Arturo Casadevall

Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans . The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology.

Microbial secretion is integral for regulating cell homeostasis as well as releasing virulence factors during infection. The genes encoding phosphatidylserine synthase ( CHO1 ) and phosphatidylserine decarboxylase ( PSD1 and PSD2 ) are Candida albicans genes involved in phospholipid biosynthesis, and mutations in these genes affect mitochondrial function, cell wall thickness, and virulence in mice. We tested the roles of these genes in several agar-based secretion assays and observed that the cho1 Δ/Δ and psd1 Δ/Δ psd2 Δ/Δ strains manifested less protease and phospholipase activity. Since extracellular vesicles (EVs) are surrounded by a lipid membrane, we investigated the effects of these mutations on EV structure, composition, and biological activity. The cho1 Δ/Δ mutant releases EVs comparable in size to wild-type EVs, but EVs from the psd1 Δ/Δ psd2 Δ/Δ strain are much larger than those from the wild type, including a population of >100-nm EVs not observed in the EVs from the wild type. Proteomic analysis revealed that EVs from both mutants had a significantly different protein cargo than that of EVs from the wild type. EVs were tested for their ability to activate NF-κB in bone marrow-derived macrophage cells. While wild-type and psd1 Δ/Δ psd2 Δ/Δ mutant-derived EVs activated NF-κB, the cho1 Δ/Δ mutant-derived EV did not. These studies indicate that the presence and absence of these C. albicans genes have qualitative and quantitative effects on EV size, composition, and immunostimulatory phenotypes that highlight a complex interplay between lipid metabolism and vesicle production.

Proof of the Germ theory of disease and acceptance of Koch’s postulates in the late 1890’s launched the fields of microbial pathogenesis and infectious diseases and provided the conceptual framework that has guided thought and research in these fields. A central tenet that emerged from studies with microbes that fulfilled Koch’s postulates was that microbes that caused disease had characteristics that allowed them to do so, with the corollary that microbes that did not cause disease lacked disease-causing determinants. This observation, which held true for many diseases that were known to cause disease in the late 19th century, such as toxin-producing and encapsulated bacteria, led to the view that the ability to cause disease rested with microbes and reflected the activity of specific determinants, or virulence factors. With the dawn of the 20th century, efforts to neutralize virulence factors were under development and ultimately translated into anti-microbial therapy in the form of antibodies targeted to toxins and polysaccharide capsules. However, the 20th century progressed, antibiotics were identified and developed as therapy for infectious diseases while other medical advances, such as specialized surgeries, intensive care units, intravenous catheters, and cytotoxic chemotherapy became commonplace in resourced nations. An unintended consequence of many of these advances was that they resulted in immune impairment. Similarly, HIV/AIDS, which emerged in the late 1970’s also produced profound immune impairment. Unexpectedly, the prevailing view that microbes were the sole perpetrators of virulence was untenable. Microbes that were rarely if ever associated with disease emerged as major causes of disease in people with impaired immunity. This phenomenon revealed that available explanations for microbial infectiveness and virulence were flawed. In this review, we discuss the question ‘what is infectiveness’ based on the tenets of the Damage-response framework.

Human infection with Cryptococcus neoformans, a common fungal pathogen, follows deposition of yeast spores in the lung alveoli. The subsequent host–pathogen interaction can result in eradication, latency, or extrapulmonary dissemination. Successful control of C. neoformans infection is dependent on host macrophages, but macrophages display little ability to kill C. neoformans in vitro. Recently, we reported that ingestion of C. neoformans by mouse macrophages induces early cell cycle progression followed by mitotic arrest, an event that almost certainly reflects host cell damage. The goal of the present work was to understand macrophage pathways affected by C. neoformans toxicity. Infection of macrophages by C. neoformans was associated with alterations in protein translation rate and activation of several stress pathways, such as hypoxia-inducing factor-1-α, receptor-interacting protein 1, and apoptosis-inducing factor. Concomitantly we observed mitochondrial depolarization in infected macrophages, an observation that was replicated in vivo. We also observed differences in the stress pathways activated, depending on macrophage cell type, consistent with the nonspecific nature of C. neoformans virulence known to infect phylogenetically distant hosts. Our results indicate that C. neoformans infection impairs multiple host cellular functions and undermines the health of these critical phagocytic cells, which can potentially interfere with their ability to clear this fungal pathogen.

From the viewpoint of fungal virulence in mammals, thermal tolerance can be defined as the ability to grow in the 35°C to 40°C range, which is essential for inhabiting these hosts. We used archival information in a fungal collection to analyze the relationship between thermal tolerance and genetic background for over 4,289 yeast strains belonging to 1,054 species. Fungal genetic relationships were inferred from hierarchical trees based on pairwise alignments using the rRNA internal transcribed spacer and large subunit rDNA (LSU) sequences. In addition, we searched for correlations between thermal tolerance and other archival information including antifungal susceptibility, carbon sources, and fermentative capacity. Thermal tolerance for growth at mammalian temperatures was not monophyletic, with thermally tolerant species being interspersed among families that include closely related species that are not thermal tolerant. Thermal tolerance and resistance to antifungal drugs were not correlated, suggesting that these two properties evolved independently. Nevertheless, the ability to grow at higher temperatures did correlate with origin from lower geographic latitudes, capacity for fermentation and assimilation of certain carbon sources. Thermal tolerance was significantly more common among ascomycetous than basidiomycetous yeasts, suggesting an explanation for the preponderance of ascomycetous yeasts among human pathogenic fungi. Analysis of strain maximum tolerable temperature as a function of collection time suggested that basidiomycetous yeasts are rapidly adapting to global warming. The analysis identified genera with a high prevalence of the thermal-tolerant species that could serve as sources of emerging pathogenic fungi.

Since proof of the germ theory of disease in the late 19^th century a major focus of the fields of microbiology and infectious diseases has been to seek differences between pathogenic and non-pathogenic microbes and the role that the host plays in microbial pathogenesis. Remarkably, despite the increasing recognition that host immunity plays a role in microbial pathogenesis, there has been little discussion about what constitutes a host. Historically, hosts have been viewed in the context of their fitness or immunological status, and characterized by adjectives such as immune, immunocompetent, immunosuppressed, immunocompromised, or immunologically impaired. However, in recent years it has become apparent that the microbiota has profound effects on host homeostasis and susceptibility to microbial diseases in addition to its effects on host immunity. This raises the question of how to incorporate the microbiota into defining a host. This definitional problem is further complicated because neither host nor microbial properties are adequate to predict the outcome of host-microbe interaction because this outcome exhibits emergent properties. In this essay we revisit the ‘damage-response framework’ (DRF) of microbial pathogenesis and demonstrate how it can incorporate the rapidly accumulating information being generated by the microbiome revolution. We use the tenets of the DRF to put forth the following definition of a host: a host is an entity that houses an associated microbiome/microbiota and interacts with microbes such that the outcome results in damage, benefit, or indifference thus resulting in the states of symbiosis, colonization, commensalism, latency and disease.

Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.