Adaptive resistance mechanisms of Aspergillus fumigatus biofilms

Rajendran, Ranjith (2013) Adaptive resistance mechanisms of Aspergillus fumigatus biofilms. PhD thesis, University of Glasgow.

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Abstract

Biofilm formation is one of several significant virulence factors associated with life threatening pulmonary infections in immunocompromised individuals caused by Aspergillus fumigatus. Previous studies have demonstrated phase dependant antifungal activity against A. fumigatus biofilms. Antifungal resistance associated with fungal biofilms is a complex multifactorial phenomenon, and it remains unclear specifically how this manifests itself in A. fumigatus. This study therefore aimed to investigate adaptive resistance mechanisms in A. fumigatus biofilms. Different phases of A. fumigatus biofilms were grown for 8, 12, 24 and 48h in polystyrene plates in RPMI media. Functional efflux pump activity was subsequently assessed using an Ala-Nap fluorescent uptake assay. Extracellular material was extracted from each phase and the level of extracellular DNA (eDNA) was quantifiedusing a microplate fluorescence assay. The minimum inhibitory concentrations (MIC) of different classes of antifungals were assessed in the presence and absence of different inhibitors using a checkerboard assay, or with a fixed concentration, by the broth microdilution method to assess synergism, antagonism, or otherwise. The presence of eDNA and phenotypic changes in biofilm caused by antifungal agents and inhibitors were assessed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) techniques. The resultant biofilm biomass for different experiments was evaluated using a crystal violet assay. SYBR green qRT-PCR was used to assess the expression of different genes implicated in biofilm resistance (AfuMDR 1-4, ChiA-E, HSP90 and Fks1) over the period of multicellular development, using a diffusion chamber in a murine model and a Galleria mellonella infection model. The results from this study demonstrated phase dependant expression of efflux pumps in A. fumigatus biofilm populations, which actively contributes to azole resistance. Moreover, voriconazole treatment induced efflux pump expression in both in vitro and in vivo models.These data suggest that A. fumigatus efflux pump proteins, which evolved to become integral to their natural physiological function, have inadvertently induced resistance to azole drugs, albeit in the early phases of biofilm development. Assessment of A. fumigatus biofilm extracellular matrix (ECM), associated with maturing biofilms, showed that eDNA is an important architectural component of the biofilm, helping to maintain its stability. The antifungal sensitivity of different phases of A. fumigatus growth decreased significantly in the presence of DNase, indicating that decreased susceptibility to antifungals in the A. fumigatus is mediated in part by eDNA.Its release was shown to correlate withchitinase activity, a marker of autolysis, suggestive that autolysis was associated with eDNA release. It was hypothesised that heat shock protein 90 (HSP90) was involved in this autolytic pathway. Therefore, when HSP90 was pharmacologically inhibited this led to a decrease in matrix eDNA level, providing a compelling mechanism through which HSP90 might regulate biofilm antifungal resistance. To test whether these mechanisms of adaptive resistance had any bearing clinically, a G. mellonella model was developed. It was shown that each of the key genes were expressed during infection, both in control and antifungal treated larvae. This validates the potential use of this insect model for resistance and virulence studies. Overall, this study establishes several novel adaptive resistance mechanisms regulating biofilm drug resistance in A. fumigatus biofilms. Moreover, it highlights the potential to target these mechanisms as a therapeutic strategy for managing and improving clinical outcomes in these hard-to-treat infections.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Aspergillus fumigatus, biofilm, drug resistance, antifungals, efflux pump, eDNA
Subjects: Q Science > QR Microbiology
R Medicine > R Medicine (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Funder's Name: UNSPECIFIED
Supervisor's Name: Ramage, Prof. Gordon and Lappin, Dr. David and Williams, Prof. Craig
Date of Award: 2013
Depositing User: Dr Ranjith Rajendran
Unique ID: glathesis:2013-4333
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 06 Jun 2013 07:54
Last Modified: 06 Jun 2013 09:28
URI: http://theses.gla.ac.uk/id/eprint/4333

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