Using Aspergillus nidulans To Identify Antifungal Drug Resistance Mutations

Abstract

Systemic fungal infections contribute to at least 10% of deaths in hospital settings. Most antifungal drugs target ergosterol (polyenes) or its biosynthetic pathway (azoles and allylamines), or beta-glucan synthesis (echinocandins). Antifungal drugs that target proteins are prone to the emergence of resistant strains. Identification of genes whose mutations lead to targeted resistance can provide new information on those pathways. We used Aspergillus nidulans as a model system to exploit its tractable sexual cycle and calcofluor white as a model antifungal agent to cross-reference our results with other studies. Within 2 weeks from inoculation on sublethal doses of calcofluor white, we isolated 24 A. nidulans adaptive strains from sectoring colonies. Meiotic analysis showed that these strains had single-gene mutations. In each case, the resistance was specific to calcofluor white, since there was no cross-resistance to caspofungin (echinocandin). Mutation sites were identified in two mutants by next-generation sequencing. These were confirmed by reengineering the mutation in a wild-type strain using a gene replacement strategy. One of these mutated genes was related to cell wall synthesis, and the other one was related to drug metabolism. Our strategy has wide application for many fungal species, for antifungal compounds used in agriculture as well as health care, and potentially during protracted drug therapy once drug resistance arises. We suggest that our strategy will be useful for keeping ahead in the drug resistance arms race.

An Effect of Biofield Treatment on Multidrug-resistant Burkholderia cepacia: A Multihost Pathogen

“…Due to continuous new drug discovery in antimicrobials, rate of MDR microorganism increased causing serious health problems. Cell membrane alterations in MDR microorganism results in decreased uptake of antimicrobials [23], overexpression of drug target enzymes results in mutation [24], and drug efflux pumps remains the predominant mechanism in multi-drug resistant organisms [25]. Nowadays, B. cepacia acquires resistance against broad range of antibiotics, so it was very difficult to start drug therapy in chronically infected patients [26].…”

Multidrug Resistance: An Emerging Crisis

“…Cell wall, in both bacteria and fungi, plays a crucial role in their survival. As discussed above, drugs inhibit the cell wall synthesis by binding with the peptidoglycan layer in bacteria or affecting ergosterol synthesis (e.g., polyenes [ 38 ]) in fungi, thus, blocking the cell growth and division. These organisms undergo certain chromosomal mutations [ 39 ] or exchange of extrachromosomal DNA elements through conjugation or transformation (horizontal gene transfer) such as in K. pneumoniae [ 4 ], which can cause alteration in the cell membrane composition (e.g., a reduction in the ergosterol content in fungal plasma membrane) resulting in decreased permeability and uptake of drugs into the cell [ 1 , 5 , 37 , 40 ].…”

“…These organisms undergo certain chromosomal mutations [ 39 ] or exchange of extrachromosomal DNA elements through conjugation or transformation (horizontal gene transfer) such as in K. pneumoniae [ 4 ], which can cause alteration in the cell membrane composition (e.g., a reduction in the ergosterol content in fungal plasma membrane) resulting in decreased permeability and uptake of drugs into the cell [ 1 , 5 , 37 , 40 ]. Altered membrane composition (such as β -1,3-glucan and lipid content in fungal cell membrane) also leads to lack of active target sites for the drugs (e.g., echinocandins in fungi [ 38 ]) to bind. Mutations in the genes encoding for the target cause modifications at the molecular level and retain cellular function by reducing susceptibility to inhibition [ 1 , 5 , 37 , 41 ].…”

An Evaluation of Biofield Treatment on Susceptibility Pattern of Multidrug Resistant Stenotrophomonas maltophilia: An Emerging Global Opportunistic Pathogen

“…MDR is an unavoidable natural phenomenon which compels continuous discovery of newer drugs causing serious public health problems. Various mechanisms involved in MDR include alteration in the cell membrane composition of microorganism resulting in decreased permeability and uptake of drugs into the cell [20], overexpression of drug target enzymes or altered the drug target through mutation [21], and drug efflux pumps remains the predominant mechanism in MDRO [22]. Now a days, S. maltophilia acquires resistance against broad range of antimicrobials, including trimethoprim/sulfamethoxazole, β-lactam antibiotics, macrolides, cephalosporins, fluoroquinolones, aminoglycosides, carbapenems, chloramphenicol, tetracyclines, and polymyxins.…”