Deciphering the Association among Pathogenicity, Production and Polymorphisms of Capsule/Melanin in Clinical Isolates of Cryptococcus neoformans var. grubii VNI

Abstract

Background: Cryptococcus neoformans is an opportunistic fungal pathogen that can cause meningitis in immunocompromised individuals. The objective of this work was to study the relationship between the phenotypes and genotypes of isolates of clinical origin from different cities in Colombia.

Methods: Genome classification of 29 clinical isolates of C. neoformans var. grubii was performed using multilocus sequence typing (MLST), and genomic sequencing was used to genotype protein-coding genes. Pathogenicity was assessed in a larval model, and melanin production and capsule size were evaluated in vitro and in vivo.

Results: Eleven MLST sequence types (STs) were found, the most frequent being ST69 (n = 9), ST2, ST93, and ST377 (each with n = 4). In the 29 isolates, different levels of pigmentation, capsule size and pathogenicity were observed. Isolates classified as highly pathogenic showed a tendency to exhibit larger increases in capsule size. In the analysis of polymorphisms, 48 non-synonymous variants located in the predicted functional domains of 39 genes were found to be associated with capsule size change, melanin, or pathogenicity.

Conclusions: No clear patterns were found in the analysis of the phenotype and genotype of Cryptococcus. However, the data suggest that the increase in capsule size is a key variable for the differentiation of pathogenic isolates, regardless of the method used for its induction.

Keywords: Cryptococcus neoformans; MLST; capsule; melanin; polymorphism.

Figure 1

Hierarchical grouping of the 29 clinical isolates of C. neoformans var. grubii based on the MLST allelic profile. The figure shows the relationship among the STs of the clinical isolates. The evolutionary history, as inferred from the MLST data, was reconstructed via the maximum likelihood method and the Jukes–Cantor model using concatenated nucleotide sequences of 7 loci. Bootstrap values are displayed for each branch (1000 repeats). Representative clinical variables are listed on the right of the image. The most frequent sequence types are highlighted in colors. ND: no data.

Figure 2

Survival curves of the 29 clinical isolates of C. neoformans with (a) low, (b) intermediate, and (c) high pathogenicity in G. mellonella. The most frequent STs are shown in green (ST2), red (ST69), purple (ST95) and blue (ST377). After injection with 1.5 × 10⁸ cells/mL, Curves were constructed using the Kaplan–Meier method, and then the curves were compared using the log-rank (Mantel–Cox) test. The data are expressed as survival percentages. No larval death was observed in control larvae injected with an equivalent volume of PBS.

Figure 3

Capsule size pre- and post-inoculation in G. mellonella of the 21 clinical isolates of C. neoformans var. grubii belonging to the most frequent sequence types: (a) ST69, (b) ST2, (c) ST95, and (d) ST377. Boxes are colored according to isolates’ pathogenicity: blue (low), black (intermediate) and red (high). The error bars represent the standard deviation. p < 0.001 (***); p > 0.0001 (****).

Figure 4

Capsule induction in vitro; figure constructed using the isolates with the greatest changes in capsule size. Bright green represents the basal capsule size, and dark green represents the size after capsule induction. The bottom figure shows the photographic record of the isolates. p < 0.0001 (****). The scale at the bottom right of the images represents 10 µm.

Figure 5

Summary of the main genotypic and phenotypic results obtained in 29 clinical isolates of C. neoformans var. grubii: Phylogenetic analysis, pathogenicity in G. mellonella, capsule size, and melanin production. The evolutionary history was derived using the maximum-likelihood method based on the Jukes–Cantor model using concatenated nucleotide sequences of 7 loci. Bootstrap values are displayed for each branch (1000 repeats).

Figure 6

Coordinates calculated via principal component analysis for each isolate and membership of isolates in the proposed categories. A positive correlation is observed; the variable that has the most weight in the analysis is the difference in capsular induction, followed by the variables corresponding to both the in vitro induction in capsular induction medium and the in vivo model. B, I and A correspond to low, intermediate and high pathogenicity, respectively. The modulation of the capsule size can be seen in the blue halo. Filled circles are colored red for active variables and blue for active observations.

Figure 7

Principal component analysis for the genomic variants of 322 genes related to capsule, melanin, and pathogenicity. The figure shows the coordinates for each of the isolates as well as inclusion in the groups proposed for change in capsule size (red: isolates with high change; blue: with low change). The groups or clusters formed appear in circles.

Figure 8

Gene variants associated with phenotypic groups and STs. The reference sequence is in bold (H99). The evolutionary history as inferred from the MLST data was reconstructed via the maximum likelihood method and the Jukes–Cantor model using concatenated nucleotide sequences of 7 loci. From phylogenetic analysis of pathogenicity in G. mellonella, capsule size and melanin production, the 39 genes that grouped the 48 non-synonymous variants identified are shown.