Biology subjects

Harper, C. P.

Publications and source records attributed to Harper, C. P..

4 recordsLinked to original sources

bioRxiv2026

Extracellular vesicle-mediated suppression of macrophage STING signaling promotes immune dysfunction in dedifferentiated liposarcoma

BackgroundDedifferentiated liposarcoma (DDLPS) is characterized by abundant immune cell infiltration yet derives limited benefit from immune checkpoint blockade and stimulator of interferon genes (STING) agonist-based strategies, suggesting tumor-mediated suppression of antitumor immunity. Tumor-associated macrophages are the most abundant immune populations in DDLPS, but the factors regulating their function remain incompletely understood. MethodsExtracellular vesicles (EVs) were isolated from two DDLPS cell lines and serum from 16 DDLPS patients and 13 healthy donors. EVs impact on cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) -induced macrophage activation was assessed by cytokine secretion, surface markers, functional assays and macrophage-T-cell coculture. Proteomics was performed in EV-treated and EV-untreated macrophages from three donors. Pathway and protein interaction analyses were integrated with The Cancer Genome Atlas (TCGA) DDLPS transcriptomic and survival data. ResultsWe show that EVs released by DDLPS cells suppress macrophage responsiveness to classic STING agonist cGAMP. EVs derived from DDLPS attenuated cGAMP-induced expression of type I interferon-associated cytokines and chemokines, reduced IFN-{beta} secretion, and impaired phosphorylation of STING, TBK1 and IRF3. Functionally, DDLPS EV exposure shifted macrophages toward an immunoregulatory phenotype, restrained phagocytic activity, and attenuated macrophage-dependent T-cell proliferation while promoting T-cell exhaustion. Proteomic profiling revealed extensive macrophage reprogramming characterized by suppression of STING-associated signaling, antigen processing and presentation associated pathways and proteins targeted by miR-16-5p. Consistent with these findings, STING expression was associated with prolonged overall survival in DDLPS, while reduced expression of miR-16-5p target proteins was associated with attenuated STING pathway activity and immunostimulatory macrophage signatures. ConclusionsThese findings identify EV-mediated suppression of macrophage STING signaling as a mechanism of immune dysfunction in DDLPS and provide a framework for understanding immune resistance in this disease.

cancer biology↗

bioRxiv2024

Investigating the biosynthesis and roles of the auxin phenylacetic acid during Pseudomonas syringae-Arabidopsis thaliana pathogenesis

Several plant-associated microbes synthesize the auxinic plant growth regulator phenylacetic acid (PAA) in culture; however, the role of PAA in plant-pathogen interactions is not well understood. In this study, we investigate the role of PAA during interactions between the phytopathogenic bacterium Pseudomonas syringae strain PtoDC3000 (PtoDC3000) and the model plant host, Arabidopsis thaliana. Previous work demonstrated that indole-3-acetaldehyde dehydrogenase A (AldA) of PtoDC3000 converts indole-3-acetaldehyde (IAAld) to the auxin indole-3-acetic acid (IAA). Here, we further demonstrate the biochemical versatility of AldA, as it can use both IAAld and phenylacetaldehyde as substrates to produce IAA and PAA, respectively. We also show that during infection AldA-dependent synthesis of either IAA or PAA by PtoDC3000 does not contribute significantly to the increase in auxin levels in A. thaliana leaves. Using available arogenate dehydratase (adt) mutant lines of A. thaliana compromised for PAA synthesis, we observed that a reduction in PAA-Asp and PAA-Glu is correlated with elevated levels of IAA and increased susceptibility. These results provide evidence that PAA/IAA homeostasis in A. thaliana influences the outcome of plant-microbial interactions.

microbiology↗

bioRxiv2024

Critical analysis of polycyclic tetramate macrolactam biosynthetic cluster phylogeny and functional diversity

Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggests their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. Towards addressing the origins of this complexity and understanding its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs, and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery. IMPORTANCEPolycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically-important bacteria, including Streptomyces and Lysobacter spp. Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggests new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize practical limitations to genomics-based discovery by exposing hidden complexity.

microbiology↗

bioRxiv2019

Dual role of auxin in regulating plant defense and bacterial virulence gene expression during Pseudomonas syringae PtoDC3000 pathogenesis.

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen Pseudomonas syringae strain PtoDC3000 produces the plant hormone auxin (Indole-3-acetic acid, or IAA) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth, demonstrating that host auxin signaling is required for normal susceptibility to PtoDC3000, and this phenotype was dependent on SA-mediated defenses. However, despite exhibiting decreased auxin perception, tir1 afb1 afb4 afb5 quadruple mutant plants lacking four of the six known auxin co-receptors supported increased levels of bacterial growth. This mutant also exhibited elevated IAA levels, suggesting that the increased IAA in these plants may promote PtoDC3000 growth independent of host auxin signaling, perhaps through a direct effect on the pathogen. In support of this, we found that IAA directly impacted the pathogen, by modulating expression of bacterial virulence genes, both in liquid culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

plant biology↗