did not change with dDAVP infusion. In conclusion, chronic dDAVP infusion decreases the expression and activity of P2Y2-R in IM. This may be due to a direct effect of dDAVP or dDAVP-induced increase in medullary tonicity.