Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis
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Summary: The description of how faster melanization is associated with LC3-mediated phagocytosis evasion, virulence and outcomes in humans is interesting and does provide some new information. In general, the study has been executed well, with clear articulation of the results and appending conclusions. However, the work falls short of investigating any substantive mechanistic basis for the observations and how they relate to the broader metabolism in Cryptococcus. .
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Abstract
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.
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Reviewer #2:
The authors quantify virulence factors in Cryptococcus neoformans and C. gattii in a large number of clinical isolates and correlate these virulence factors to survival in a g. mellonella infection model and to the clinical outcome. The authors found a correlation between secreted laccases and disease outcome in patients. In addition, the authors show that a faster melanization rate in C. neoformans correlated with phagocytosis evasion, virulence in the g. mellonella model and worse prognosis in humans.
The manuscript is well structured with an appropriate abstract summing the main findings, a clear introduction, well described methods section and appropriate number of figures and tables. The results are clearly described.
The authors identify and acknowledge the most important limitation of the study: line 365-366 the …
Reviewer #2:
The authors quantify virulence factors in Cryptococcus neoformans and C. gattii in a large number of clinical isolates and correlate these virulence factors to survival in a g. mellonella infection model and to the clinical outcome. The authors found a correlation between secreted laccases and disease outcome in patients. In addition, the authors show that a faster melanization rate in C. neoformans correlated with phagocytosis evasion, virulence in the g. mellonella model and worse prognosis in humans.
The manuscript is well structured with an appropriate abstract summing the main findings, a clear introduction, well described methods section and appropriate number of figures and tables. The results are clearly described.
The authors identify and acknowledge the most important limitation of the study: line 365-366 the patients were treated with different regimens in distinct health services. This reviewers agrees this is a limitation. However, to get a feeling about the impact of these differences the authors should indicate how the patients were treated and whether there were differences in patients that died and survived. Without this information clearly presented, I cannot interpret the correlations between virulence factors and outcome found in this study. Perhaps the authors can show how many patients that were included in the phenotype-survival analysis, that died and survived were treated according to Brazilian guidelines.
The melanin production evaluation assay is an important tool that the authors use in this study and the measurements from these assays were correlated with G. mellonella and patients survival and thus are essential to the conclusions of the study. The method is well standardized, and the authors show elegantly that the outcomes are highly reproducible. Can the authors describe when melanization occurs: does it occur in mature colonies and may growth rate itself may influence the measurements? Do isolates with a high growth rate/colony maturation have a low T-HMM or high melanization Top. Have the final colonies of different species have a different final cell number after 7 days incubation and how does this correlate to melanization? And how does the growth rate/ budding rate/ colony maturation/ correlate to G. mellonella survival?
The figures 1-5 give a clear picture of the wide distribution and variation of virulence parameters e.g. the distribution of melanization kinetics parameters, the distribution of capsule sizes, GXM secretion and LC3 phagocytosis. But what does this distribution mean, it only shows that the isolates are not the same but does not contribute majorly to the final conclusion. Can the authors think of a way to give more meaning to these figures: e.g. indicate with colors which isolates were retrieved from patients that eventually died and which survived (although this may be inappropriate as not all clinical information is available. Figure 6 really gives meaning to the numbers displayed in figure 1-5. Perhaps move some figures to the supplementary file.
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Reviewer #1:
The manuscript describes the characterization of in total 85 Cryptococcus spp. clinical isolates with regard to virulence phenotypes including a Galleria mellonella infection model for cryptococcosis. The authors determined the melanization kinetics of all strains, measured the whole-cell and extracellular laccase activity, the capsule thickness, and the concentration of the cell wall polysaccharide glucuronoxylomannan. In addition, during macrophage interaction the proportion of Cryptococcus-containing LC3-positive phagosomes for each strain was determined as well as the survival of G. mellonella after infection with selected Cryptococcus strains. Finally, regression analyses were performed to estimate the relationship between the risk of death in crytptococcosis patients and the phenotypes of the isolated Cryptococcus …
Reviewer #1:
The manuscript describes the characterization of in total 85 Cryptococcus spp. clinical isolates with regard to virulence phenotypes including a Galleria mellonella infection model for cryptococcosis. The authors determined the melanization kinetics of all strains, measured the whole-cell and extracellular laccase activity, the capsule thickness, and the concentration of the cell wall polysaccharide glucuronoxylomannan. In addition, during macrophage interaction the proportion of Cryptococcus-containing LC3-positive phagosomes for each strain was determined as well as the survival of G. mellonella after infection with selected Cryptococcus strains. Finally, regression analyses were performed to estimate the relationship between the risk of death in crytptococcosis patients and the phenotypes of the isolated Cryptococcus strains. A major finding was that the risk of death in patients with disseminated cryptococcosis increased with the level of extracellular laccase activity and the time for half-maximum melanization in the Cryptococcus isolates. This suggests that the melanization rate, more than the total amount of melanin, impacts the outcome of a Cryptococcus infection.
General assessment:
The study is based on carefully performed experiments. However, the scientific significance of this work is moderate. Melanin and the laccases that are involved in its synthesis are known virulence factors of Cryptococcus spp. for many years and similar studies have already been published elsewhere (e.g. Samarasinghe et al. 2018). The major new finding of the presented work is that the speed of melanization has an impact on the virulence of Cryptococcus spp. rather than total amount of melanin. The shortcoming of the manuscript is that the author's hypothesis is mainly based on regression analyses, but the final proof based on a genetically well-defined background is missing. Therefore, the study only provides little new insight into fundamental mechanisms of Cryptococcus virulence but includes associations with patients and therefore might be more suited for a journal specialized in pathogenic fungi.
Following points should be considered:
The authors show the association between faster Cryptococcus melanization and more effective evasion from host immunity. However, the author cannot totally exclude other factors that are associated with host evasion. It would be more appropriate to either create a mutant (e.g. overexpression of LAC1), which showed faster melanization in comparison to a wildtype strain or to perform multilocus sequence typing (including the LAC1 locus) to capture the genetic variation of the clinical isolates and to find come correlations with the speed of melanization. The interesting question is which genetic factors contribute to the difference in the melanization rate.
The authors should critically discuss the suitability of their Galleria mallonella infection model. It is a known fact that temperature has an influence on the melanization in Cryptococcus spp.. Laccasse activity is significantly inhibited at temperatures of 37°C and higher. The Galleria model can only be used at lower temperatures.
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Summary: The description of how faster melanization is associated with LC3-mediated phagocytosis evasion, virulence and outcomes in humans is interesting and does provide some new information. In general, the study has been executed well, with clear articulation of the results and appending conclusions. However, the work falls short of investigating any substantive mechanistic basis for the observations and how they relate to the broader metabolism in Cryptococcus. .
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