Mapping and editing animal mitochondrial genomes: can we overcome the challenges?

The animal mitochondrial genome, although small, can have a big impact on health and disease. Non-pathogenic sequence variation among mitochondrial DNA (mtDNA) haplotypes influences traits including fertility, healthspan and lifespan, whereas pathogenic mutations are linked to incurable mitochondrial diseases and other complex conditions like ageing, diabetes, cancer and neurodegeneration. However, we know very little about how mtDNA genetic variation contributes to phenotypic differences. Infrequent recombination, the multicopy nature and nucleic acid-impenetrable membranes present significant challenges that hamper our ability to precisely map mtDNA variants responsible for traits, and to genetically modify mtDNA so that we can isolate specific mutants and characterize their biochemical and physiological consequences. Here, we summarize the past struggles and efforts in developing systems to map and edit mtDNA. We also assess the future of performing forward and reverse genetic studies on animal mitochondrial genomes. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.This work is funded by Wellcome Trust grant 203767/Z/16/Z to AK and 202269/Z/16/Z to HM

A battle for transmission: the cooperative and selfish animal mitochondrial genomes.

The mitochondrial genome is an evolutionarily persistent and cooperative component of metazoan cells that contributes to energy production and many other cellular processes. Despite sharing the same host as the nuclear genome, the multi-copy mitochondrial DNA (mtDNA) follows very different rules of replication and transmission, which translate into differences in the patterns of selection. On one hand, mtDNA is dependent on the host for its transmission, so selections would favour genomes that boost organismal fitness. On the other hand, genetic heterogeneity within an individual allows different mitochondrial genomes to compete for transmission. This intra-organismal competition could select for the best replicator, which does not necessarily give the fittest organisms, resulting in mito-nuclear conflict. In this review, we discuss the recent advances in our understanding of the mechanisms and opposing forces governing mtDNA transmission and selection in bilaterians, and what the implications of these are for mtDNA evolution and mitochondrial replacement therapy

Selections that isolate recombinant mitochondrial genomes in animals.

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germline and revealed somatic recombination. When the recombination partner was a diverged Drosophila melanogaster genome or a genome from a different species such as Drosophila yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome

A Genome-wide Screen Reveals that Reducing Mitochondrial DNA Polymerase Can Promote Elimination of Deleterious Mitochondrial Mutations.

A mutant mitochondrial genome arising amid the pool of mitochondrial genomes within a cell must compete with existing genomes to survive to the next generation. Even weak selective forces can bias transmission of one genome over another to affect the inheritance of mitochondrial diseases and guide the evolution of mitochondrial DNA (mtDNA). Studies in several systems suggested that purifying selection in the female germline reduces transmission of detrimental mitochondrial mutations [1-7]. In contrast, some selfish genomes can take over despite a cost to host fitness [8-13]. Within individuals, the outcome of competition is therefore influenced by multiple selective forces. The nuclear genome, which encodes most proteins within mitochondria, and all external regulators of mitochondrial biogenesis and dynamics can influence the competition between mitochondrial genomes [14-18], yet little is known about how this works. Previously, we established a Drosophila line transmitting two mitochondrial genomes in a stable ratio enforced by purifying selection benefiting one genome and a selfish advantage favoring the other [8]. Here, to find nuclear genes that impact mtDNA competition, we screened heterozygous deletions tiling ∼70% of the euchromatic regions and examined their influence on this ratio. This genome-wide screen detected many nuclear modifiers of this ratio and identified one as the catalytic subunit of mtDNA polymerase gene (POLG), tam. A reduced dose of tam drove elimination of defective mitochondrial genomes. This study suggests that our approach will uncover targets for interventions that would block propagation of pathogenic mitochondrial mutations

Two mitochondrial DNA polymorphisms modulate cardiolipin binding and lead to synthetic lethality

Genetic screens have been used extensively to probe interactions betweennuclear genes and their impact on phenotypes. Probing interactions betweenmitochondrial genes and their phenotypic outcome, however, has not beenpossible due to a lack of tools to map the responsible polymorphisms. Here,using a toolkit we previously established in Drosophila, we isolate over 300recombinant mitochondrial genomes and map a naturally occurring polymorphism at the cytochrome c oxidase III residue 109 (CoIII109) that fully rescues the lethality and other defects associated with a point mutation incytochrome c oxidase I (CoIT300I). Through lipidomics profiling, biochemicalassays and phenotypic analyses, we show that the CoIII109 polymorphismmodulates cardiolipin binding to prevent complex IV instability caused by theCoIT300I mutation. This study demonstrates the feasibility of genetic interactionscreens in animal mitochondrial DNA. It unwraps the complex intra-genomicinterplays underlying disorders linked to mitochondrial DNA and how theyinfluence disease expression

Direct cell-to-cell spread of a pathogenic yeast.

BACKGROUND: Cryptococcosis, a fatal fungal infection of the central nervous system, is one of the major killers of AIDS patients and other immunocompromised hosts. The causative agent, Cryptococcus neoformans, has a remarkable ability to 'hide' and proliferate within phagocytic cells of the human immune system. This intracellular phase is thought to underlie the ability of the pathogen to remain latent for long periods of time within infected individuals. RESULTS: We now report that Cryptococcus is able to undergo 'lateral transfer' between phagocytes, moving directly from infected to uninfected macrophages. This novel process was observed in both C. neoformans serotypes (A and D) and occurs in both immortalised cell lines and in primary human macrophages. Lateral transfer is independent of the initial route of uptake, since both serum-opsonised and antibody-opsonised C. neoformans are able to undergo direct cell-to-cell transfer. CONCLUSION: We provide the first evidence for lateral transfer of a human fungal pathogen. This rare event may occur repeatedly during latent cryptococcal infections, thereby allowing the pathogen to remain concealed from the immune system and protecting it from exposure to antifungal agents

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak

Transmission of Hypervirulence traits via sexual reproduction within and between lineages of the human fungal pathogen cryptococcus gattii.

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal