size-matters-measurement-capsule-diameter-cryptococcus

Size Matters: Measurement of Capsule Diameter in <em>Cryptococcus neoformans</em>

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

Middle Tennessee State University

Cryptococcus neoformans (Cn) is a pathogenic yeast and the cause of cryptococcal meningitis. Prevalence of disease between males and females is skewed, with males having an increased incidence of disease. Based on the reported gender susceptibility differences to Cn in the literature, we used clinical isolates from Botswanan HIV-infected patients to test the hypothesis that different gender environments exerted different selective pressures on Cn. When we examined this data set, we found that men had significantly higher risk of death despite having significantly higher CD4(+) T lymphocyte counts upon admittance to the hospital. These observations suggested that Cn strains are uniquely adapted to different host gender environments and that the male immune response may be less efficient in controlling Cn infection. To discriminate between these possibilities, we tested whether there were phenotypic differences between strains isolated from males and females and whether there was an interaction between Cn and the host immune response. Virulence phenotypes showed that Cn isolates from females had longer doubling times and released more capsular glucoronoxylomannan (GXM). The presence of testosterone but not 17-β estradiol was associated with higher levels of GXM release for a laboratory strain and 28 clinical isolates. We also measured phagocytic efficiency, survival of Cn, and amount of killing of human macrophages by Cn after incubation with four isolates. While macrophages from females phagocytosed more Cn than macrophages from males, male macrophages had a higher fungal burden and showed increased killing by Cn. These data are consistent with the hypothesis that differential interaction between Cn and macrophages within different gender environments contribute to the increased prevalence of cryptococcosis in males. This could be related to differential expression of cryptococcal virulence genes and capsule metabolism, changes in Cn phagocytosis and increased death of Cn-infected macrophages.

The role of host gender in the pathogenesis of Cryptococcus neoformans infections.

Fungal melanins are important in the virulence of many pathogenic fungi. In this study, we examined the role of melanin in the interaction between Cryptococcus neoformans and the invertebrate host, Galleria mellonella. C. neoformans was able to melanize in the presence of G. mellonella homogenate, indicating the presence of melanin substrates. Melanization was confirmed by the recovery of acid-resistant particles that were recognized by anti-melanin antibodies. In addition, we tested the effect of fungal melanization on virulence. Surprisingly, G. mellonella larvae infected with melanized fungal cells lived longer than those infected with non-melanized fungi. When the cellular immune response of G. mellonella to melanized and non-melanized cells was compared, inflammatory nodules were observed in both groups. However the response was stronger in larvae infected with melanized cells. These results suggest that fungal melanin activates the immune response of G. mellonella, thereby resulting in the decreased virulence observed with melanized cells.

Reduced virulence of melanized Cryptococcus neoformans in Galleria mellonella.

Cryptococcus neoformans (Cn) is a common facultative intracellular pathogen that can cause life-threatening fungal meningitis in immunocompromised individuals. Shortly after infection, Cn is detectable as both extra- and intracellular yeast particles, with Cn being capable of establishing long-lasting latent infections within host macrophages. Although recent studies have shown that shed capsular polysaccharides and intact extracellular Cn can compromise macrophage function through modulation of NF-κB signaling, it is currently unclear whether intracellular Cn also affects NF-κB signaling. Utilizing live cell imaging and computational modeling, we find that extra- and intracellular Cn support distinct modes of NF-κB signaling in cultured murine macrophages. Specifically, in RAW 264.7 murine macrophages treated with extracellular glucuronoxylomannan (GXM), the major Cn capsular polysaccharide, LPS-induced nuclear translocation of p65 is inhibited, whereas in cells with intracellular Cn, LPS-induced nuclear translocation of p65 is both amplified and sustained. Mathematical simulations and quantification of nascent protein expression indicate that this is a possible consequence of Cn-induced "translational interference," impeding IκBα resynthesis. We also show that long term Cn infection induces stable nuclear localization of p65 and IκBα proteins in the absence of additional pro-inflammatory stimuli. In this case, nuclear localization of p65 is not accompanied by TNFα or inducible NOS (iNOS) expression. These results demonstrate that capsular polysaccharides and intact intracellular yeast manipulate NF-κB via multiple distinct mechanisms and provide new insights into how Cn might modulate cellular signaling at different stages of an infection.

Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans.

The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.

A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals.

Studies show there is an increasing rate of fungal infections, especially in immunocompromised patients and treatments for fungal genera, such as,, and, carry significant cytotoxicity with an increasing prevalence of antifungal resistance. We have previously reported a high-throughput assay for identifying peptoids with antimicrobial properties from combinatorial libraries. Here we report the application of this assay in identifying a peptoid with antifungal properties against. Termed AEC5, this peptoid has comparable potency to existing clinical antifungal agents, excellent stability, and minimal cytotoxicity in mammalian cells.

Discovery and Characterization of a Peptoid with Antifungal Activity against.

Pathogenic cryptococci are encapsulated yeast that can cause severe meningoencephalitis. Existing therapeutic options are dated and there is a growing need for new alternative antifungal agents for these fungi. Here we report novel inhibition of pathogenic cryptococci by the antimicrobial lectin Scytovirin. Inhibition was most potent againstvarand, with MFC values of 500 nM. Scytovirin binding was localized to the cell wall and shown to affect capsule size and release. No effect was observed on melanization or with cells grown in the presence the cell wall stressor Congo red. Synergy with existing antifungals was indicated, most strongly for amphotericin B. Overall, Scytovirin serves as a much needed new avenue for anticryptococcal development.

Article	Year
The role of host gender in the pathogenesis of Cryptococcus neoformans infections. PloS one\| PubMed ID: 23741297	2013
Reduced virulence of melanized Cryptococcus neoformans in Galleria mellonella. Virulence\| PubMed ID: 24846144	2014
Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans. The Journal of biological chemistry\| PubMed ID: 27231343	2016
A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals. PLoS pathogens\| PubMed ID: 27583447	2016
Discovery and Characterization of a Peptoid with Antifungal Activity against. ACS medicinal chemistry letters\| PubMed ID: 27994753	2016
Novel Antifungal Activity for the Lectin Scytovirin: Inhibition ofand. Frontiers in microbiology\| PubMed ID: 28536555	2017

Erin E. McClelland

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