Biology subjects

Bussan, H. E.

Publications and source records attributed to Bussan, H. E..

4 recordsLinked to original sources

bioRxiv2024

Impacts of perR on oxygen sensitivity, gene expression, and murine infection in Clostridioides difficile 630Δerm

Clostridioides difficile infection (CDI), characterized by colitis and diarrhea, afflicts approximately half a million people in the United States every year, burdening both individuals and the healthcare system. C. difficile 630{Delta}erm is an erythromycin-sensitive variant of the clinical isolate C. difficile 630 and is commonly used in the C. difficile research community due to its genetic tractability. 630{Delta}erm possesses a point mutation in perR, an autoregulated transcriptional repressor that regulates oxidative stress resistance genes. This point mutation results in a constitutively de-repressed PerR operon in 630{Delta}erm. To address the impacts of perR on phenotypes relevant for oxygen tolerance and relevant to a murine model of CDI, we corrected the point mutant to restore PerR function in 630{Delta}erm (herein, 630{Delta}erm perRWT). We demonstrate that there is no difference in growth between 630{Delta}erm and a 630{Delta}erm perRWT under anaerobic conditions or when exposed to concentrations of O2 that mimic those found near the surface of the colonic epithelium. However, 630{Delta}erm perRWT is more sensitive to ambient oxygen than 630{Delta}erm, which coincides with alterations in expression of a variety of perR-dependent and perR-independent genes. Finally, we show that 630{Delta}erm and 630{Delta}erm perRWT do not differ in their ability to infect and cause disease in a well-established murine model of CDI. Together, these data support the hypothesis that the perR mutation in 630{Delta}erm arose as a result of exposure to ambient oxygen and that the perR mutation in 630{Delta}erm is unlikely to impact CDI-relevant phenotypes in laboratory studies. ImportanceClostridioides difficile is a diarrheal pathogen and a major public health concern. To improve humans understanding of C. difficile, a variety of C. difficile isolates are used in research, including C. difficile 630{Delta}erm. 630{Delta}erm is a derivative of the clinical isolate 630 and is commonly studied because it is genetically manipulable. Previous work showed that a mutation in perR in 630{Delta}erm results in a dysregulated oxidative stress response, but no work has been done to characterize perR-dependent effects on the transcriptome or to determine impacts of perR during infection. Here, we identify transcriptomic differences between 630{Delta}erm and 630{Delta}erm perRWT exposed to ambient oxygen and demonstrate that there is no strain-based difference in burdens in murine C. difficile infection.

microbiology↗

bioRxiv2022

Complete sequencing of a cynomolgus macaque major histocompatibility complex haplotype

Macaques provide the most widely used nonhuman primate models for studying immunology and pathogenesis of human diseases. While the macaque major histocompatibility complex (MHC) region shares most features with the human leukocyte antigen (HLA) region, macaques have an expanded repertoire of MHC class I genes. Although a chimera of two rhesus macaque MHC haplotypes was first published in 2004, the structural diversity of MHC genomic organization in macaques remains poorly understood due to a lack of adequate genomic reference sequences. We used ultra-long Oxford Nanopore and high-accuracy PacBio HiFi sequences to fully assemble the [~]5.2 Mb M3 haplotype of an MHC-homozygous, Mauritian-origin cynomolgus macaque (Macaca fascicularis). The MHC homozygosity allowed us to assemble a single MHC haplotype unambiguously and avoid chimeric assemblies that hampered previous efforts to characterize this exceptionally complex genomic region in macaques. The high quality of this new assembly is exemplified by the identification of an extended cluster of six Mafa-AG genes that contains a recent duplication with a remarkably similar [~]48.5 kb block of sequence. The MHC class II region of this M3 haplotype is similar to the previously sequenced rhesus macaque haplotype and HLA class II haplotypes. The MHC class I region, in contrast, contains 13 MHC-B genes, four MHC-A genes, and three MHC-E genes (versus 19 MHC-B, two MHC-A, and one MHC-E in the previously sequenced haplotype). These results provide an unambiguously assembled single contiguous cynomolgus macaque MHC haplotype with fully curated gene annotations that will inform infectious disease and transplantation research.

genomics↗

bioRxiv2020

Consistent ultra-long DNA sequencing with automated slow pipetting

BackgroundOxford Nanopore Technologies instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing. ResultsWe have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding one hundred kilobases in length and increased its libraries average read length over manual slow pipetting. ConclusionsSNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.

genomics↗

bioRxiv2019

Characterization of 100 extended major histocompatibility complex haplotypes in Indonesian cynomolgus macaques

Many medical advancements - including improvements to anti-rejection therapies in transplantation and vaccine development - rely on pre-clinical studies conducted in cynomolgus macaques (Macaca fascicularis). Major histocompatibility complex (MHC) class I and class II genes of cynomolgus macaques are orthologous to human leukocyte antigen complex (HLA) class I and class II genes, respectively. Both encode cell-surface proteins involved in cell recognition and rejection of non-host tissues. MHC class I and class II genes are highly polymorphic, so comprehensive genotyping requires the development of complete databases of allelic variants. Our group used PacBio circular consensus sequencing of full-length cDNA amplicons to characterize MHC class I and class II transcript sequences for a cohort of 295 Indonesian cynomolgus macaques (ICM) in a large, pedigreed breeding colony. These studies allowed us to expand the existing database of Macaca fascicularis (Mafa) alleles by identifying an additional 141 MHC class I and 61 class II transcript sequences. In addition, we defined co-segregating combinations of allelic variants as regional haplotypes for 70 Mafa-A, 78 Mafa-B and 45 Mafa-DRB gene clusters. Finally, we defined class I and class II transcripts that are associated with 100 extended MHC haplotypes in this breeding colony by combining our genotyping analyses with short tandem repeat (STR) patterns across the MHC region. Our sequencing analyses and haplotype definitions improve the utility of these ICM for transplantation studies as well as infectious disease and vaccine research.

genetics↗