Melanization is associated with virulence in Cn and other fungi. Melanins are dark pigments composed of a polymer of indole and phenol subunits of unknown secondary structure. They absorb light across the UV and visible spectrum, have high physical and chemical strength and can resist degradation, even by strong acids. Lastly, melanins have redox properties and can scavenge free radicals (Hill, 1992; Nosanchuk & Casadevall, 2003b).

In Cn, melanin is observed as an electron-dense layer in the cell wall (Nosanchuk & Casadevall, 2003a). Treatment of melanized cells with strong acids and denaturants yields particles of melanin that retain the original cell shape. Because of this they are referred to as melanin 'ghosts' (Wang et al., 1996). High-resolution microscopy of melanin architecture in Cn shows a granular surface and multiple layers (Eisenman et al., 2005). Melanin in Cn has pores that presumably allow melanized cells to obtain nutrients and export products. NMR cryporometry measurements show that melanin ghosts contain pores of 10-50 A, with the porosity decreasing with time of melanization (Eisenman et al., 2005). Using size exclusion chromatography, Jacobsen and Ikeda (2005) found that Cn melanin has pores with a maximum size of 4-16 nm. Together, these studies show that rather than being a solid layer, melanin in Cn has a complex architecture that includes pores large enough to accommodate nutrients.

The development of monoclonal antibodies to Cn melanin demonstrates that melanin is a target of the immune response (Nosanchuk et al., 1998). In addition, the antibodies are useful in studying Cn melanin. The antibodies bind to yeast in the lung and brain tissue of animals infected with Cn, indicating that Cn is melanized in these tissues (Rosas et al., 2000). Interestingly, the antibodies are able to inhibit growth of Cn in vitro under melanizing conditions and they also protect mice from Cn infection (Rosas et al., 2001).

In Cn, melanin polymerization is catalyzed by the enzyme laccase, encoded by the CNLAC1 and LAC2 genes (Missall et al., 2005; Pukkila-Worley et al., 2005; Williamson, 1994). Of the two genes, LAC1 is the major contributor to melanin production. As shown by insertional mutagenesis, a number of other genes are required for melanization, including genes involved in metal transport and metabolism and cell wall biosynthesis (Walton et al., 2005; Zhu & Williamson, 2003).

Melanization of Cn in vitro requires the addition of exogenous laccase substrate. In vitro melanization can be induced by the addition of catecholamine neurotransmitters to the culture medium. These include l-dopa, norepinephrine, and dopamine, for example. Comparison of melanin particles produced from different substrates reveals significant differences between the pigments produced by Cn. The structure and amount of incorporation of the different substrates into pigment varies. Furthermore, not all of the pigments have all the hallmarks of melanins, such as a stable free radical signal, or specific oxidative breakdown products (Garcia-Rivera et al., 2005a). It is assumed that, in vivo, Cn uses host neurotransmitters to produce melanin. However, this has yet to be shown. The substrates used by Cn in the environment to produce melanin are likewise unknown. However, recent work suggests that catecholamines produced by other microbes may be a source of substrates as Cn produces melanin when cocultured with the bacterium, Klebsiella aerogenes (Frases et al., 2006a). In addition, Cn has been shown to be able to use the bacterial melanin precursor homogentisic acid for melanization (Frases et al., 2006b).

The question of in vivo melanization and its importance to virulence is the subject of some debate and recent studies address this issue. Laccase is expressed during infection, consistent with in vivo melanization (Garcia-Rivera et al., 2005b; Waterman et al., 2006). Melanized Cn is detectable in tissue samples from infected patients. Furthermore, melanin particles isolated from these tissues by chemical extraction are similar to in vitro melanized Cn when viewed by electron microscopy (Nosanchuk et al., 2000). Similar results are obtained in experimental mouse infections (Rosas et al., 2000). The results of other in vivo studies highlight the importance of melaniza-tion to virulence. Experimental cryptococcosis in a mouse model can be improved by treatment with glyphosate, an inhibitor of melanization (Nosanchuk et al., 2001). Together, these data show that melanization of Cn occurs in vivo.

A number of in vitro and in vivo studies focus on the role of melanin in virulence. Melanin is a negatively charged, hydrophobic surface capable of binding many substances including antibiotics and heavy metals (Nosanchuk & Casadevall, 2003b). Cn melanin binds to a number of proteins, as well antimicrobial peptides normally produced by the host. Melanin protects Cn from the antimicrobial activity of the peptides protegrin and defensin, by binding and sequestering them (Doering et al., 1999). When wild type and CNLAC1 deletion strains are used to infect mice, there is no difference in lung clearance between the wild type and deletion strains. However, the fungal burden in the brain and spleen is higher for wild type Cn, showing that laccase, and likely melanization, is required for dissemination and/or survival outside the lung (Noverr et al., 2004).

Alternate functions for laccase in virulence, including protection from oxidative stress are proposed. One study found that strains lacking LAC1 are more susceptible to killing by alveolar macrophages, even in the absence of l-dopa, the substrate for melanization (Liu et al., 1999). Oxidation of iron from Fe2+ to Fe3+ by laccase may protect Cn from free radicals generated by Fe2+, suggesting an alternate function for laccase. Also, the LAC1 and LAC2 genes have altered transcription in response to oxidative and nitrosative stress, consistent with the view that laccase has an important antioxidant function in Cn. Finally, LAC2 has genetic interactions with the thiol-specific antioxidant TSA1 (Missall et al., 2005). The role of antioxi-dant genes in Cn virulence is discussed in depth below. A second connection between laccase and response to stress exists as expression of laccase is regulated in part by the heat-shock proteins Hsp70 and HSF (Zhang et al., 2006).

Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

The term vaginitis is one that is applied to any inflammation or infection of the vagina, and there are many different conditions that are categorized together under this ‘broad’ heading, including bacterial vaginosis, trichomoniasis and non-infectious vaginitis.

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