Biology subjects

O'Brien, C. E.

Publications and source records attributed to O'Brien, C. E..

8 recordsLinked to original sources

bioRxiv2025

Recurrent acquisition of nuclease-protease pairs in antiviral immunity

Antiviral immune systems diversify by integrating new genes into existing pathways, creating new mechanisms of viral resistance. We identified genes encoding a predicted nuclease paired with a trypsin-like protease repeatedly acquired by multiple, otherwise unrelated antiviral immune systems in bacteria. Cell-based and biochemical assays revealed the nuclease is a proenzyme that cleaves DNA only after activation by its partner protease. Phylogenetic analysis showed that two distinct immune systems, Hachiman and AVAST, use the same mechanism of proteolytic activation despite their independent evolutionary origins. Examination of nuclease-protease inheritance patterns identified caspase-nuclease (canu) genomic loci that confer antiviral defense in a pathway reminiscent of eukaryotic caspase activation. These results uncover the coordinated activities of pronucleases and their activating proteases within different immune systems and show how coevolution enables defense system innovation.

microbiology↗

bioRxiv2025

Proline Stickland fermentation supports C. difficile spore maturation

Clostridioides difficile is an anaerobic pathogen that thrives in the metabolically diverse intestinal environment. C. difficile is readily transmitted due to its transformation into a dormant spore form that is highly resistant to heat and disinfectants. Nutrient limitation is a key driver of spore formation; however, few metabolites have been directly shown to influence the regulation of C. difficile sporulation. A distinct aspect of C. difficile biology is the fermentation of amino acids through Stickland metabolism pathways, which are critical sources of energy for this pathogen. We hypothesized that as a preferred energy source, the amino acid proline may serve as a signal that regulates the initiation of sporulation or the development of spores. Using mutants in the proline reductase gene, prdA, and the proline-dependent regulator, prdR, we examined the impact of proline on C. difficile physiology and differentiation. Our results demonstrate that proline reductase is important for the development of mature spores and that excess proline can repress C. difficile sporulation through PrdR regulation. Further, we discovered that the end product of proline reduction, 5-aminovalerate, can support the growth of C. difficile through an unidentified, PrdR-dependent mechanism. IMPORTANCEC. difficile is an anaerobic intestinal pathogen that disseminates in the environment as dormant, resilient spores. Nutrient limitation is known to stimulate spore production, but the contribution of specific nutrients to sporulation is poorly understood. In this study, we examined the contribution of proline and proline fermentation to spore formation. Our results demonstrate the effect of proline fermentation on spore quality and the importance of the proline reductase pathway on spore maturation.

physiology↗

bioRxiv2021

Loss of the GARP but not EARP protein complex drives Golgi sterol overload during dendrite remodeling

Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, we find that both complexes are required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, we investigated genes that regulate sterols and related lipids at the TGN. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.

cell biology↗

bioRxiv2021

Identification of a novel Candida metapsilosis isolate suggests ongoing hybridization.

Candida metapsilosis is a member of the C. parapsilosis species complex, a group of opportunistic human pathogens. Of all the members of this complex, C. metapsilosis is the least virulent, and accounts for a small proportion of invasive Candida infections. Previous studies established that all C. metapsilosis isolates are hybrids, originating from a single hybridization event between two lineages, parent A and parent B. Here, we use MinION and Illumina sequencing to characterize a C. metapsilosis isolate that originated from a separate hybridization. One of the parents of the new isolate is very closely related to parent A. However, the other parent (parent C) is not the same as parent B. Unlike C. metapsilosis AB isolates, the C. metapsilosis AC isolate has not undergone introgression at the Mating Type-like Locus. In addition, the A and C haplotypes are not fully collinear. The C. metapsilosis AC isolate has undergone Loss of Heterozygosity (LOH) with a preference for haplotype A, indicating that this isolate is in the early stages of genome stabilization.

genomics↗

bioRxiv2020

Population genomics of the pathogenic yeast Candida tropicalis identifies hybrid isolates in environmental samples.

Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates C. tropicalis isolates from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2 - 6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36 - 49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/ heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source. Author summaryCandida tropicalis is an important fungal pathogen, which is particularly common in the Asia-Pacific and Latin America. There is currently very little known about the diversity of genotype and phenotype of C. tropicalis isolates. By carrying out a phylogenomic analysis of 77 isolates, we find that C. tropicalis genomes range from very homozygous to highly heterozygous. We show that the heterozygous isolates are hybrids, most likely formed by mating between different parents. Unlike other Candida species, the hybrids are more common in environmental than in clinical niches, suggesting that for this species, hybridization is not associated with virulence. We also explore the range of phenotypes, and we identify a genomic variant that is required for growth on valine and isoleucine as sole nitrogen sources.

genomics↗

bioRxiv2020

Polymorphic centromere locations in the pathogenic yeast Candida parapsilosis

Centromeres pose an evolutionary paradox: strongly conserved in function, but rapidly changing in sequence and structure. However, in the absence of damage, centromere locations are usually conserved within a species. We report here that isolates of the pathogenic yeast species Candida parapsilosis exhibit within-species polymorphism for the location of centromeres on two of its eight chromosomes. Its old centromeres have an inverted-repeat (IR) structure, whereas its new centromeres have no obvious structural features, but are located within 30 kb of the old site. Centromeres can therefore move naturally from one chromosomal site to another, apparently spontaneously and in the absence of any significant changes in DNA sequence. Our observations are consistent with a model where all centromeres are genetically determined, such as by the presence of short or long IRs, or the ability to form cruciforms. We also find that centromeres have been hotspots for genomic rearrangements in the C. parapsilosis clade.

genomics↗

bioRxiv2020

Spatial proximity of homologous centromere DNA sequences facilitated karyotype diversity and seeding of evolutionary new centromeres

Centromeres of Candida albicans form on unique and different DNA sequences but a closely related species, Candida tropicalis, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of centromere-type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of C. tropicalis by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in C. tropicalis. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of C. albicans and C. tropicalis. Identification of putative centromeres in closely related Candida sojae, Candida viswanathii and Candida parapsilosis indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in C. albicans. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.

genetics↗

bioRxiv2018

Coverage-versus-Length plots, a simple quality control step for de novo yeast genome sequence assemblies

Illumina sequencing has revolutionized yeast genomics, with prices for commercial draft genome sequencing now below $200. The popular SPAdes assembler makes it simple to generate a de novo genome assembly for any yeast species. However, whereas making genome assemblies has become routine, understanding what they contain is still challenging. Here, we show how graphing the information that SPAdes provides about the length and coverage of each scaffold can be used to investigate the nature of an assembly, and to diagnose possible problems. Scaffolds derived from mitochondrial DNA, ribosomal DNA, and yeast plasmids can be identified by their high coverage. Contaminating data, such as cross-contamination from other samples in a multiplex sequencing run, can be identified by its low coverage. Scaffolds derived from the bacteriophage PhiX174 and Lambda DNAs that are frequently used as molecular standards in Illumina protocols can also be detected. Assemblies of yeast genomes with high heterozygosity, such as interspecies hybrids, often contain two types of scaffold: regions of the genome where the two alleles assembled into two separate scaffolds and each has a coverage level C, and regions where the two alleles co-assembled (collapsed) into a single scaffold that has a coverage level 2C. Visualizing the data with Coverage-versus-Length (CVL) plots, which can be done using Microsoft Excel or Google Sheets, provides a simple method to understand the structure of a genome assembly and detect aberrant scaffolds or contigs. We provide a Python script that allows assemblies to be filtered to remove contaminants identified in CVL plots.\n\n100-word article summaryWe describe a simple new method, Coverage-versus-Length plots, for examining de novo genome sequence assemblies. These plots enable researchers to detect scaffolds that have unusually high or unusually low coverage, which allows contaminants, and scaffolds that come from atypical parts of the organisms DNA complement, to be detected. We show that contaminants are common in yeast genomes sequenced in multiplex Illumina runs. We provide instructions for making plots using Microsoft Excel or Google Sheets, and software for filtering assemblies to remove contaminants. Contaminants can be detected and removed, even without knowing their source.

genomics↗