Biology subjects

Callahan, B.

Publications and source records attributed to Callahan, B..

7 recordsLinked to original sources

bioRxiv2026

Determinants in gammaretroviral Env dictate the production of neutralizing antibodies

Protective immune responses are shaped by the nature of the infectious pathogen. Some viral infections are self-limiting and stimulate lasting immunity, while others activate poor responses that fail to control the infection or prevent development of disease. Little is known concerning the specific viral determinants that control whether a given viral infection will result in protective immunity. Here, we demonstrate that strains of the gammaretrovirus, murine leukemia virus, differ in their ability to stimulate neutralizing antibody production via a noncanonical pathway. Additionally, we show that virus-specific production of neutralizing immune responses is unique to infection, suggesting that the activation of alternative antibody responses is triggered by the infectious process rather than the unique nature of specific viral antigens. Viral chimeras indicate that activation of neutralizing antiviral antibody production is determined by the receptor-binding domain, and that the surface subunit controls binding to and infection of various cell types. The ability of specific viral factors to determine the outcome of infection and the induction of distinctive immune responses between closely related retroviral strains provides an ideal model for the dissection of both host and viral determinants underlying the control of viral infection and the production of neutralizing antibodies. SUMMARYWe report here that retroviral envelope glycoprotein determines stimulation of GC B cell responses and the production of neutralizing antibodies in the absence of IFN{gamma}-signaling, thus defining the factors that stimulate non-canonical immune responses and further developing the knowledge required for vaccine development.

immunology↗

bioRxiv2026

Perturbations shift the composition of bacterial DNA carried by virus-like particles in the murine gut microbiome

Horizontal gene transfer (HGT) is a driving force in microbial evolution that allows community members to rapidly evolve to cope with environmental stressors and competition. Despite the importance of HGT for the generation of genetic diversity, little is known about the specific mechanisms or dynamics of transfer in complex communities. Transductomics is a sequencing based technique which identifies potential HGT by bacteriophages (transduction) through sequencing of the transductome - the DNA carried by bacteriophages and other virus-like particles in a sample. We analyzed the murine gut transductome before and after perturbations with antibiotics and Clostridioides difficile infection (CDI). We found that several bacterial families - the Oscillospiraceae, Butyricoccaceae, and Turicibactericeae - disproportionally contributed to the transductome. Some families, like the Butyricicoccaceae, were frequent transducers in both the baseline and perturbed murine gut microbiome while other taxa displayed condition-specific transduction indicating that there may be specific transducing subpopulations or regulatory mechanisms controlling transduction frequency. Additionally, we found a diversity of highly abundant and enriched mobile genetic elements (MGEs) in the transductome including plasmids, integrative conjugative elements, phage satellites and transposons. The detection of MGEs containing conjugative elements suggest that some MGEs may spread through both transduction and conjugation. Overall, our work reveals a complex network of gene exchange occurring through transduction in the gut microbiome.

microbiology↗

bioRxiv2026

TLR7-independent control of retroviral infection

Development of effective immune responses against any pathogen requires efficient activation of the innate immune system followed by induction of an appropriate adaptive immune response using specific signaling pathways tailored to each infection. The sole innate immune receptor implicated in the induction of adaptive immunity to retroviral infection is Toll like receptor 7 (TLR7). We have found that germinal center responses, neutralizing antibody production, and clearance of murine leukemia virus (MLV) infection in BALB.J and C57BL/6N (B6N) mice occurs in the absence of TLR7 signaling. This suggests that a previously unknown alternative sensing pathway exists for the activation of protective immune responses upon retroviral infection. Genetic crosses indicate that the ability to control retroviral infection in the absence of TLR7 signaling is determined by the same recessive mechanism in both B6N and BALB.J mice, suggesting that TLR7-independent responses do not result from a gain-of-function of an alternative pattern recognition receptor. Additionally, we observed that TLR7-deficient BALB.J mice produce neutralizing antibodies against mouse mammary tumor virus (MMTV) infection, indicating that this alternative sensing pathway is active against retroviruses of multiple genera. Finally, we determined that the alternative sensing pathway is also independent of both MyD88 and STING signaling. The ability of mice of two genetic backgrounds to control retroviral infection in the absence of TLR7 signaling provides a valuable tool for the identification of a novel mechanism of retrovirus control. The dissection of this pathway has the potential to alter our understanding of the requirements for the stimulation of antigen-specific neutralizing immunity. Significance StatementInnate immune sensors are required for induction of pathogen-specific immune responses, and the development of novel vaccines requires an understanding of the basic mechanisms by which the immune system detects and responds to pathogens. While multiple innate immune receptors have been implicated in the sensing of retroviral infection, only Toll-like receptor 7 (TLR7) has been found to upregulate adaptive immune responses. Here, we demonstrate that an additional TLR7-independent mechanism for the activation of antiretroviral antibody responses is present in inbred mice from two genetic backgrounds. This finding has implications for the selection of mouse models for the study of antiviral immune responses and has the potential to alter our understanding of the requirements for the stimulation of antigen-specific neutralizing immunity.

microbiology↗

bioRxiv2026

Gut Microbiome Alterations in Canine Idiopathic Epilepsy: A Pairwise Case-Control Study

