A role for vesicular transport of macromolecules across cell walls in fungal pathogenesis

Abstract

In our recent work, we have shown that fungal species from different phyla produce extracellular vesicles. The vesicles are heterogeneous and morphologically similar to mammalian exosomes, with intact bilayered membranes. Proteomic analyses reveal that the vesicles contain a broad array of macromolecules, many of which are associated with fungal virulence. Further, the biological import of the extracellular fungal vesicles is supported by their presence during murine cryptococcosis and the immunoreactivity of convalescent serum from patients with Cryptococcus neoformans or Histoplasma capsulatum vesicle protein extracts.

In contrast to most eukaryotic cells, fungi have complex cell walls, that could in theory provide a significant barrier to the secretion of large molecules. The discovery of trans-cell wall vesicular transport in fungi provides a solution to the problem of extracellular transport of macromolecules. Identifying similar vesicles in ascomycetes and basidiomycetes suggest that the shuttle system is ancient, predating the divergence of these branches 0.5–1.0 billion years ago. Importantly, the discovery of this trans-cell wall vesicular transport system also poses new, interesting questions for future investigations.

Eukaryotic and prokaryotic organisms have several pathways for secretion.¹ However, the best understood mechanism in eukaryotes utilizes vesicular migration from the endoplasmic reticulum to the trans face of the Golgi followed by loading into the trans-Golgi reticulum where compounds are sorted into transport vesicles that subsequently move to and fuse with the plasma membrane where the contents are released by exocytosis.² It is well established that fungi can utilize this conventional pathway for secretion.³

One alternative mechanism for secretion involves the release of vesicles into the extracellular space. The process is characterized by the formation of small vesicles formed by invagination of the limiting membrane of endocytic compartments (endosomes), and some endosomes develop internal vesicles to form multivesicular bodies. Although multivesicular bodies typically fuse with lysosomes in degradation pathways, they can also fuse with the plasma membrane where the internal vesicles are released extracellularly (exosomes).⁴

Current Knowledge of Fungal Extracellular Vesicles

At the inception of our studies with Cryptococcus neoformans, a basidiomyces, we hypothesized that the fungus utilized vesicular transport given that polysaccharide synthesis occurred intracellularly in vesicles^5,6 and that the polysaccharide was a large macromolecule with a mass of 1–7 million Daltons.⁷ The ascomycetes Histoplasma capsulatum, Candida albicans, Candida parapsilosis, Sporothrix schenckii and Saccharomyces cerevisiae are also known to produce diverse exocellular compounds^8,9 and we similarly hypothesized that these fungi utilize vesicles for transport of macromolecules. Our recent work^10–12 demonstrates that these diverse fungi produce heterogeneous extracellular vesicles that contain lipids, carbohydrates and proteins, many of which are associated with fungal virulence.

Glucuronoxylomannan (GXM) is a key factor for virulence in C. neoformans and we demonstrated that certain extracellular vesicles contained the polysaccharide.¹⁰ This finding extended the observation by Yoneda and Doering⁶ showing that a C. neoformans strain defective in the production of Sav1p, a homolog of the S. cerevisiae protein Sec4p, accumulated exocytic vesicles containing GXM at the yeast cell septum and bud during cell division. In addition to polysaccharide, well established virulence factors of C. neoformans such as glucosylceramide, urease and laccase were also vesicle associated.¹¹ Further supporting the biological relevance of the vesicles, we observed vesicles within the cell wall and extracellular vesicles associated with the fungal surface during murine cryptococcosis.¹⁰

However, the most striking finding was the identification of many diverse proteins within the extracellular vesicles. Proteomic analysis of C. neoformans and H. capsulatum vesicles identified 76 and 206 proteins, respectively.^11,12 Notably, numerous proteins associated with virulence were identified, and heat shock proteins, superoxide dismutase, thiol-specific anti-oxidants and catalases were found in both fungi. Furthermore, roughly a third of the proteins characterized in the fungal vesicles corresponded to proteins previously identified in mammalian exosomes. To test whether such vesicles were made in during infection, we examined whether H. capsulatum vesicular proteins were recognized by hyperimmune human serum and found that the serum strongly reacted with diverse vesicular proteins, including the virulence associated proteins Hsp60,^13,14 and histone 2B.¹⁵ Hence, proteins transported via vesicles appear to be involved in host-pathogen interactions.

