Convergent microevolution of Cryptococcus neoformans hypervirulence in the laboratory and the clinic

Samantha D M Arras,Benjamin L Schulz,Samantha R Stowasser,Milos Tanurdzic,Ross D Blundell,James A Fraser,Monica I Espinosa,Alex C Carpenter,Kate L Ormerod,Paige E Erpf

doi:10.1038/s41598-017-18106-2

Samantha D M Arras, Benjamin L Schulz + Show 8 more

Open Access

https://doi.org/10.1038/s41598-017-18106-2

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Journal: Scientific Reports	Publication Date: Dec 1, 2017
Citations: 36	License type: open-access

Affiliation: University of Queensland

Abstract

Reference strains are a key component of laboratory research, providing a common background allowing for comparisons across a community of researchers. However, laboratory passage of these strains has been shown to lead to reduced fitness and the attenuation of virulence in some species. In this study we show the opposite in the fungal pathogen Cryptococcus neoformans, with analysis of a collection of type strain H99 subcultures revealing that the most commonly used laboratory subcultures belong to a mutant lineage of the type strain that is hypervirulent. The pleiotropic mutant phenotypes in this H99L (for “Laboratory”) lineage are the result of a deletion in the gene encoding the SAGA Associated Factor Sgf29, a mutation that is also present in the widely-used H99L-derived KN99a/α congenic pair. At a molecular level, loss of this gene results in a reduction in histone H3K9 acetylation. Remarkably, analysis of clinical isolates identified loss of function SGF29 mutations in C. neoformans strains infecting two of fourteen patients, demonstrating not only the first example of hypervirulence in clinical C. neoformans samples, but also parallels between in vitro and in vivo microevolution for hypervirulence in this important pathogen.

Highlights

Reference strains are an essential element of laboratory research that provide a common genotype to enable comparison of scientific observations in any given species across a community of researchers
Laboratory passage often leads to reduced fitness; in the past subculturing has led to the attenuation of virulence in isolates of pathogens including Burkholderia pseudomallei[9], Mycobacterium tuberculosis[10], Cryptococcus deneoformans[11] and Cryptococcus neoformans[12]
Given that loss of SGF29 results in hypervirulence, we investigated if mutations equivalent to that observed in H99L occur in the clinical setting

Summary

Introduction

Reference strains are an essential element of laboratory research that provide a common genotype to enable comparison of scientific observations in any given species across a community of researchers. An analysis of K-12 stored in a stab culture for 30 years revealed the development of massive heterogeneity even in this state of very slow growth; of 118 colonies purified from this stab, 68 different insertion sequence-associated RFLP patterns were observed[6]. While genetic diversity such as this may be dismissed as a consequence of the storage method, mutations have been described in K-12 subcultures obtained from stock centres where strains are frozen[7,8]. The impact of the disease is significant, especially in those populations with restricted access to health care, leading to an estimated 600,000 deaths per year[16,17]

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