Biology subjects

Freimark, L.

Publications and source records attributed to Freimark, L..

3 recordsLinked to original sources

bioRxiv2025

Complex exchanges among plasmids and clonal expansion of lineages shape the population structure and virulence of Borrelia burgdorferi

The many plasmids (commonly >20 per strain) of Borrelia burgdorferi (Bb) pose challenges for studies of Lyme disease. The genetic content of most plasmids cannot be resolved using short-read sequencing. We generated long-read assemblies (LRA) of 183 isolates of human-derived Bb and analyzed patterns of genome variation in the Lyme spirochete. LRAs confirm that populations consist of strongly-structured genotypes with nearly-clonal structure and tight-knit blocks of accessory genome elements. Notably, these patterns of linkage were statistical but not physical, with linkage blocks distributed across multiple plasmids. A consequence of this structure is that plasmid name and/or plasmid subtype does not capture strain-specific genetic content. We used network methods to characterize patterns of genetic linkage. We demonstrate that co-occurring gene networks, here termed genetic modules, are the fundamental unit of genome variation in Bb and designate genetic modules consisting of co-occurring genes. We linked genetic modules to Bb phenotype by identifying modules that influence dissemination in humans. This modular decomposition clarifies previously observed associations between strain and virulence. For example, virulent RST1/OspC type A strains are distinguished by the presence of virulence-associated modules containing gene content from lp28-1, lp56, and the chromosome along with the absence of gene content on lp28-1 and lp28-4 associated with localized disease. LRAs also demonstrate that the well-established statistical linkage between physical unlinked genetic markers (e.g. RST and OspC) is a general pattern among accessory genome elements. In summary, genetic modules containing genes linked across multiple replicons, rather than strain-defining plasmids, organize the Bb accessory genome and the strain-specific variation responsible for differences in human virulence.

microbiology↗

bioRxiv2023

Whole genome sequencing of Borrelia burgdorferi isolates reveals linked clusters of plasmid-borne accessory genome elements associated with virulence.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived B. burgdorferi sensu stricto (Bbss) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bbss isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bbss isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of [~]800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm across Bbss isolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

microbiology↗

bioRxiv2020

Enhanced virus detection and metagenomic sequencing in patients with meningitis and encephalitis

Meningitis and encephalitis are leading causes of central nervous system (CNS) disease and often result in severe neurological compromise or death. Traditional diagnostic workflows largely rely on pathogen-specific diagnostic tests, sometimes over days to weeks. Metagenomic next-generation sequencing (mNGS) is a high-throughput platform that profiles all nucleic acid in a sample. We prospectively enrolled 68 patients from New England with known or suspected CNS infection and performed mNGS from both RNA and DNA to identify potential pathogens. Using a computational metagenomic classification pipeline based on KrakenUniq and BLAST, we detected pathogen nucleic acid in cerebrospinal fluid (CSF) from 22 subjects. This included some pathogens traditionally diagnosed by serology or not typically identified in CSF, including three transmitted by Ixodes scapularis ticks (Powassan virus, Borrelia burgdorferi, Anaplasma phagocytophilum). Among 24 subjects with no clinical diagnosis, we detected enterovirus in two subjects and Epstein Barr virus in one subject. We also evaluated two methods to enhance detection of viral nucleic acid, hybrid capture and methylated DNA depletion. Hybrid capture nearly universally increased viral read recovery. Although results for methylated DNA depletion were mixed, it allowed detection of varicella zoster virus DNA in two samples that were negative by standard mNGS. Overall, mNGS is a promising approach that can test for multiple pathogens simultaneously, with similar efficacy to pathogen-specific tests, and can uncover geographically relevant infectious CNS disease, such as tick-borne infections in New England. With further laboratory and computational enhancements, mNGS may become a mainstay of workup for encephalitis and meningitis. ImportanceMeningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures. Despite intensive diagnostic efforts, 40-60% of people with meningitis or encephalitis have no clear cause of their CNS disease identified. As diagnostic uncertainty often leads to costly inappropriate therapies, the need for novel pathogen detection methods is paramount. Metagenomic next-generation sequencing (mNGS) offers the unique opportunity to circumvent these challenges using unbiased laboratory and computational methods. Here, we performed comprehensive mNGS from 68 patients with suspected CNS infection, and define enhanced methods to improve the detection of CNS pathogens, including those not traditionally identified in the CNS by nucleic acid detection. Overall, our work helps elucidate how mNGS can become a mainstay in the diagnostic toolkit for CNS infections.

neuroscience↗