Future Questions for Consideration

Numerous interesting questions have arisen from the discovery of vesicular trans-cellular transport in fungi, including “what are the functions, morphologies and contents of the various types of vesicles?”; “what is the mechanism for trans-cellular passage?”; and “what are the relationships between vesicles and host cells?”. For instance, our microscopy studies suggest that at least four types of vesicles are released by fungi.^10–12 This is especially important due to the potential role of extracellular vesicles as ‘virulence factor bags’. We hypothesize that vesicular transport has been developed as a mechanism for efficient delivery of factors that combat host effector responses (such as catalases and superoxide dismutases) and are toxic to host cells (such as GXM, urease and laccase) with an end result of the vesicles significantly facilitating fungal pathogenesis. However, capsular synthesis also appears to depend on vesicular delivery of GXM, which raises the question of whether the fungus can target vesicles to itself versus host cells. At present, it is not clear whether individual vesicles contain products toxic to host and factors involved in maintenance of the fungal cell (such as GXM or glucosylceramide) or whether they are independent. The current methods we use to isolate vesicles do not permit discrimination of their origin or content, but differences in size, density and enzymatic contents should permit fractionation in future studies. We do not know if vesicles are formed by processes similar to mammalian exosomes or multivesicular bodies or if they derive via membrane budding or other processes. Clearly, the finding of vesicles will lead to the development of new methods for separation and analysis that will shed light on these questions.

As noted above, the fungal cell wall is remarkably complex and the mechanism for vesicle trans-cell wall transport is unknown. Data from Yoneda and Doering⁶ suggests that vesicles with GXM are directed to the cell surface in post-Golgi vesicles or through endosome recycling. More recently, Hu et al.¹⁶ showed that C. neoformans lacking Vps34 (vacuolar protein sorting 34) had marked reduction in melanin formation suggesting that laccase-containing vesicles derive from the endocytic pathway. S. cerevisiae expresses the molecular machinery associated with multivesicular body formation and sorting,¹⁷ but their role in extracellular vesicle production has not been shown. Notably, we and others have reported the presence of pores in fungal cell walls that can be sufficient to allow the shuttling of vesicles.¹⁸ Myosin and similar motor proteins can be involved in vesicle transportation¹⁹ and a myosin analog has identified in the cell wall of Aspergillus fumigatus.²⁰ However, there is no information regarding the functionality of motor proteins within a fungal cell wall. This area of study is particularly important as the transport mechanisms are potential targets for novel antifungal drugs or vaccines, especially if the pathway is broadly conserved in fungi. Given that the fungal cell wall is significantly different from mammalian cellular structures, it is suspected that such therapeutics would have high specificity for fungal cells and low host toxicity.

Summary

We have demonstrated that diverse fungi produce extracellular vesicles that contain numerous carbohydrates, lipids and proteins.^10–12 Concentration of pathogenic determinants into vesicles appears to represent an efficient mechanism for the release of virulence factors into host tissues. Pathogenic mechanisms and secretory processes in microbes are closely associated and we advocate that the understanding of secretory mechanisms and their regulation in microbial pathogens may represent a promising strategy for the design of new drugs and prophylactic agents. The discovery of vesicles in cryptococcal culture supernatants has opened an exciting, broad avenue for exploration of fungal cell biology that impacts our understanding of macromolecular export and pathogenesis.

Addendum to: Rodrigues ML, Nakayasu ES, Oliveira DL, Nimrichter L, Nosanchuk JD, Almeida IC, Casadevall A. Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence. Eukaryot Cell. 2008;7:58–67.

Albuquerque PC, Nakayasu ES, Rodrigues ML, Frases S, Casadevall A, Zancope-Oliveira RM, Almeida IC, Nosanchuk JD. Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol. 2008;10:1695–1710.