Aspergillus fumigatus and aspergillosis: From basics to clinics

Abstract

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A. fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP 51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP 51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A. fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A. fumigatus. This review paper comprehensively discusses the current clinical challenges caused by A. fumigatus and provides insights on how to address them.

Keywords: Aspergillus fumigatus; Azole-resistance; Drug-resistance mechanism; Invasive aspergillosis.

Fig. 1

Cladogram of the genus Aspergillus and the relationship between sections and subgenera. A selection of the species mentioned in the text are given in brackets in bold font after the section name. Adopted from Houbraken et al. (2020) with permission.

Fig. 2

The ergosterol pathway involves multiple enzymes and is tightly regulated by the key rate-limiting enzyme Hmg1 producing mevalonate. Lanosterol produced by ERG6 is the substrate catalysed by CYP51A and CYP51B, while azoles competitively occupy the catalytic site and hence reduce the ergosterol synthesis. Adopted from Moreno-Velásquez et al. (2017), with permission.

Fig. 3

Molecular mechanisms contributing to triazole resistance observed in A. fumigatus. The scheme represents an A. fumigatus cell - with particular focus on the nucleus (N), the endoplasmic reticulum (ER), and the plasma membrane (PM), which depicts the most relevant mechanisms of triazole resistance in this fungus. In A. fumigatus, a major role in ergosterol biosynthesis is played by the sterol demethylase CYP51A (a1). The CYP51A gene is regulated positively by the SrbA protein, which activates its expression by binding to two Sterol Regulatory Elements (SRE) in the promoter region. When ergosterol biosynthesis is repressed, the access of SrbA to SREs is prevented by both the CBC complex and the HapX transcription factor binding to regulatory elements located downstream of SREs, resulting in negative regulation of CYP51A expression. The sterol demethylase CYP51A, whose native substrate is eburicol, an intermediate of ergosterol biosynthesis, is the target of azole drugs (a2). As a result, changes in CYP51A sequence or expression are associated with increased MIC to triazoles. Amino acid substitutions in either the ligand binding site or the catalytic site (b1) modulates triazole binding affinity to CYP51A (b2). A different mutation that can be found in combination with SNPs in the CYP51A gene is the presence of tandem repeats (TRs) in the promoter region, resulting in an expansion of the SREs, unimpeded SrbA binding, and ultimately hyper-activation of CYP51A expression (c1). The same outcome was observed in the case of the P88L mutation in the HapE subunit of the CCAAT-binding complex (CBC) complex, which diminishes CBC binding affinity and its negative regulation of CYP51A expression, although this genotype had only been observed in the clinical isolates in which it was first described (d1). In both cases, the increased amount of the CYP51A enzyme prevents saturation by triazoles and sustains ergosterol biosynthesis (c2 and d2). As for other pathogenic fungi, overexpression of either ATP-binding cassettes (ABC) or Major Facilitator Superfamily (MFS) type drug efflux pumps had been observed among triazole-resistant clinical isolates, which prevents the accumulation of active concentration of drug in the cell. In particular, the transcription factor AtrR positively regulates the expression of the ABC transporter CDR1B (d1 and d2). Notably, AtrR is also involved in the positive regulation of CYP51A. A clinically relevant mutation of a different kind is the one affecting the Hmg-CoA reductase encoded by hmg1, which takes part in ergosterol biosynthesis by converting Hmg-CoA into Mevalonate. Hmg1 has a conserved Sterol Sensing Domain (SSD) involved in regulation of sterol biosynthesis. Mutations in the SSD result in a dysregulation of the sterol pathway that eventually translates to an increased cellular ergosterol production and triazole resistance (f1 and f2).

Fig. 4

CRISPR-Cas9 technology in A. fumigatus. The vignettes illustrate representative methods for CRISPR–Cas9 genetic manipulation of A. fumigatus. (a) MMEJ can be used to integrate an HPH cassette into a desired locus by using short homology arms. The strain is first transformed with a plasmid expressing Cas9 and containing a PYR4 marker and then with an in vitro transcribed sgRNA and the HPH cassette. (b) The need to set up a suitable system to express the CRISPR elements can be circumnavigated by using CRISPR RNPs, in which two different crRNAs and tracrRNA are assembled in vitro with Cas9 and then transformed into the cells to target the upstream and downstream regions of YFG. MMEJ results in the integration of a HPH cassette into the targeted gene. (c) Ballard and colleagues tweaked the systems developed by into a two-plasmid system for introducing SNPs into a clinical isolate without marker integration. The AMA1 sequence supports the replication of the plasmid harbouring CAS9 in A. fumigatus, which confers resistance to hygromycin. A different plasmid carries a cassette for the expression of a ribozyme-flanked sgRNA from a A. nidulans RNA pol II promoter; after expression in A. fumigatus, the self-splicing activity of the rybozymes releases the mature sgRNA. This plasmid contains a PTRA split marker, interrupted by the same protospacer sequence that is being targeted on the gene of interest. After transformation of both plasmids and a RT containing the SNP to introduce in YFG, Cas9 targets both the protospacer on the desired locus in the genome and the twin protospacer interrupting the split marker. HDR then simultaneously mediates the insertion of the SNP into YFG and the reconstitution of the PTRA marker, thus allowing for selection of the transformants on pyrithiamine without marker integration. (d) The RNP system can also be exploited to affect gene expression, and it was recently used to replace a native promoter with a constitutive hspA promoter by transforming the cells with the RNP particle and a repair template carrying HPH, hspA, and homology arms flanking the insertion site. Af, A. fumigatus; CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR-RNA; HDR, homology-directed repair; HDV, human hepatitis delta virus; HH, hammerhead; MMEJ, microhomology-mediated end joining; RNA–Cas9 protein complex; sgRNA, single- guide RNA; tracrRNA, trans-activating RNA; YFG, your favourite gene. HA, Homology Arms; RT, Repair Template. Panels a and b were adopted from Morio et al. (2020) with permission.