Secretion stress and fungal pathogenesis

“For the rain had ceased at last, and a sickly autumn sun shone upon a land which was soaked and sodden with water. Wet and rotten leaves reeked and festered under the foul haze which rose from the woods. The fields were spotted with monstrous fungi of a size and color never matched before—scarlet and mauve and liver and black. It was as though the sick earth had burst into foul pustules; mildew and lichen mottled the walls and with that filthy crop, Death sprang also from the water-soaked earth. Men died, and woman and children … and all died the same death of corruption.”

From: Sir Nigel, Chapter 1, by Sir Arthur Conan Doyle, 1906.

This oft-used quote by the famous author of Sherlock Holmes illustrates two points relevant to the exciting results regarding fungal pathogenesis presented by Richie et al. in this issue of Virulence. First, throughout human history, fungi have been associated with death and decay. These mysterious, curiously shaped organisms often appear on gloomy, rain-soaked days in dark and inaccessible places, the very ambience associated with death, as described so brilliantly in the excerpt from Sir Nigel. We now know, of course, that fungi do play a major role in death by recycling carbon and nitrogen sources from detritus throughout the environment. Thus, perhaps ironically, fungi breathe life into the complex and diverse ecosystems of planet Earth.

Fungi's ability to decay and recycle organic material comes from secretion of hydrolytic enzymes into the environment and subsequent absorption of the delectable products produced from these enzymatic reactions. Thus, protein secretion is key to the fungal lifestyle. Protein secretion is a complex process that requires multiple control checkpoints that ensure proteins are properly folded and delivered to appropriate sites in the fungal cell. The endoplasmic reticulum is a critical organelle in eukaryotic cells being the entry point of proteins into the secretory pathway. Research in both yeast and vertebrate model systems has revealed a central role for the ER in maintaining cellular homeostasis (reviewed in ref. 1). Two key pathways that link the ER and protein secretion with maintenance of cellular homeostasis in the face of external and internal stresses, are the unfolded protein response (UPR) and the endoplasmic reticulum associated degradation pathway (ERAD). In this issue of Virulence, Richie et al. provide new insights into the role of the UPR and ERAD in the biology and virulence potential of the human fungal pathogen Aspergillus fumigatus.

The second relation between the quoted text and the results presented in the Richie et al. study relates more broadly to the role of fungi as agents of infectious disease. Astute readers of Sir Conan Doyle will realize that the “death of men, woman and children” referred to in the excerpt is not the direct result of the fungi described, but rather, an outbreak of the Black Death, or bubonic plague, in England around 1348. Throughout history, major infectious disease epidemics have been associated with bacteria such as Yersinia pestis—the cause of the Black Death—or viruses such as the influenza that caused the infamous 1918 Spanish Flu. Fungi have traditionally not been considered major causes of infectious disease in the animal kingdom. Indeed, prior to the mid-late 20^th century, few references in the medical literature refer to invasive fungal infections.

However, as Bob Dylan once said, “The Times They Are A-Changin.” Over the last three decades, significant increases in often-lethal diseases caused by fungi are occurring not only in human populations, but in other animal, amphibian and insect populations throughout the world.2–6 In general, the majority of human fungal infections are caused by three fungi: two organisms that primarily live as yeast, Candida albicans and Cryptococcus neoformans, and one that is the proverbial “mold,” Aspergillus fumigatus. The frequency of occurrence of these three organisms as agents of infectious disease has led to the hypothesis that they are more “virulent” than other fungal organisms. The burden of these infections on patients and health care systems around the world has stimulated intense research into understanding the potential virulence attributes of these fungi.

Of course, the definition of virulence is controversial. Often, fungal pathogens are described with the caveat that they are “opportunistic.” This caveat essentially conveys the idea that most fungi do not cause disease in a mammalian host with an intact immune system, and thus are not virulent. However, as Casadevall and Pirofski have argued, disease always occurs in the context of host susceptibility.7,8 In this light, one can argue that all microbial pathogens are opportunistic, and moreover, that studies on fungal pathogenesis have enriched our understanding of infectious diseases by calling increased attention to “microbial attributes” that are critical for adaptation to the mammalian host environment (and thus not “traditional” virulence factors). Without the ability to utilize the complex substrates encountered in the host as an energy source, fungi would be incapable of causing human disease.

