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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 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.

Suppressing cytokine responses has frequently been shown to have promising therapeutic effects for many chronic inflammatory and autoimmune diseases. However, the severe side effects associated with the long-term use of current treatments, such as allergic reactions and increased risk of stroke, have focused attention towards the targeting of intracellular signaling mechanisms, such as NF-κB, that regulate inflammation. We synthesized a series of non-natural aurone derivatives and investigated their ability to suppress pro-inflammatory signaling in human monocyte (THP-1) and murine macrophage-like (RAW 267.4) cell lines. One of these derivatives, (Z)-2-((5-(hydroxymethyl) furan-2-yl) methylene) benzofuran-3(2H)-one (aurone 1), was found to inhibit LPS-induced secretion of the pro-inflammatory cytokines, tumor-necrosis factor α (TNFα), interleukin 1β (IL-1β), and IL-8 by THP-1 cells. To investigate the mechanism, we probed the effect of aurone 1 on LPS-induced MAPK and NF-κB signaling in both THP-1 and RAW264.7. While aurone 1 pre-treatment had no effect on the phosphorylation of ERK, JNK, or p38 MAPK, it strongly suppressed activation of IKK-β, as indicated by attenuation of Ser176/180 phosphorylation, resulting in decreased phosphorylation of p65 (ser536) as well as phosphorylation (ser32) and degradation of IκBα. Consistent with this, aurone 1 significantly reduced LPS-stimulated nuclear translocation of p65-containing NF-κB transcription factors and expression of an mCherry reporter of TNFα gene transactivation in RAW264.7 cells. Inhibition of TNFα expression at the transcription level was also demonstrated in THP-1 by qRT-PCR. In addition to its effects on cytokine expression, aurone 1 pre-treatment decreased expression of iNOS, a bona fide NF-κB target gene and marker of macrophage M1 polarization, resulting in decreased NO production in RAW264.7 cells. Together, these data indicate that aurone 1 may have the potential to function as a pharmacological agent for the treatment of chronic inflammation disorders.

Suppression of LPS-induced NF-κB activity in macrophages by the synthetic aurone, (Z)-2-((5-(hydroxymethyl) furan-2-yl) methylene) benzofuran-3(2H)-one.

David E. Nelson

Department of Biology

Middle Tennessee State University

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

David E. Nelson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

Middle Tennessee State University

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

Department of Biology