MLST and Whole-Genome-Based Population Analysis of Cryptococcus gattii VGIII Links Clinical, Veterinary and Environmental Strains, and Reveals Divergent Serotype Specific Sub-populations and Distant Ancestors

Abstract

The emerging pathogen Cryptococcus gattii causes life-threatening disease in immunocompetent and immunocompromised hosts. Of the four major molecular types (VGI-VGIV), the molecular type VGIII has recently emerged as cause of disease in otherwise healthy individuals, prompting a need to investigate its population genetic structure to understand if there are potential genotype-dependent characteristics in its epidemiology, environmental niche(s), host range and clinical features of disease. Multilocus sequence typing (MLST) of 122 clinical, environmental and veterinary C. gattii VGIII isolates from Australia, Colombia, Guatemala, Mexico, New Zealand, Paraguay, USA and Venezuela, and whole genome sequencing (WGS) of 60 isolates representing all established MLST types identified four divergent sub-populations. The majority of the isolates belong to two main clades, corresponding either to serotype B or C, indicating an ongoing species evolution. Both major clades included clinical, environmental and veterinary isolates. The C. gattii VGIII population was genetically highly diverse, with minor differences between countries, isolation source, serotype and mating type. Little to no recombination was found between the two major groups, serotype B and C, at the whole and mitochondrial genome level. C. gattii VGIII is widespread in the Americas, with sporadic cases occurring elsewhere, WGS revealed Mexico and USA as a likely origin of the serotype B VGIII population and Colombia as a possible origin of the serotype C VGIII population. Serotype B isolates are more virulent than serotype C isolates in a murine model of infection, causing predominantly pulmonary cryptococcosis. No specific link between genotype and virulence was observed. Antifungal susceptibility testing against six antifungal drugs revealed that serotype B isolates are more susceptible to azoles than serotype C isolates, highlighting the importance of strain typing to guide effective treatment to improve the disease outcome.

Advances in the understanding of the Cryptococcus neoformans and C. gattii species complexes and cryptococcosis

“…In addition, in both species, the same genotypes have been found to be recovered from patients and from the patients' environment 19,22 , which supports the theory of the acquisition of the infection from environmental sources and reaffirms the saprophytic and arboreal sources of these yeasts. Both species have also been isolated frequently from air, and swabs from trees, from decaying wood in hollows of many tree species, either individually or together 14,23 .…”

Metatranscriptomics as a tool to identify fungal species and subspecies in mixed communities

“…The major advantages of metatranscriptomics over other HTS technologies include the selective sequencing of living organisms and the ability to detect a wide range of microorganisms in one step, which has multiple applications in biological research, surveillance and diagnosis. There is an increasing literature reporting that virulence and antimicrobial tolerance traits vary within species (Firacative et al 2016; Rizzetto et al 2013; Schauwvlieghe et al 2017; Strope et al 2015) and that multiple strains or species can co-infect a host (Desnos-Ollivier et al 2010; Gupta et al 2014; Seki et al 2014; Soll et al 1988; Tati et al 2016). The high discriminatory power obtained for closely-related lineages of Cryptococcus provides a good example of where metatranscriptomics would be valuable in precision medicine, where therapy practices are defined according to strain-specific pathogenicity and drug susceptibility traits.…”

The Case for Adopting the “Species Complex” Nomenclature for the Etiologic Agents of Cryptococcosis

“…The total number of monophyletic clades recognized within the C. neoformans/C. gattii species complex is increasing as more strains collected globally are being included in phylogenetic analyses (18). The major monophyletic clades for the two species have most commonly been designated molecular types VNI (AFLP1), VNII (AFLP1A/IB), VNIII (AFLP3), and VNIV (AFLP2) for C. neoformans and molecular types VGI (AFLP4), VGII (AFLP6), VGIII (AFLP5), and VGIV (AFLP6) for C. gattii .…”

“…A strict, accepted phylogenetic species concept defines a species as a single lineage of ancestor-descendant populations which maintains its identity from other such lineages and which has its own evolutionary tendencies and historic fate (21–23). With this definition, even the smallest diagnosable cluster of individual strains that form a monophyletic group in a phylogenetic tree can be considered deserving of species recognition (24), and the number of cryptococcosis agents with a species status will continue to increase (18). For this reason, the proposed taxonomy is likely to prove to be unstable.…”