The study by Richie et al. in this issue of Virulence increases our knowledge of fungal virulence attributes by continuing previous work by this group to understand the role of the endoplasmic reticulum's (ER) stress homeostasis mechanisms in the biology of A. fumigatus. As mentioned, protein secretion is key to the fungal lifestyle. Whether it is growth in an organic debris pile or in a susceptible mammalian host, fungi must secrete proteins into their environment to acquire nutrients and adapt to their local microenvironments. Given the enormous importance of protein secretion on fungal growth, it is not surprising that key regulatory mechanisms exist to maintain cellular homeostasis in the face of intense demand on the secretory machinery. Previously, Richie et al. reported that the UPR was a pathogenesis and clinically relevant pathway in A. fumigatus.9 A null mutant of the key transcription factor that mediates effectors of the UPR in fungi, hacA, displays several pathogenesis relevant phenotypes including a reduction, but not loss, in fungal virulence and increases in susceptibility to azole, polyene and echinocandin antifungal drugs. Consistent with the increased understanding of the UPR in governing vertebrate cell homeostasis, the UPR in Aspergillus affects protein secretion, apical growth, membrane homeostasis and cell wall integrity.

In the current study, Richie et al. discover that in the absence of a functional ERAD response, A. fumigatus compensates by activating the UPR. To study the role of ERAD in A. fumigatus, a derA null mutant was generated. DerA is a key component of a multi-protein complex found in the ER membrane required for ERAD. Surprisingly, the derA null mutant displayed little in the way of significant phenotypes. However, Richie et al. discovered that levels of the UPR target gene bipA were significantly elevated in the derA null mutant. This result suggested that the UPR was compensating for loss of ERAD. To test this hypothesis, a derA/hacA double null mutant was generated. The combined loss of these two key proteins involved in ERAD and the UPR resulted in a strain with a severe reduction in hyphal growth, antifungal drug resistance, protease secretion and importantly fungal virulence.

These results offer new, and potentially exploitable, insights into filamentous fungal pathogenesis. Primarily, these results strongly suggest that for A. fumigatus, and likely other molds, the simple act of growing as a hyphal organism places significant stress on the secretory system of the fungus. While it is well established that microbial organisms, not just fungi, face significant in vivo microenvironmental stresses during pathogenesis, the idea that polarized growth itself generates a significant burden on fungal cellular homeostasis is quite novel and exciting. Given that loss of UPR and ERAD essentially resulted in a fully avirulent fungal strain, an important question to be further explored is the mechanism behind the virulence attenuation. The alterations in the cell wall, a key pathogen associated molecular pattern (PAMP), in the UPR/ERAD-deficient strain may trigger a successful host response even in an immuno-compromised animal. Alternatively, and perhaps likely given the in vitro growth results, the complexity of host tissue composition may place a significant stress on the invading fungus. Moreover, it may be possible that the secretion of important fungal effector molecules, or virulence-related molecules, is affected in the mutant strains. Regardless, it seems clear that in the absence of secretion stress homeostasis mechanisms, the fungus may simply not be able to survive in the host environment.

An intriguing question is whether unicellular fungal pathogens such as Cryptococcus neoformans or Candida albicans in its yeast form also require the UPR and ERAD to cause disease. In addition, it will be interesting to determine whether the UPR and ERAD are important in the mycelial-to-yeast transition in important endemic fungi such as Histoplasma capsulatum and Blastomyces dermatitidis. Interestingly, in C. albicans HAC1 has been observed to be required for cell wall integrity and the yeast to hyphal transition.10 This might suggest that the UPR is important for C. albicans to cause disease, however, whether the UPR as mediated by HAC1 is required for C. albicans virulence has not been tested. With regard to C. neoformans, a basidiomycete yeast that does not grow via polarized growth in vivo, it will be interesting to see if the UPR and ERAD have the same importance for pathogenesis. Clearly, given the importance of protein secretion in the fungal lifestyle, further exploration of the UPR and ERAD is warranted in the context of discovering a way to exploit these pathways for therapeutic purposes.

With regard to antifungal therapies, the increased susceptibility to all three main classes of antifungal drugs in the derA/hacA double mutant is also an exciting finding that adds to the attraction of further research on these pathways in A. fumigatus and other human, plant and insect fungal pathogens. While the mechanism behind the increased susceptibility to antifungal agents is not known, and likely multifactorial, this finding raises the intriguing idea of being able to potentiate the efficacy of currently used antifungal drugs by affecting the UPR/ERAD pathways. While these pathways are highly conserved in mammals, further study of upstream regulators and downstream effectors of the UPR/ERAD pathways in fungi may yield exploitable targets for which novel therapeutic agents can be designed. In the end, the study by Richie et al., much like Sherlock Holmes himself, has provided “elementary” new clinically relevant insights into fungal pathogenesis and the fungal lifestyle.

Acknowledgements

R.A.C. would like to thank Drs. Andrew Alspaugh and John R. Perfect at Duke University Medical Center for constructive comments on the editorial.