Cryptococcus strains with different pathogenic potentials have diverse protein secretomes.

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.

Nuclear dynamics in a fungal chimera.

A fungal colony is a syncytium composed of a branched and interconnected network of cells. Chimerism endows colonies with increased virulence and ability to exploit nutritionally complex substrates. Moreover, chimera formation may be a driver for diversification at the species level by allowing lateral gene transfer between strains that are too distantly related to hybridize sexually. However, the processes by which genomic diversity develops and is maintained within a single colony are little understood. In particular, both theory and experiments show that genetically diverse colonies may be unstable and spontaneously segregate into genetically homogenous sectors. By directly measuring patterns of nuclear movement in the model ascomycete fungus Neurospora crassa, we show that genetic diversity is maintained by complex mixing flows of nuclei at all length scales within the hyphal network. Mathematical modeling and experiments in a morphological mutant reveal some of the exquisite hydraulic engineering necessary to create the mixing flows. In addition to illuminating multinucleate and multigenomic lifestyles, the adaptation of a hyphal network for mixing nuclear material provides a previously unexamined organizing principle for understanding morphological diversity in the more-than-a-million species of filamentous fungi.

Physiological significance of network organization in fungi.

The evolution of multicellularity has occurred in diverse lineages and in multiple ways among eukaryotic species. For plants and fungi, multicellular forms are derived from ancestors that failed to separate following cell division, thus retaining cytoplasmic continuity between the daughter cells. In networked organisms, such as filamentous fungi, cytoplasmic continuity facilitates the long-distance transport of resources without the elaboration of a separate vascular system. Nutrient translocation in fungi is essential for nutrient cycling in ecosystems, mycorrhizal symbioses, virulence, and substrate utilization. It has been proposed that an interconnected mycelial network influences resource translocation, but the theory has not been empirically tested. Here we show, by using mutants that disrupt network formation in Neurospora crassa (Δso mutant, no fusion; ΔPrm-1 mutant, ∼50% fusion), that the translocation of labeled nutrients is adversely affected in homogeneous environments and is even more severely impacted in heterogeneous environments. We also show that the ability to share resources and genetic exchange between colonies (via hyphal fusion) is very limited in mature colonies, in contrast to in young colonies and germlings that readily share nutrients and genetic resources. The differences in genetic/resource sharing between young and mature colonies were associated with variations in colony architecture (hyphal differentiation/diameters, branching patterns, and angles). Thus, the ability to share resources and genetic material between colonies is developmentally regulated and is a function of the age of a colony. This study highlights the necessity of hyphal fusion for efficient nutrient translocation within an N. crassa colony but also shows that established N. crassa colonies do not share resources in a significant manner.

Genes encoding a striatin-like protein (ham-3) and a forkhead associated protein (ham-4) are required for hyphal fusion in Neurospora crassa.

Cell-cell fusion during fertilization and between somatic cells is an integral process in eukaryotic development. In Neurospora crassa, the hyphal anastomosis mutant, ham-2, fails to undergo somatic fusion. In both humans and Saccharomyces cerevisiae, homologs of ham-2 are found in protein complexes that include homologs to a striatin-like protein and a forkhead-associated (FHA) protein. We identified a striatin (ham-3) gene and a FHA domain (ham-4) gene in N. crassa; strains containing mutations in ham-3 and ham-4 show severe somatic fusion defects. However, ham-3 and ham-4 mutants undergo mating-cell fusion, indicating functional differences in somatic versus sexual fusion events. The ham-2 and ham-3 mutants are female sterile, while ham-4 mutants are fertile. Homozygous crosses of ham-2, ham-3 and ham-4 mutants show aberrant meiosis and abnormally shaped ascospores. These data indicate that, similar to humans, the HAM proteins may form different signaling complexes that are important during both vegetative and sexual development in N. crassa.

Cell fusion in the filamentous fungus, Neurospora crassa.

Hyphal fusion occurs at different stages in the vegetative and sexual life cycle of filamentous fungi. Similar to cell fusion in other organisms, the process of hyphal fusion requires cell recognition, adhesion, and membrane merger. Analysis of the hyphal fusion process in the model organism Neurospora crassa using fluorescence and live cell imaging as well as cell and molecular biological techniques has begun to reveal its complex cellular regulation. Several genes required for hyphal fusion have been identified in recent years. While some of these genes are conserved in other eukaryotic species, other genes encode fungal-specific proteins. Analysis of fusion mutants in N. crassa has revealed that genes previously identified as having nonfusion-related functions in other systems have novel hyphal fusion functions in N. crassa. Understanding the molecular basis of cell fusion in filamentous fungi provides a paradigm for cell communication and fusion in eukaryotic organisms. Furthermore, the physiological and developmental roles of hyphal fusion are not understood in these organisms; identifying these mechanisms will provide insight into environmental adaptation.