Nitrogen concentration affects amphotericin B and fluconazole tolerance of pathogenic cryptococci

Abstract

Environmental stress often causes phenotypic changes among pathogenic cryptococci, such as altered antifungal susceptibility, changes in capsule and melanin formation, as well as altered levels of the membrane sterol and antifungal target, ergosterol. We therefore hypothesised that nitrogen limitation, a prevalent environmental stress in the natural habitat of these yeasts, might affect virulence and antifungal susceptibility. We tested the effect of different nitrogen concentrations on capsule, melanin and ergosterol biosynthesis, as well as amphotericin B (AmB) and fluconazole (FLU) susceptibility. This was achieved by culturing cryptococcal strains representing Cryptococcus neoformans and Cryptococcus gattii in media with high (0.53 g/l), control (0.42 g/l) and low (0.21 g/l) NH4Cl concentrations. India ink staining was used to determine capsule thickness microscopically, while melanin and ergosterol content were determined spectrophotometrically. We found that lower nitrogen concentrations enhanced both ergosterol and capsule biosynthesis, while a variable effect was observed on melanisation. Evaluation of drug tolerance using time-kill methodology, as well as tests for FLU heteroresistance, revealed that the low nitrogen cultures had the highest survival percentages in the presence of both AmB and FLU, and showed the highest frequency of FLU heteroresistance, suggesting that nitrogen concentration may indeed influence drug tolerance.

Directed Evolution Detects Supernumerary Centric Chromosomes Conferring Resistance to Azoles in Candida auris

“…Another major upregulated gene, MEP2 , the ammonia transporter, is shown to be influenced by GAT1 , a transcription factor, and positively regulates the filamentation growth of C. albicans cells under nitrogen-starved conditions via activation of the MAP kinase and cAMP-dependent signaling pathway ( 26 ). In Cryptococcus neoformans , lower nitrogen concentrations promote ergosterol and capsule biosynthesis ( 27 ). In C. albicans , the higher survival percentage in the presence of antifungal drugs under nitrogen-starved cultures suggests that nitrogen starvation influences the drug susceptibili/ty of cells ( 28 ).…”

Aspergillus terreus and the Interplay with Amphotericin B: from Resistance to Tolerance?

“…1 and 2 ). Polyene antifungal activity may be influenced by medium composition ( 44 , 56 , 57 ). Besides this, nutrient-limited media affects fungal growth and, in consequence, tolerance ( 33 , 38 ).…”

Prevalence, Genetic Structure, and Antifungal Susceptibility of the Cryptococcus neoformans/C. gattii Species Complex Strains Collected from the Arboreal Niche in Poland

“…The detailed studies over the sources of this envi-ronmentally distributed complex are essential in order to define its geographical extent, population structure, and mode and risk of transmission. It was also proven that certain environmental conditions, among them those associated with arboreal ancestry of these fungi, may be responsible for the acquisition of virulence factors allowing them to establish infection within human/animal hosts [30][31][32][33][34]. In Europe, several studies concerning the arboreal source of the CNGSC were performed, the majority of which were related to western and southern regions of the continent as well as to Mediterranean Basin [13,17,[35][36][37][38].…”

“…The significant impact of factors, which may be present in the natural habitat of the CNGSC, including nutrient limitation (among them nitrogen limitation), temperature, ultraviolet radiation, enzymatic degradation on the susceptibility of the CNGSC to FLU, and AMB, has been described [34,52,[60][61][62][63]. The above-listed factors are probably able to activate adaptive processes, favoring the survival of the microorganism in the presence of the drugs; however, their role in the development of persistent, mutation-dependent resistance cannot be excluded.…”