Biology subjects

Johnson, T. L.

Publications and source records attributed to Johnson, T. L..

6 recordsLinked to original sources

bioRxiv2026

Identification of novel inhibitors of Mycobacterium smegmatis growth through genome-wide overexpression of Cluster P3 mycobacteriophage Xavia genes

We examined whether genes encoded by the mycobacteriophage Xavia disrupt growth of Mycobacterium smegmatis, a widely used mycobacterial model. Seventy-one Xavia genes were individually expressed using an inducible plasmid system and assessed for effects on colony formation. Two genes were lethal even without induction, indicating toxicity under basal expression. Induction of sixteen additional genes reduced bacterial growth, spanning structural proteins, lysogeny regulators, DNA-associated enzymes, a lysis protein, and several genes with no known function. These findings expand functional insights into mycobacteriophage gene repertoires and identify candidates for future mechanistic studies. AbstractBacteriophage genomes encode large numbers of genes with no known function, and many of these genes affect essential host processes when expressed in a heterologous system. For mycobacteriophages, genome-wide overexpression in Mycobacterium smegmatis provides a direct way to identify proteins that impair growth and to determine which mycobacterial pathways are sensitive to phage gene products. To evaluate the cytotoxic potential of the Cluster P3 phage Xavia, a lineage that has not undergone functional screening, we constructed an arrayed pExTra library containing 71 predicted Xavia genes under control of the anhydrotetracycline inducible promoter pTet. All constructs were sequence-verified and transformed into M. smegmatis, and induction allowed measurement of gene-specific effects on growth. Two genes prevented recovery of transformants, suggesting toxicity under basal promoter leakiness. Inducible expression of 16 additional genes impaired growth, and these inhibitory proteins include structural components, regulators of lysogeny, enzymes of DNA metabolism, a lysis factor, and several proteins with no known function. Four of the strongest inhibitors were genes with no known function. These results extend functional screening into the previously untested P3 branch of Actinobacteriophages and identify new proteins that require mechanistic analysis.

microbiology↗

bioRxiv2025

Identification of Amblyomma americanum antigens after vaccination with tick extracellular vesicles in white-tailed deer

Anti-tick vaccines represent a promising alternative to chemical acaricides for the management of ticks on wildlife; however little progress has been made to produce a vaccine effective in wild hosts that are critical for tick reproduction, such as the white-tailed deer (Odocoileus virginianus; WTD). To date, most tick antigens have been tested using laboratory models (i.e. rabbits); however, their expression in wild hosts has not been confirmed. We recently tested Amblyomma americanum salivary (SG) and midgut (MG) extracellular vesicles (EVs) as vaccine candidates in WTD, which resulted in on-host female tick mortality. Using a proteomic approach, we show that these SG- and MG-EVs contain a "core-cargo" enriched in chaperones, small GTPases, actin and actin-related proteins, and other proteins previously reported in small EVs. Label-free quantitative proteomics showed significant differences in protein cargo between MG and SG-EVs (333 proteins out of 516). Pre-vaccinated and day 57 post-injection serum samples from three vaccinated and one control WTD were used to immuno-precipitate antigenic proteins from SG- and MG-EV preparations. Proteomic analysis of immunoprecipitated proteins identified thirty antigens with potential for use in anti-tick vaccines, seven of which we have categorized as high priority. These proteins represent promising candidates for anti-tick vaccine design in WTD and other wildlife hosts.

immunology↗

bioRxiv2024

Evaluation of tick salivary and midgut extracellular vesicles as anti-tick vaccines in White-tailed deer (Odocoileus virginianus)

Current tick control measures are focused on the use of synthetic acaricides and personal protective measures. However, the emergence of acaricide resistance and the maintenance of tick populations in wildlife has precluded the efficient management of ticks. Thus, host-targeted, non-chemical control measures are needed to reliably reduce ticks parasitizing sylvatic reservoirs. This project aimed to evaluate extracellular vesicles (EVs) from Amblyomma americanum as vaccine candidates for white-tailed deer (Odocoileus virginianus; WTD). Salivary gland (SG) and midgut (MG) EVs were isolated by ultracentrifugation. Three deer were vaccinated with SG and MG EVs and received two boosters at days 28 and 50. Two control deer were injected with adjuvant and PBS only. On day 58, WTD were infested with 100 A. americanum nymphs, 50 females, and 50 males that were allowed to feed to repletion. On-host and off-host mortality, tick engorgement weight, nymph molting, time to oviposition, and egg hatchability were evaluated. Serum samples were recovered every seven days until the last day of tick drop off, and then at one year (Y1) and 1-year and 1-month (Y1M1). Vaccination resulted in seroconversion and significant increases in total IgG levels that remained significantly higher than controls and pre-vaccination levels at Y1 and Y1M1. No negative effects were observed in nymphs, but on-host mortality of female A. americanum was significantly higher in vaccinated animals. No effects were observed on reproductive parameters. These results indicate that proteins within female tick SG and MG vesicles are not good candidates for vaccine design against nymphs; however, the on-host adult mortality suggests that tick EVs harbor protective antigens against A. americanum females.

