Biology subjects

Collman, R. G.

Publications and source records attributed to Collman, R. G..

3 recordsLinked to original sources

bioRxiv2025

Optimizing methods for virome analysis based on studies of a synthetic viral community

Studies of whole viral populations--the "virome"--are yielding exciting new insights into biological systems, but methods are still being optimized. Here we describe generation and use of a synthetic viral community to assess several technical challenges important in virome analysis. Our mock community was comprised of phages lambda, T4, M13, MS2, and phi6, together with adeno-associated virus (AAV), murine hepatitis virus (MHV), and vaccinia virus (VV). We spiked the mock community into different human sample types, including stool, saliva, oropharyngeal (OP) wash, and bronchoalveolar lavage (BAL), then passed the samples through different virus enrichment protocols and analyzed by Illumina sequencing. Compared to direct metagenomic sequencing, VLP enrichment protocols greatly increased viral read yields from virus-rich samples such as from stool and saliva. Three VLP enrichment work flows were compared, and each was found to have strengths and weaknesses. Four methods for DNA amplification were compared, with three showing over-amplification of small circular ssDNA viruses, most notably GenomiPhi. Studies of viral particle stability in the presence of nuclease showed that most viral genomes were stable when protected in viral particles, but phage MS2 RNA was unexpectedly labile under some of the conditions tested. Comparison of Illumina 1000-cycle sequencing versus 300-cycle sequencing showed that longer reads supported generation of longer viral genome assemblies. Bacteriophage DNA can be modified by at least 12 different chemistries, raising the question of whether these modifications might block recovery in virome analytical protocols. We tested bacteriophage T4 DNA modified with glucosyl-hydroxymethylcytosine (ghmC) and hydroxymethylcytosine (hmC), and found that both were readily detected, though the recovery of ghmC-modified DNA was reduced. These studies together with published data help provide guidance for virome researchers optimizing analytical protocols.

microbiology↗

bioRxiv2023

Comparison of SARS-CoV-2 variants in primary human nasal cultures indicates Delta as most cytopathic and Omicron as fastest replicating

The SARS-CoV-2 pandemic was marked with emerging viral variants, some of which were designated as variants of concern (VOCs) due to selection and rapid circulation in the human population. Here we elucidate functional features of each VOC linked to variations in replication rate. Patient-derived primary nasal cultures grown at air-liquid-interface (ALI) were used to model upper-respiratory infection and human lung epithelial cell lines used to model lower-respiratory infection. All VOCs replicated to higher titers than the ancestral virus, suggesting a selection for replication efficiency. In primary nasal cultures, Omicron replicated to the highest titers at early time points, followed by Delta, paralleling comparative studies of population sampling. All SARS-CoV-2 viruses entered the cell primarily via a transmembrane serine protease 2 (TMPRSS2)-dependent pathway, and Omicron was more likely to use an endosomal route of entry. All VOCs activated and overcame dsRNA-induced cellular responses including interferon (IFN) signaling, oligoadenylate ribonuclease L degradation and protein kinase R activation. Among the VOCs, Omicron infection induced expression of the most IFN and IFN stimulated genes. Infections in nasal cultures resulted in cellular damage, including a compromise of cell-barrier integrity and loss of nasal cilia and ciliary beating function, especially during Delta infection. Overall, Omicron was optimized for replication in the upper-respiratory system and least-favorable in the lower-respiratory cell line; and Delta was the most cytopathic for both upper and lower respiratory cells. Our findings highlight the functional differences among VOCs at the cellular level and imply distinct mechanisms of pathogenesis in infected individuals. ImportanceComparative analysis of infections by SARS-CoV-2 ancestral virus and variants of concern including Alpha, Beta, Delta, and Omicron, indicated that variants were selected for efficiency in replication. In infections of patient-derived primary nasal cultures grown at air-liquid-interface to model upper-respiratory infection, Omicron reached highest titers at early time points, a finding that was confirmed by parallel population sampling studies. While all infections overcame dsRNA-mediated host responses, infections with Omicron induced the strongest interferon and interferon stimulated gene response. In both primary nasal cultures and lower-respiratory cell line infections by Delta were most damaging to the cells as indicated by syncytia formation, loss of cell barrier integrity and nasal ciliary function.

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa Elicits Sustained IL-1β Upregulation in Alveolar Macrophages from Lung Transplant Recipients

BackgroundIsolation of Pseudomonas aeruginosa (PsA) is associated with increased BAL (bronchoalveolar lavage) inflammation and lung allograft injury in lung transplant recipients (LTR). However, the effect of PsA on macrophage responses in this population is incompletely understood. We examined human alveolar macrophage (AM) responses to PsA and Pseudomonas dominant microbiome in healthy lung transplant recipients (LTR). MethodsWe stimulated THP-1 derived macrophages (THP-1M) and human AM from LTR with different bacteria and LTR BAL derived microbiome characterized as Pseudomonas-dominant. Macrophage responses were assessed by high dimensional flow cytometry, including their intracellular production of cytokines (TNF-, IL-6, IL-8, IL-1{beta}, IL-10, IL-1RA, and TGF-{beta}). Pharmacological inhibitors were utilized to evaluate the role of the inflammasome in PsA-macrophages interaction. ResultsWe observed upregulation of pro-inflammatory cytokines (TNF-, IL-6, IL-8, IL-1{beta}) following stimulation by PsA compared to other bacteria (Staphylococcus aureus, Prevotella melaninogenica, Streptococcus pneumoniae) in both THP-1 derived and LTR AM, predominated by IL-1{beta}. IL-1{beta} production from THP-1 was sustained after PsA stimulation for up to 96 hours and 48 hours in LTR AM. Treatment with the inflammasome inhibitor BAY11-7082 abrogated macrophage IL-1{beta} and IL-18 production after PsA exposure. BAL Pseudomonas-dominant microbiota elicited an increased IL-1{beta}, similar to PsA, an effect abrogated by the addition of antibiotics. ConclusionPsA and PsA-dominant lung microbiota induce sustained IL-1{beta} production in LTR AM. Pharmacological targeting of the inflammasome reduces PsA-macrophage-IL1{beta} responses, underscoring their use in lung transplant recipients.

immunology↗