Isolation and Characterization of
            <i>Cryptococcus neoformans</i>
            Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

Botts, Michael R.; Giles, Steven S.; Gates, Marcellene A.; Kozel, Thomas R.; Hull, Christina M.

doi:10.1128/ec.00352-08

Cited by 81 publications

(100 citation statements)

References 52 publications

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“…No sterigmata on the basidia were observed in these crosses, similar to the findings for C. neoformans. Different C. gattii molecular types were compared to spores produced by serotype A and D strains (see findings are in accord with data from previous and recent studies (3,47) and reveal additional insights into spore morphology. Spore germination signals.…”

Section: Resultssupporting

confidence: 70%

Spores as Infectious Propagules ofCryptococcus neoformans

Velagapudi¹,

Hsueh²,

et al. 2009

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Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite-or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (ϳ95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules.

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Section: Resultssupporting

confidence: 70%

Spores as Infectious Propagules ofCryptococcus neoformans

Velagapudi¹,

Hsueh²,

et al. 2009

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“…The resulting plasmid encodes a histone 3 promoter-driven enhanced green fluorescent protein (EGFP) with an 80-amino-acid NLS and a nourseothricin resistance (NAT r ) cassette. This integrating plasmid was then transformed via bombardment into C. neoformans B3502 to generate strain CHY3135 (47). This strain produces bright GFP that is localized to the nucleus.…”

Section: Methodsmentioning

confidence: 99%

“…All were handled using standard techniques and media as described previously (45,46). Spores were purified from crosses using density gradient purification as described previously (47). Briefly, mixed haploid yeast cells of opposite mating types (CHY3515 and CHY3516) were spotted onto V8 solid medium at a ratio of 1:1 and a total optical density at 600 nm (OD 600 ) of 1.0.…”

Section: Methodsmentioning

confidence: 99%

A Zebrafish Model of Cryptococcal Infection Reveals Roles for Macrophages, Endothelial Cells, and Neutrophils in the Establishment and Control of Sustained Fungemia

et al. 2016

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Cryptococcal meningoencephalitis is a fungal infection that predominantly affects immunocompromised patients and is uniformly fatal if left untreated. Timely diagnosis is difficult, and screening or prophylactic measures have generally not been successful. Thus, we need a better understanding of early, asymptomatic pathogenesis. Inhaled cryptococci must survive the host immune response, escape the lung, and persist within the bloodstream in order to reach and invade the brain. Here we took advantage of the zebrafish larval infection model to assess the process of cryptococcal infection and disease development sequentially in a single host. Using yeast or spores as infecting particles, we discovered that both cell types survived and replicated intracellularly and that both ultimately established a sustained, low-level fungemia. We propose that the establishment and maintenance of this sustained fungemia is an important stage of disease progression that has been difficult to study in other model systems. Our data suggest that sustained fungemia resulted from a pattern of repeated escape from, and reuptake by, macrophages, but endothelial cells were also seen to play a role as a niche for cryptococcal survival. Circulating yeast collected preferentially in the brain vasculature and eventually invaded the central nervous system (CNS). As suggested previously in a mouse model, we show here that neutrophils can play a valuable role in limiting the sustained fungemia, which can lead to meningoencephalitis. This early stage of pathogenesis-a balanced interaction between cryptococcal cells, macrophages, endothelial cells, and neutrophils-could represent a window for timely detection and intervention strategies for cryptococcal meningoencephalitis. R ecent data from Uganda and Tanzania indicate that there are over 10,000 cases of HIV-related cryptococcosis per year in these countries alone (1, 2). Of those, roughly 5,000 to 8,000 (54 to 79%) are fatal. At a global scale, the survival rate is similarly poor, and over 600,000 deaths are attributed to Cryptococcus neoformans every year (3). The high mortality rate is in part due to difficulties in timely diagnosis; even in susceptible hosts, clinical signs of the initial pulmonary infection can be subtle or essentially absent (4). Studies in this population using serum cryptococcal antigen testing suggest that antigenemia (which may or may not represent actual fungemia) is a harbinger of meningoencephalitis but can precede it by weeks or more (5, 6). Once the infection has progressed to clinical meningoencephalitis, the prognosis is grim even with aggressive treatment. Beyond the HIV-infected population, a growing transplant and otherwise immunosuppressed population is also vulnerable, and even an immunocompetent host may be susceptible to infection and disease caused by the closely related species Cryptococcus gattii (7). An understanding of the events prior to fulminant disease is critical to better management of patients at risk for cryptococcal meningoencephalitis.While ...

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“…grubii strains (4). Our initial biochemical characterization of C. neoformans spores revealed that they possessed a cell surface composition different from yeast but physical properties similar to other fungal spores (4). These observations led us to speculate that, like other pathogenic fungi, the infectious life cycle of C. neoformans might include an infectious spore form that transitions into a pathogenic form in the lungs.…”

mentioning

confidence: 99%

Elucidating the Pathogenesis of Spores from the Human Fungal PathogenCryptococcus neoformans

et al. 2009

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Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal ␤-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.

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Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

Cited by 81 publications

References 52 publications

Spores as Infectious Propagules ofCryptococcus neoformans

Spores as Infectious Propagules ofCryptococcus neoformans

A Zebrafish Model of Cryptococcal Infection Reveals Roles for Macrophages, Endothelial Cells, and Neutrophils in the Establishment and Control of Sustained Fungemia

Elucidating the Pathogenesis of Spores from the Human Fungal PathogenCryptococcus neoformans

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