Daryl L. Richie

A role for the unfolded protein response (UPR) in virulence and antifungal susceptibility in Aspergillus fumigatus

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

Unexpected Link between Metal Ion Deficiency and Autophagy in Aspergillus fumigatus

ABSTRACT Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The ΔAfatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ΔAfatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ΔAfatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the ΔAfatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress

We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic‐like programmed cell death. Single metacaspase‐deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase‐deficient mutant, ΔcasA/ΔcasB, was constructed by disrupting both genes. Stationary phase cultures of wild‐type A.  fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the ΔcasA/ΔcasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild‐type virulence and showed no difference in sensitivity to a range of pro‐apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the ΔcasA/ΔcasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic‐like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.

HacA-independent functions of the ER stress sensor IreA synergize with the canonical UPR to influence virulence traits in Aspergillus fumigatus.

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacA i, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireA Δ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus.

Divergent Protein Kinase A isoforms co-ordinately regulate conidial germination, carbohydrate metabolism and virulence in Aspergillus fumigatus

The genome of Aspergillus fumigatus encodes two isoforms of the catalytic subunit of the cAMP‐dependent Protein Kinase (PKA). Although deletion of the class I isoform, pkaC1, leads to an attenuation of virulence, the function of the class II subunit, PkaC2, was previously uninvestigated. In this report, we demonstrate that both isoforms act in concert to support various physiologic processes that promote the virulence of this pathogen. Whereas pkaC1 and pkaC2 single‐deletion mutants display wild‐type conidial germination, a double‐deletion mutant is delayed in germination in response to environmental nutrients. Furthermore, PkaC1 and PkaC2 interact to positively regulate flux through the carbohydrate catabolic pathway and, consequently, the ΔpkaC1ΔpkaC2 mutant is unable to grow on low glucose concentrations. Importantly, the reduced germinative capacity and inability to utilize glucose observed for the ΔpkaC1ΔpkaC2 strain correlated with an inability of the mutant to establish infection in a murine model. Conversely, overexpression of pkaC2 both promotes the in vitro growth on glucose, and restores the fungal burden and mortality associated with the ΔpkaC1 to that of the wild‐type organism. Taken together, these data demonstrate the functional capacity of pkaC2 and emphasize the importance of PKA‐mediated metabolic control in the pathogenic potential of A. fumigatus.

The virulence of the opportunistic fungal pathogen Aspergillus fumigatus requires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR)

The filamentous fungal pathogen Aspergillus fumigatus secretes hydrolytic enzymes to acquire nutrients from host tissues. The production of these enzymes exerts stress on the endoplasmic reticulum (ER), which is alleviated by two stress responses: the unfolded protein response (UPR), which adjusts the protein folding capacity of the ER, and ER-associated degradation (ERAD), which disposes of proteins that fail to fold correctly. In this study, we examined the contribution of these integrated pathways to the growth and virulence of A. fumigatus, focusing on the ERAD protein DerA and the master regulator of the UPR, HacA. A ΔderA mutant grew normally and showed no increase in sensitivity to ER stress. However, expression of the UPR target gene bipA was constitutively elevated in this strain, suggesting that the UPR was compensating for the absence of DerA function. To test this, the UPR was disrupted by deleting the hacA gene. The combined loss of derA and hacA caused a more severe reduction in hyphal growth, antifungal drug resistance and protease secretion than the loss of either gene alone, suggesting that DerA and HacA cooperate to support these functions. Moreover, the ΔderA/ΔhacA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasted the wild type virulence of ΔderA and the reduced virulence of the ΔhacA mutant. Taken together, these data demonstrate that DerA cooperates with the UPR to support the expression of virulence-related attributes of A. fumigatus.

Effects of a Defective Endoplasmic Reticulum-Associated Degradation Pathway on the Stress Response, Virulence, and Antifungal Drug Susceptibility of the Mold Pathogen Aspergillus fumigatus

ABSTRACT Proteins that are destined for release outside the eukaryotic cell, insertion into the plasma membrane, or delivery to intracellular organelles are processed and folded in the endoplasmic reticulum (ER). An imbalance between the level of nascent proteins entering the ER and the organelles ability to manage that load results in the accumulation of unfolded proteins. Terminally unfolded proteins are disposed of by ER-associated degradation (ERAD), a pathway that transports the aberrant proteins across the ER membrane into the cytosol for proteasomal degradation. The ERAD pathway was targeted in the mold pathogen Aspergillus fumigatus by deleting the hrdA gene, encoding the A. fumigatus ortholog of Hrd1, the E3 ubiquitin ligase previously shown to contribute to ERAD in other species. Loss of HrdA was associated with impaired degradation of a folding-defective ERAD substrate, CPY*, as well as activation of the unfolded-protein response (UPR). The ΔhrdA mutant showed resistance to voriconazole and reduced thermotolerance but was otherwise unaffected by a variety of environmental stressors. A double-deletion mutant deficient in both HrdA and another component of the same ERAD complex, DerA, was defective in secretion and showed hypersensitivity to ER, thermal, and cell wall stress. However, the ΔhrdA ΔderA mutant remained virulent in mouse and insect infection models. These data demonstrate that HrdA and DerA support complementary ERAD functions that promote survival under conditions of ER stress but are dispensable for virulence in the host environment.

Autophagy in the Filamentous Fungus Aspergillus fumigatus

Many breakthroughs in our understanding of the function and molecular basis of autophagy have been achieved in mammalian and yeast systems. However, we still know very little about the contribution of autophagy to the biology of filamentous fungi. A comparative analysis of autophagy between genera will expand our knowledge of the autophagy machinery and has the potential to identify novel functions that are relevant to multiple biological systems. This chapter will discuss methods that have been employed for studying autophagy in the opportunistic mold pathogen Aspergillus fumigatus. Understanding how autophagy influences the growth of this important human pathogen could lead to the development of novel antifungal drugs that restrict the growth of the fungus by manipulating the autophagy pathway.

Impaired Ribosome Biogenesis Disrupts the Integration between Morphogenesis and Nuclear Duplication during the Germination of Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37°C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a ΔcgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the ΔcgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although ΔcgrA conidia germinated normally at 22°C, they swelled excessively when incubated at 37°C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.

Secretion stress and antifungal resistance: An Achilles’ heel of Aspergillus fumigatus?

The ability of Aspergillus fumigatus to establish and maintain an infection requires a continuous supply of nutrients to fuel energy production and growth. Like other filamentous fungi, A. fumigatus acquires nutrients by absorption, a mode of nutrition that depends upon the secretion of extracellular hydrolases to degrade the complex organic polymers in host tissues into reduced forms of carbon and nitrogen. If the folding capacity of the endoplasmic reticulum (ER) is exceeded during periods of high secretory activity, a signaling pathway known as the unfolded protein response (UPR) is activated to relieve the stress on the ER. Current evidence indicates that A. fumigatus relies upon this pathway to sustain the high rate of protease secretion needed to grow optimally in mammalian tissue. In addition, the UPR strengthens the ability of the secretory system to deliver cell wall and membrane components to the hyphal apex, which promotes the invasive growth of the expanding hyphae and protects the fungus from damage caused by antifungal drugs. The important contribution of UPR-dependent functions to the pathogenesis of invasive aspergillosis and antifungal susceptibility suggests that components of this pathway could be promising new targets for antifungal therapy.