Intraspecific Comparative Genomics of Candida albicans Mitochondria Reveals Non-Coding Regions Under Neutral Evolution

The opportunistic fungal pathogen Candida albicans causes serious hematogenic hospital acquired candidiasis with worldwide impact on public health. Because of its importance as a nosocomial etiologic agent, C. albicans genome has been largely studied to identify intraspecific variation and several typing methods have been developed to distinguish closely related strains. Mitochondrial DNA can be useful for this purpose because, as compared to nuclear DNA, its higher mutational load and evolutionary rate readily reveals microvariants. Accordingly, we sequenced and assembled, with 8 fold coverage, the mitochondrial genomes of two C. albicans clinical isolates (L296 and L757) and compared these sequences with the genome sequence of reference strain SC5314. The genome alignment of 33,928 positions revealed 372 polymorphic sites being 230 in coding and 142 in non-coding regions. Three intergenic regions located between genes tRNAGly/COX1, NAD3/COB and ssurRNA/NAD4L, named IG1, IG2 and IG3 respectively, which showed high number of neutral substitutions, were amplified and sequenced from 18 clinical isolates from different locations in Latin America and 2 ATCC standard C. albicans strains. High variability of sequence and size were observed, ranging up to 56bp size difference and phylogenies based on IG1, IG2 and IG3 revealed three groups. Insertions of up to 49bp were observed exclusively in Argentinean strains relative to the other sequences which could suggest clustering by geographical polymorphism. Because of neutral evolution, high variability, easy isolation by PCR and full length sequencing these mitochondrial intergenic regions can contribute with a novel perspective in molecular studies of C. albicans isolates, complementing well established multilocus sequence typing methods.

💡 Research Summary

Candida albicans is a major opportunistic fungal pathogen responsible for life‑threatening bloodstream infections in hospitals worldwide. Because of its clinical relevance, extensive research has focused on nuclear genome variation and multilocus sequence typing (MLST) has become a standard method for discriminating closely related isolates. However, nuclear DNA evolves relatively slowly, limiting its resolution for fine‑scale epidemiology. Mitochondrial DNA (mtDNA), by contrast, exhibits a higher mutation rate and faster evolutionary dynamics, making it a promising source of high‑resolution markers.

In this study the authors sequenced and assembled the complete mitochondrial genomes of two clinical isolates (L296 and L757) with an average coverage of eight‑fold and compared them to the reference strain SC5314. The alignment of 33,928 nucleotides revealed 372 polymorphic sites: 230 located within protein‑coding genes and 142 in non‑coding regions. While coding‑region polymorphisms were largely constrained, three intergenic segments—between tRNAGly and COX1 (IG1), between NAD3 and COB (IG2), and between ssurRNA and NAD4L (IG3)—displayed a striking excess of neutral substitutions, primarily insertions/deletions and simple‑repeat variations.

To assess their epidemiological utility, IG1, IG2 and IG3 were PCR‑amplified and fully sequenced from 18 additional clinical isolates collected across Latin America and from two ATCC reference strains. The intergenic regions proved highly variable in both sequence and length, with differences up to 56 bp. Notably, isolates from Argentina carried unique insertions of up to 49 bp, suggesting a geographically restricted polymorphism. Phylogenetic analyses based on each intergenic region independently resolved three major clusters, partially mirroring MLST‑derived groups but offering finer discrimination because of the higher mutation density in the mitochondrial intergenic DNA.

The authors argue that these three mitochondrial intergenic loci possess several attributes that make them attractive molecular markers: (1) they evolve under neutral drift, minimizing the confounding effects of selection; (2) they exhibit abundant polymorphism, providing high discriminatory power; (3) they can be amplified with a single PCR reaction and sequenced in full length, simplifying laboratory workflows; and (4) they complement existing nuclear‑based typing schemes, enhancing the resolution of strain‑level epidemiology.

In conclusion, the study demonstrates that mitochondrial intergenic regions IG1, IG2 and IG3 are robust, cost‑effective tools for C. albicans strain typing. Their neutral evolution and pronounced variability enable detection of micro‑variants that nuclear markers may miss, and the observed geographic clustering (e.g., Argentinean‑specific insertions) highlights their potential for tracing transmission routes and outbreak sources. Future work should expand sampling to a broader global collection and explore additional mitochondrial non‑coding regions to build a comprehensive, high‑resolution phylogeographic map of C. albicans.