Measuring Laccase Activity and Melanin Production in Cryptococcus neoformans.

Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Cryptococcal Virulence in Humans: Learning From Translational Studies With Clinical Isolates.

Cryptococcosis, an invasive mycosis caused by Cryptococcus spp, kills between 20% and 70% of the patients who develop it. There are no vaccines for prevention, and treatment is based on a limited number of antifungals. Studying fungal virulence and how the host responds to infection could lead to new therapies, improving outcomes for patients. The biggest challenge, however, is that experimental cryptococcosis models do not completely recapitulate human disease, while human experiments are limited due to ethical reasons. To overcome this challenge, one of the approaches used by researchers and clinicians is to: 1) collect cryptococcal clinical isolates and associated patient data; 2) study the set of isolates in the laboratory (virulence and host-pathogen interaction variables, molecular markers); 3) correlate the laboratory and patient data to understand the roles fungal attributes play in the human disease. Here we review studies that have shed light on the cryptococcosis pathophysiology using these approaches, with a special focus on human disease. Isolates that more effectively evade macrophage responses, that secrete more laccase, melanize faster and have larger capsules in the cerebrospinal fluid are associated with poorer patient outcomes. Additionally, molecular studies have also shown that cryptococcal clades vary in virulence, with clinical impact. Limitations of those studies include the use of a small number of isolates or retrospectively collected clinical data. The fact that they resulted in very important information is a reflection of the impact this strategy has in understanding cryptococcosis and calls for international collaboration that could boost our knowledge.

Thioredoxin Reductase 1 Is a Highly Immunogenic Cell Surface Antigen in Paracoccidioides spp., Candida albicans, and Cryptococcus neoformans.

The increasing number of immunocompromised people has made invasive fungal infections more common. The antifungal armamentarium, in contrast, is limited to a few classes of drugs, with frequent toxicity and low efficacy pointing to the need for new agents. Antibodies are great candidates for novel antifungals, as their specificity can result in lower toxicity. Additionally, the immunomodulatory activity of antibodies could treat the underlying cause of many invasive mycoses, immune disfunction. In a previous comparative genomics study, we identified several potential targets for novel antifungals. Here we validate one of these targets, thioredoxin reductase (TRR1), to produce antibodies that could be useful therapeutic tools. Recombinant TRR1 proteins were produced by heterologous expression in Escherichia coli of genes encoding the proteins from Candida albicans, Cryptococcus neoformans, and Paracoccidioides lutzii. These proteins were then used to immunize mice, followed by detection of serum antibodies against them by ELISA and western blot. A first set of experiments in which individual mice were immunized repeatedly with TRR1 from a single species showed that all three were highly immunogenic, inducing mostly IgG1 antibodies, and that antibodies produced against one species cross-reacted with the others. In a second experiment, individual mice were immunized three times, each with the protein from a different species. The high titers of antibodies confirmed the presence of antigenic epitopes that were conserved in fungi but absent in humans. Immunofluorescence with sera from these immunized mice detected the protein in the cytoplasm and on the cell surface of fungi from all three species. These results validate TRR1 as a good target for potentially broad-spectrum antifungal antibodies.