MLST-Based Population Genetic Analysis in a Global Context Reveals Clonality amongst Cryptococcus neoformans var. grubii VNI Isolates from HIV Patients in Southeastern Brazil

Abstract

Cryptococcosis is an important fungal infection in immunocompromised individuals, especially those infected with HIV. In Brazil, despite the free availability of antiretroviral therapy (ART) in the public health system, the mortality rate due to Cryptococcus neoformans meningitis is still high. To obtain a more detailed picture of the population genetic structure of this species in southeast Brazil, we studied 108 clinical isolates from 101 patients and 35 environmental isolates. Among the patients, 59% had a fatal outcome mainly in HIV-positive male patients. All the isolates were found to be C. neoformans var. grubii major molecular type VNI and mating type locus alpha. Twelve were identified as diploid by flow cytometry, being homozygous (AαAα) for the mating type and by PCR screening of the STE20, GPA1, and PAK1 genes. Using the ISHAM consensus multilocus sequence typing (MLST) scheme, 13 sequence types (ST) were identified, with one being newly described. ST93 was identified from 81 (75%) of the clinical isolates, while ST77 and ST93 were identified from 19 (54%) and 10 (29%) environmental isolates, respectively. The southeastern Brazilian isolates had an overwhelming clonal population structure. When compared with populations from different continents based on data extracted from the ISHAM-MLST database (mlst.mycologylab.org) they showed less genetic variability. Two main clusters within C. neoformans var. grubii VNI were identified that diverged from VNB around 0.58 to 4.8 million years ago.

Fig 1. Representative flow cytometry curves of selected southeastern Brazilian Cryptococcus neoformans var. grubii VNI isolates.

Out of 143 isolates, 12 were identified as diploid. The haploid strains H99 C. neoformans var. grubii VNI and CDCR265 C. gattii VGII and the diploid hybrid strain WM 09.184 C. neoformans VNII/VNIV were included in each flow run as controls.

Fig 2. Maximum likelihood (ML) consensus tree of the global Cryptococcus neoformans var. grubii VNI dataset using the concatenated sequences of the seven MLST loci (CAP59, GPD1, LAC1, PLB1, SOD1, URA5, and the IGS1 region).

The tree with the highest log likelihood (-7,114) drawn to scale with branch lengths measuring the number of substitutions per site is shown. Bootstrap values >50% based on 1,000 replicates for the ML and the neighbour-joining (NJ) analyses, which showed similar topologies, are presented close to the branches. The analysis involved 92 nucleotide sequences with 4,010 positions revealing the two main clusters (red = major and blue = minor). The isolates are described according to the sequence type number (ST), followed by mating type (a or α) and country of isolation, which are abbreviated according to the alfa-2 code of ISO 3166–1. The colours of each country represent the continent of origin as follows: blue: Europe, brown: South America, green: Africa, orange: North America, red: Asia.

Fig 3

A) Minimum spanning tree showing the distribution of the 143 Cryptococcus neoformans var. grubii VNI isolates from southeastern Brazil according to the source of isolation. Two main clusters were identified. The first composed of the clonal complexes (CC) CC31 and CC32, which are both linked with the most frequent STs found in the study, ST93 (recovered from both clinical and environmental samples) and ST77 (recovered only from environmental samples). The second main cluster is represented by the ST289, ST63, ST23, and ST2. Each circle represents a unique ST, and the circumference is proportional to the number of isolates within each ST. Clonal complexes surrounded by black circles represent the ancestors in the network. Solid, grey and dashed branches represent at least one, two, and three differences in alleles, respectively. B) Minimum spanning tree of the isolates presented in A, and the inclusion of 181 STs identified in different continents, retrieved from the ISHAM-MLST database (http://mlst.mycologylab.org/). Fifteen CCs were identified, with the CC174 presenting a central role in the network. This picture shows the pattern of clonality of the VNI genotype, the widely distributed STs (e.g. ST5, ST23, ST32, ST63), the two main clusters, and the broad distribution of African isolates, highlighted in green. The STs are described according to the colours of the map as follows: blue: Europe, brown: South America, green: Africa, yellow: North America, red: Asia. The map also displays the pairwise F_ST tests calculated by AMOVA, with African, North American, and South American populations presenting similar populations, whereas the Asian and European populations are more differentiated.

Fig 4. Principle Components Analysis of Cryptococcus neoformans var. grubii VNI allelic profiles depicted according to: A) the southeastern Brazilian population (13 STs), B) C. neoformans population using the global dataset (92 STs), C) isolates distributed according to clinical and environmental sources (104 STs), and D) distributed according to continents (136 STs).

Dots represent STs linked by coloured lines to form clusters, proportional in sizes to the number of isolates. The two main clusters identified in the C. neoformans population are highlighted in blue (minor) and red (major). The samples recovered from clinical (Clin) and environmental (Env) sources are highlighted in blue and green respectively. The colours of each continent of origin are described as follows: blue: Europe (EU), brown: South America (SA), green: Africa (AF), orange: North America (NA), red: Asia (AS). Eigenvalues highlighting the two first components are represented in the bar plot. The p-value calculated using the Mont Carlo test is described in the upper left side.

Fig 5. Structure analyses of Cryptococcus neoformans var. grubii VNI showing that K = 3 is the actual number of subpopulations. Clusters of individuals based on prior-defined populations according to: A) major and minor main clusters identified in the phylogenetic and coalescence analyses using the expanded global dataset (92 isolates), B) clinical and environmental isolates (104 isolates), and C) according to continents (136 isolates).

Each vertical line represents one ST and the colours represent the most likely ancestry of each individual from the population. Individuals with multiple colours have admixed genotypes from the prior-defined subpopulations. One ST per region and/or per pre-defined subpopulation was used throughout the whole analyses.

Fig 6. Coalescence gene genealogy of the Cryptococcus neoformans var. grubii VNI isolates evidencing the presence of two main clusters that separated 0.29 to 2.8 million years ago.

Blue bars represent the 95% high posterior probability of the ages in the branches with a posterior probability limit of 0.5. The Bayesian posterior support values higher than 70% are described in the branches of the tree. One ST representing the VNB (ST7) and one representing the VNII (ST40) lineage were included in the analysis as out-groups. The continents of origin of the isolates are abbreviated as follows: AS: Asia, AF: Africa, EU: Europe, SA: South America, NA: North America.