Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment

Abstract

and species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, H99 and R265. Deletion of in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when or was deleted in both strains. However, when these genes were deleted in combination with , a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in mutants of both strains, but rhodamine 6G accumulation increased only in the mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. and expression was upregulated in H99 and R265 when treated with fluconazole. However, expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

Decreasing fluconazole susceptibility of clinical South African Cryptococcus neoformans isolates over a decade

“…The molecular basis of resistance includes multidrug efflux pump proteins, decreased affinity to target enzymes or overall decreased drug uptake [7]. Heteroresistance which is a phenomenon where strains express a transient adaptation to the drug also occurs [24,25]. These mechanisms of resistance will need to be investigated further in the few C. neoformans isolates with a high fluconazole MIC from our study.…”

“…The 2019 guideline also recommends that isolates from the first relapse episode be tested for fluconazole susceptibility rather than waiting for repeated PLOS NEGLECTED TROPICAL DISEASES relapses to occur before testing [19,23]. Fluconazole inhibits the lanosterol 14α-demethylase enzyme encoded by ERG11 in the ergosterol biosynthesis pathway and targets the same cell processes as amphotericin B [6,24]. The molecular basis of resistance includes multidrug efflux pump proteins, decreased affinity to target enzymes or overall decreased drug uptake [7].…”

Fatores microbiológicos da criptococose e sua importância na carga da doença em pacientes atendidos em um hospital de referência do Estado de São Paulo

“…Esta enzima é codificada pelo ERG11, presente no cromossomo 1, e mutações neste gene ocasionam alterações de aminoácidos desta proteína, gerando modificações estruturais na enzima alvo, o que diminui sua afinidade aos azóis, principalmente ao FCZ (PFALLER, 2012;SAGATOVA et al, 2016;SANGLARD, 2016). A superexpressão desta enzima, devido a mecanismos regulatórios do ERG11, leva ao aumento da produção da molécula alvo e como consequência da menor efetividade do FCZ, à resistência (SANGLARD et al, 1998; PRASAD e RAWAL, COWEN et al, 2015) (POSTERARO et al, 2003;SANGUINETTI et al, 2006;CHANG et al, 2018). O aumento destas bombas pode ser avaliado por expressão gênica e também pelo acúmulo de substratos fluorescentes específicos para estes transportadores, como a rodamina 6G para CnAFR1 e CnMDR1 em células de C. neoformans (THORNEWELL et al, 1997;POSTERARO et al, 2003;CANNON et al, 2016).…”

“…Outro mecanismo molecular de resistência ao FCZ descrito em C. neoformans é o efluxo do antifúngico provocado por bombas de efluxo. Embora numerosos genes já tenham sido associados a bombas de efluxo, atualmente apenas um dos transportadores ABC, o AFR1, tem sido atribuído ao FCZ (POSTERARO et al, 2003;CHANG et al, 2018). Alterações fenotípicas têm sido observadas em células resistentes a FCZ, como diminuição na taxa de crescimento além do aumento do tamanho celular, provavelmente decorrente da duplicação de genes e do conteúdo de DNA (SMITH et al, 2015;ALTAMIRANO et al, 2017).…”

“…A estratégia fúngica do aumento de bombas de expulsão de FCZ em populações heterorresistentes também foi sugerida em estudos prévios (SIONOV et al, 2012;CHANG et al, 2018), tendo sido comprovada neste estudo através dos testes utilizando rodamina 6G como marcador por citometria de fluxo. Em C. neoformans, este marcador é o único substrato semelhante ao FCZ descrito para as bombas de efluxo ABC CnAFR1 e CnMDR1 e a diminuição da sua fluorescência indica o aumento da expressão destas bombas na membrana celular (POSTERARO et al, 2003;LAMPING et al, 2007;CANNON et al, 2016).…”