BackgroundIdiopathic epilepsy (IE) is the most common chronic nervous system disorder of dogs, and its cause is poorly understood. Emerging evidence suggests that microbiome alterations can occur with IE via the microbiota-gut-brain axis. Therefore, we analyzed the fecal microbiomes of 98 dogs (49 IE, 49 control) in a pairwise case-control observational study using 16S rRNA gene sequencing. ResultsAlthough the microbial community was mostly similar between groups, IE was associated with a modest but significant shift in Weighted-Unifrac distance (P = 0.042). We used six differential abundance (DA) methods to identify differentially abundant amplicon sequencing variants (ASVs) between IE and control groups. Notably, one Collinsella ASV was found to be significantly more abundant in IE dogs by all six methods. The gut microbial compositions varied drastically across households (accounting for about 69% of the total variation), but did not have significant differences between sex, age, or breed. Phenobarbital administration in IE dogs had a significant effect on seizure control, and was not associated with changes in the microbiome. ConclusionOur findings suggest a relationship between gut microbiomes and IE. However, the specific mechanism needs to be further investigated.

microbiology↗

bioRxiv2026

TrIdent - An R package to automate transductomics analysis of virus-like particle mediated DNA mobilization

BackgroundTransduction is a form of horizontal gene transfer in which bacterial DNA is packaged and transferred by virus-like particles (VLPs). Transductomics is a sequencing-based method used to detect DNA carried by VLPs. During transductomics analysis, reads from a samples ultra-purified VLPs are mapped to metagenomic contigs assembled from the same samples whole-community. The read mapping produces coverage patterns that require a time-consuming manual inspection and classification process which makes the methods use unfeasible for datasets with many samples. ResultsWe developed a novel algorithm, TrIdent (Transduction Identification), that uses pattern-matching to automate the transductomics data analysis and that is available as an R package (https://jlmaier12.github.io/TrIdent/). There is no software equivalent to TrIdent so we compared TrIdents classifications of transductomics datasets to classifications made by human classifiers. TrIdents classifications were generally comparable to the manual classifications on a previously generated, manually classified transductomics dataset. When applied to newly generated transductomics data from the murine microbiota, TrIdent agreed with two independent human classifiers as much as the two independent human classifications agreed with each other. TrIdent classified transductomics datasets in a fraction of the time needed by human classifiers, and the classifications produced by TrIdent are fully reproducible. We used TrIdent to explore three murine gut transductomes and found that bacterial DNA associated with the Oscillospiraceae and Turicibacteraceae families was highly enriched in the DNA packaged by VLPs as compared to the whole community metagenomes. ConclusionsThe TrIdent software is a more accessible, more efficient, and more reproducible alternative to the manual inspection of read coverage patterns previously required for transductomics data analysis. To demonstrate the application of TrIdent, we analyzed transductomics datasets from murine fecal pellets and showed that specific low abundance bacterial families appear to be heavily involved in transduction.

genomics↗

bioRxiv2024

Pseudo-pac site sequences used by phage P22 in generalized transduction of Salmonella

Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction. Typically, the P22 DNA packaging machinery initiates packaging when a short sequence of DNA, known as the pac site, is recognized on the P22 genome. However, sequences similar to the pac site in the host genome, called pseudo-pac sites, lead to erroneous packaging and subsequent generalized transduction of Salmonella DNA. While the general genomic locations of the Salmonella pseudo-pac sites are known, the sequences themselves have not been determined. We used visualization of P22 sequencing reads mapped to host Salmonella genomes to define regions of generalized transduction initiation and the likely locations of pseudo-pac sites. We searched each genome region for the sequence with the highest similarity to the P22 pac site and aligned the resulting sequences. We built a regular expression (sequence match pattern) from the alignment and used it to search the genomes of two P22-susceptible Salmonella strains-LT2 and 14028S- for sequence matches. The final regular expression successfully identified pseudo-pac sites in both LT2 and 14028S that correspond with generalized transduction initiation sites in mapped read coverages. The pseudo-pac site sequences identified in this study can be used to predict locations of generalized transduction in other P22-susceptible hosts or to initiate generalized transduction at specific locations in P22-susceptible hosts with genetic engineering. Furthermore, the bioinformatics approach used to identify the Salmonella pseudo-pac sites in this study could be applied to other phage-host systems. ImportanceBacteriophage P22 has been a genetic tool and a key model for the study of generalized transduction in Salmonella since the 1950s, yet certain components of the generalized transduction molecular mechanism remain unknown. Specifically, the locations and sequences of pseudo-pac sites, hypothesized to facilitate packaging of Salmonella DNA by P22, to date have not been determined. In this study, we identified the specific locations and sequences of the pseudo-pac sites frequently recognized by P22 in Salmonella genomes. The identification of highly efficient pseudo-pac sites in Salmonella provides fundamental insights into the sequence specificity necessary for P22 pac site recognition and opens the door to more targeted use of generalized transduction with P22.

microbiology↗

bioRxiv2022

Bioluminescence-based reporters for characterizing inhibitors and activators of human Sonic Hedgehog protein autoprocessing in live cells at high throughput.

The Sonic hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and cholesterylate the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursors enzymatic domain. Here we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing non-invasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a wild-type (WT) SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. A conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the substrate activity of cholesterol. Inducibility of the D46A reporter was established using a synthetic sterol, 2- carboxy cholestanol, designed to bypass the defect through intra-molecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel membrane-impermeable nanoluciferase substrate, CLZ-2P. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/497760v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@63c641org.highwire.dtl.DTLVardef@6cee38org.highwire.dtl.DTLVardef@18bd310org.highwire.dtl.DTLVardef@cdfdc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