Entry for:The Peer Prize for Women in Science 2017
1. Please give a brief summary of your work.
Fungi account for a large number of infections that are extremely difficult to treat. Half of the deaths from hospital-acquired infections are due to fungi and 1.5 million people die from invasive fungal infections yearly. Treatment is challenging due to the ability of fungi to rapidly adapt to the host environment, enabling avoidance of the immune system and the generation of resistance to antifungal drugs. One potential mechanism for adaptation is the process of microevolution, a change in gene frequencies occurring within a population over a short period of time. Microevolution can select for novel traits that facilitate the ability to cause disease. These traits are generated by mutations in the DNA sequence, arising from chromosomal changes, DNA replication errors or from environmental damage. This study investigates the role of mutations arising from DNA replication errors during microevolution in one of the world’s most common and deadliest fungal pathogens.
2. Describe your approach and broader findings.
Transmission of an organism’s genetic material through mitosis is extremely accurate due to the activity of a number of systems that detect the introduction of incorrect nucleotides during DNA replication. However, mutations that arise through errors in replication or from environmental stresses are biologically important since they provide variation upon which selection can act. Those which confer fitness defects are removed by natural selection, whereas, others provide an opportunity for adaptive evolution to occur. The number and type of mutations generated is determined by the effectiveness of DNA repair pathways. Therefore, despite conventional thinking that DNA damage is detrimental for an organism, specific types of DNA damage and the subsequent mutations generated play an important role in enabling fungal pathogens to undergo microevolution in order to adapt to the host environment and cause disease.
This study aimed to identify isolates of the fungal pathogen Cryptococcus neoformans which exhibit a higher than normal mutation rate (a ‘mutator’ phenotype) and investigate whether this phenotype leads to rapid microevolution during infection. Whole genome sequence analysis of two clinical isolates with a mutator phenotype revealed these isolates possessed mutations in the MSH2 gene of the mismatch DNA repair pathway. The mismatch repair pathway corrects single nucleotide errors arising during DNA synthesis. Re-introduction of the wildtype MSH2 gene into the C. neoformans isolates returned the mutation rate to normal suggesting that the MSH2 mutations are responsible for the mutator phenotype of these clinical isolates.
The study proceeded to analyze C. neoformans strains containing deletions of genes encoding mismatch repair pathway components. The deletion of three of these genes (MSH2, MLH1 and PMS1) results in an elevated mutation rate (~200 fold) due to an increase in mutations in homopolymeric tracts (strings of the same nucleotide in the DNA sequence) and transitions (interchanges of purines or pyrimidines), specifically guanine to adenine. This elevated mutation rate resulted in only minor sensitivity to stress and was not detrimental to growth in vitro and in vivo, with no decrease in colonization or virulence observed in a mouse infection model. Importantly, microevolution occurred more rapidly in the mismatch repair mutants compared to wild type and introduced phenotypic variation in traits associated with the ability to grow in the host such as growth at 37ºC, resistance to oxidative stress, alterations in the cell wall and melanization. In addition, the mismatch mutants all showed an increase in the frequency of spontaneously occurring resistance to antifungal agents.
3. What is the wider contribution, or impact, to your scientific field(s)?
C. neoformans isolates possessing mutations in mismatch repair pathway components have an opportunity for enhanced microevolution during infection. However, as no difference in colonization or virulence was observed in a murine infection model, the findings suggest that rather than having a direct impact on the virulence composite of isolates, defects in mismatch repair of DNA replication errors is more likely to be a significant factor in allowing outbreaks to occur via the generation of hypervirulent strains from less virulent ancestors, and by allowing recurrence of cryptococcal meningitis through the generation of antifungal resistance.
More than 90% of all reported fungal deaths result from species that belong to one of four genera: Cryptococcus, Candida, Aspergillus and Pneumocystis. Cryptococcosis, caused by C. neoformans or C. gattii, is a significant fungal disease worldwide, with high incidence and morbidity. The highest prevalence is in sub-Saharan Africa where an estimated 624,000 deaths were attributed to cryptococcosis yearly, more than tuberculosis. Treated patients often relapse due to gain of resistance to antifungal drugs, which leads to a mortality rate as high as 30%. Given the widespread use of long-term antifungal treatment to prevent recurrence, the potential development of drug resistance poses a serious threat to the future management of cryptococcal disease. The findings of this study reveal that defects in mismatch repair of DNA replication errors enhance microevolution and the rapid generation of resistance to antifungal drugs. Therefore the probability of the emergence of resistance to antifungal drugs in clinical isolate carrying mutations in mismatch repair components is high and this will have a direct bearing on clinical treatment. The significance of these discoveries is the better control of fungal diseases.
4. Are there any potential ideas you would like to explore to take this research further?
In bacterial microbial populations, retaining a proportion of mutator cells in the population is thought to provide a selective advantage in vivo. This suggests that this adaptive mechanism may be conserved in a broad variety of pathogens, including fungi. This study has shown that 2/11 clinical strains of C. neoformans are msh2 mutants. However, analysis of a larger number of strains is needed to determine if this is a general feature of the pathogen population. I plan to assess mutation rate in a collection of 198 C. neoformans clinical strains from Kampala, Uganda collected between 2010-2014. Whole genome sequencing will be performed on those with increased mutation rate to determine if there are mutations in DNA repair components. The minimum inhibitory concentrations (MICs) for antifungal drugs have been previously determined for all of these strains, allowing determination of the proportion of C. neoformans clinical strains exhibiting a mutator phenotype and mutations in mismatch repair components and if this correlates to a higher level of resistance to antifungal drugs.