immunology↗

bioRxiv2024

Synaptic vesicle release regulates pre-myelinating oligodendrocyte-axon interactions in a neuron subtype-specific manner

Oligodendrocyte-lineage cells are central nervous system (CNS) glia that perform multiple functions including the selective myelination of some but not all axons. During myelination, synaptic vesicle release from axons promotes sheath stabilization and growth on a subset of neuron subtypes. In comparison, it is unknown if pre-myelinating oligodendrocyte process extensions selectively interact with specific neural circuits or axon subtypes, and whether the formation and stabilization of these neuron-glia interactions involves synaptic vesicle release. In this study, we used fluorescent reporters in the larval zebrafish model to track pre-myelinating oligodendrocyte process extensions interacting with spinal axons utilizing in vivo imaging. Monitoring motile oligodendrocyte processes and their interactions with individually labeled axons revealed that synaptic vesicle release regulates the behavior of subsets of process extensions. Specifically, blocking synaptic vesicle release decreased the longevity of oligodendrocyte process extensions interacting with reticulospinal axons. Furthermore, blocking synaptic vesicle release increased the frequency that new interactions formed and retracted. In contrast, tracking the movements of all process extensions of singly-labeled oligodendrocytes revealed that synaptic vesicle release does not regulate overall process motility or exploratory behavior. Blocking synaptic vesicle release influenced the density of oligodendrocyte process extensions interacting with reticulospinal and serotonergic axons, but not commissural interneuron or dopaminergic axons. Taken together, these data indicate that alterations to synaptic vesicle release cause changes to oligodendrocyte-axon interactions that are neuron subtype specific.

neuroscience↗

bioRxiv2020

Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling

Viruses must effectively remodel host cellular pathways to replicate and evade immune defenses, and they must do so with limited genomic coding capacity. Targeting post-translational modification (PTM) pathways provides a mechanism by which viruses can broadly and rapidly transform a hostile host environment into a hospitable one. We used quantitative proteomics to measure changes in two PTM types - phosphorylation and ubiquitination - in response to HIV-1 infection with viruses harboring targeted deletions of a subset of HIV-1 genes. PTM analysis revealed a requirement for Aurora kinase A activity in HIV-1 infection and furthermore revealed that AMP-activated kinase activity is modulated during infection via HIV-1 Vif-mediated degradation of B56-containing protein phosphatase 2A (PP2A). Finally, we demonstrated that the Cullin4A-DDB1-DCAF1 E3 ubiquitin ligase ubiquitinates histone H1 somatic isoforms and that HIV-1 Vpr inhibits this process, leading to defects in DNA repair. Thus, global PTM profiling of infected cells serves as an effective tool for uncovering specific mechanisms of host pathway modulation.

microbiology↗

bioRxiv2019

Extensive splicing across the Saccharomyces genome

Pre-mRNA splicing is vital for the proper function and regulation of eukaryotic gene expression. Saccharomyces cerevisiae has been used as a model organism for studies of RNA splicing because of the striking conservation of the spliceosome components and its catalytic activity. Nonetheless, there are relatively few annotated alternative splice forms, particularly when compared to higher eukaryotes. Here, we describe a method to combine large scale RNA sequencing data to accurately discover novel splice isoforms in Saccharomyces cerevisiae. Using our method, we find extensive evidence for novel splicing of annotated intron-containing genes as well as genes without previously annotated introns and splicing of transcripts that are antisense to annotated genes. By incorporating several mutant strains at varied temperatures, we find conditions which lead to differences in alternative splice form usage. Despite this, every class and category of alternative splicing we find in our datasets is found, often at lower frequency, in wildtype cells under normal growth conditions. Together, these findings show that there is widespread splicing in Saccharomyces cerevisiae, thus expanding our view of the regulatory potential of RNA splicing in yeast.\n\nAuthor SummaryPre-mRNA splicing is a fundamental step in eukaryotic gene expression. Saccharomyces cerevisiae, also known as brewers yeast, is a model organism for the study of pre-mRNA splicing in eukaryotes. Through the process of pre-mRNA splicing, a single gene is capable of encoding multiple mature mRNA products, but it is often difficult to identify the splice events that lead to these mRNA products. Here, we describe a method to accurately discover novel splice events in Saccharomyces cerevisiae and find evidence for extensive splicing in Saccharomyces. By utilizing a variety of strains and growth conditions, we are able to characterize many splice forms and correlate cellular conditions with prevalence of novel splice events.

molecular biology↗