Hansong Ma

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Expulsion of Live Pathogenic Yeast by Macrophages

Phagocytic cells, such as neutrophils and macrophages, perform a critical role in protecting organisms from infection by engulfing and destroying invading microbes . Although some bacteria and fungi have evolved strategies to survive within a phagocyte after uptake, most of these pathogens must eventually kill the host cell if they are to escape and infect other tissues . However, we now demonstrate that the human fungal pathogen Cryptococcus neoformans is able to escape from within macrophages without killing the host cell by a novel expulsive mechanism. This process occurs in both murine J774 cells and primary human macrophages. It is extremely rapid and yet can occur many hours after phagocytosis of the pathogen. Expulsion occurs independently of the initial route of phagocytic uptake and does not require phagosome maturation . After the expulsive event, both the host macrophage and the expelled C. neoformans appear morphologically normal and continue to proliferate, suggesting that this process may represent an important mechanism by which pathogens are able to escape from phagocytic cells without triggering host cell death and thus inflammation .

The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation

In 1999, the population of Vancouver Island, Canada, began to experience an outbreak of a fatal fungal disease caused by a highly virulent lineage of Cryptococcus gattii. This organism has recently spread to the Canadian mainland and Pacific Northwest, but the molecular cause of the outbreak remains unknown. Here we show that the Vancouver Island outbreak (VIO) isolates have dramatically increased their ability to replicate within macrophages of the mammalian immune system in comparison with other C. gattii strains. We further demonstrate that such enhanced intracellular parasitism is directly linked to virulence in a murine model of cryptococcosis, suggesting that this phenotype may be the cause of the outbreak. Finally, microarray studies on 24 C. gattii strains reveals that the hypervirulence of the VIO isolates is characterized by the up-regulation of a large group of genes, many of which are encoded by mitochondrial genome or associated with mitochondrial activities. This expression profile correlates with an unusual mitochondrial morphology exhibited by the VIO strains after phagocytosis. Our data thus demonstrate that the intracellular parasitism of macrophages is a key driver of a human disease outbreak, a finding that has significant implications for a wide range of other human pathogens.

Ancient Dispersal of the Human Fungal Pathogen Cryptococcus gattii from the Amazon Rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Virulence in Cryptococcus species.

Cryptococcus neoformans and Cryptococcus gattii are the cause of life-threatening meningoencephalitis in immunocompromised and immunocompetent individuals respectively. The increasing incidence of cryptococcal infection as a result of the AIDS epidemic, the recent emergence of a hypervirulent cryptococcal strain in Canada and the fact that mortality from cryptococcal disease remains high have stimulated intensive research into this organism. Here we outline recent advances in our understanding of C. neoformans and C. gattii, including intraspecific complexity, virulence factors, and key signaling pathways. We discuss the molecular basis of cryptococcal virulence and the interaction between these pathogens and the host immune system. Finally, we discuss future challenges in the study and treatment of cryptococcosis.

Transmission of mitochondrial mutations and action of purifying selection in Drosophila melanogaster

It is not known how selection affects mutations in the multiple copies of the mitochondrial genome. We transferred cytoplasm between D. melanogaster embryos carrying mitochondrial mutations to create heteroplasmic lines transmitting two mitochondrial genotypes. Increased temperature imposed selection against a temperature-sensitive mutation affecting cytochrome oxidase, driving decreases in the abundance of the mutant genome over successive generations. Selection did not influence the health or fertility of the flies but acted during midoogenesis to influence competition between the genomes. Mitochondria might incur an advantage through selective localization, survival or proliferation, yet timing and insensitivity to park mutation suggest that preferential proliferation underlies selection. Selection drove complete replacement of the temperature-sensitive mitochondrial genome by a wild-type genome but also stabilized the multigenerational transmission of two genomes carrying complementing detrimental mutations. While they are so balanced, these stably transmitted mutations have no detrimental phenotype, but their segregation could contribute to disease phenotypes and somatic aging.

Sex-Dependent Resistance to the Pathogenic Fungus Cryptococcus neoformans

Sex differences occur in most species and affect a variety of biological traits including morphology, behavior, and life history. The nematode Caenorhabditis elegans exists as a population of self-fertile hermaphrodites with occasional males, which differ anatomically and behaviorally from hermaphrodites. Here we show that male C. elegans also differ from hermaphrodites in their susceptibility to a fungal pathogen, Cryptococcus neoformans. Wild-type males show greater resistance than hermaphrodite animals to killing by this pathogen and this resistance can be induced in hermaphrodite animals by inappropriate activation of the male sex-determination pathway. Resistance is molecularly determined, rather than resulting from behavioral changes or reproductive differences, and requires the activity of the stress-response transcription factor DAF-16. Finally, we demonstrate that resistance to C. neoformans correlates broadly with longevity within the Caenorhabditis genus. Our results hint at an overlap between the pathways controlling immunity and longevity and raise the possibility that differential regulation of these pathways may contribute to sex-dependent and species-dependent variation.

Transmission of Hypervirulence Traits via Sexual Reproduction within and between Lineages of the Human Fungal Pathogen Cryptococcus gattii

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal.

Mitochondria and the regulation of hypervirulence in the fatal fungal outbreak on Vancouver Island

In our recent paper, we demonstrated that the hypervirulence exhibited by a lineage of the fatal fungal pathogen Cryptococcus gattii is associated with its mitochondrial gene expression and an unusual mitochondrial morphology. As an important organelle, the mitochondrion has been linked to various cellular activities, but its role in modulating virulence of pathogens remains unclear. In this addendum, the potential role of mitochondria in determining virulence in eukaryotic pathogens is discussed along with future experiments that may lead to an improved understanding of this topic.

Selfish drive can trump function when animal mitochondrial genomes compete

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection. In contrast, matchups between distantly related genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome, leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes showed that the noncoding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, in each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection, promoting change in the sequences influencing transmission.