Biology subjects

Newman, J. D.

Publications and source records attributed to Newman, J. D..

6 recordsLinked to original sources

bioRxiv2026

Structural and biochemical characterisation of an iterative GCN5-related N-acetyltransferase required for fungal siderophore tailoring

Siderophore-mediated iron acquisition is essential for fungal survival, particularly under iron-limiting conditions. In Aspergillus fumigatus, SidG, a member of the GCN5-related N-acetyltransferase (GNAT) superfamily, catalyses the final step in the biosynthesis of the extracellular siderophore triacetylfusarinine C (TAFC) through sequential acetylation of the precursor fusarinine C (FsC). However, the timing, catalytic mechanism, and functional significance of this modification are not fully understood. Here, we reconstituted SidG activity in vitro and combined native mass spectrometry, X-ray crystallography, molecular dynamics simulations, and site-directed mutagenesis to investigate its catalytic properties. Our analyses demonstrate that SidG selectively binds acetyl-CoA from the cellular milieu and iteratively acetylates the FsC scaffold prior to iron chelation. Structural, biochemical, and molecular dynamics analyses support a direct transfer mechanism, identify key catalytic residues, and demonstrate the strict selectivity of SidG for short-chain acyl-CoA donors. Together, these findings establish the molecular basis for SidG-dependent siderophore tailoring and expand our understanding of GNAT-catalysed transformations in fungal natural product biosynthesis.

biochemistry↗

bioRxiv2023

SARS-CoV-2 infection triggers pro-atherogenic inflammatory 1 responses in human coronary vessels

COVID-19 patients present higher risk for myocardial infarction (MI), acute coronary syndrome, and stroke for up to 1 year after SARS-CoV-2 infection. While the systemic inflammatory response to SARS-CoV-2 infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques to locally promote inflammation remains unknown. Here, we report that SARS-CoV-2 viral RNA (vRNA) is detectable and replicates in coronary atherosclerotic lesions taken at autopsy from patients with severe COVID-19. SARS-CoV-2 localizes to plaque macrophages and shows a stronger tropism for arterial lesions compared to corresponding perivascular fat, correlating with the degree of macrophage infiltration. In vitro infection of human primary macrophages highlights that SARS-CoV-2 entry is increased in cholesterol-loaded macrophages (foam cells) and is dependent, in part, on neuropilin-1 (NRP-1). Furthermore, although viral replication is abortive, SARS-CoV-2 induces a robust inflammatory response that includes interleukins IL-6 and IL-1{beta}, key cytokines known to trigger ischemic cardiovascular events. SARS-CoV-2 infection of human atherosclerotic vascular explants recapitulates the immune response seen in cultured macrophages, including pro-atherogenic cytokine secretion. Collectively, our data establish that SARS-CoV-2 infects macrophages in coronary atherosclerotic lesions, resulting in plaque inflammation that may promote acute CV complications and long-term risk for CV events.

immunology↗

bioRxiv2021

Thiophenesulfonamides are specific inhibitors of quorum sensing in pathogenic Vibrios

Vibrio bacteria are pathogens of fish, shellfish, coral, and humans due to contaminated seafood consumption. Vibrio virulence factors are controlled by the cell-to-cell communication called quorum sensing, thus this signaling system is a promising target for therapeutic design. We screened a compound library and identified nine compounds, including several 2-thiophenesulfonamides, that inhibit the master quorum sensing transcription factor LuxR in Vibrio campbellii but do not affect cell growth. We synthesized a panel of 50 thiophenesulfonamide compounds to examine the structure-activity relationship effects on quorum sensing in vivo. The most potent molecule identified, PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole), specifically inhibits LuxR homologs in multiple strains of Vibrio vulnificus, Vibrio parahaemolyticus, and V. campbellii with sub-micromolar concentrations. PTSP efficacy is driven by amino acid conservation in the binding pocket, which is accurately predicted using in silico modeling of inhibitors. Our results underscore the potential for developing thiophenesulfonamides as specific quorum sensing-directed treatments for Vibrio infections.

microbiology↗

bioRxiv2020

The DNA binding domain of the Vibrio vulnificus SmcR transcription factor is flexible and recognizes diverse DNA sequences

The quorum-sensing regulon in vibrios is controlled by the LuxR/HapR family of transcriptional regulators. In Vibrio vulnificus, this regulator is called SmcR, and it controls expression of numerous virulence behaviors, including biofilm formation and elastase production. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic, as is common for regulators that bind as dimers with helix-turn-helix N-terminal DNA binding domains. However, the LuxR/HapR/SmcR consensus site is highly degenerate and asymmetric with variations in sequence at each promoter. To determine the mechanism of DNA site recognition, we generated separation-of-function mutants of SmcR that either repress or activate transcription but not both. The SmcR N55I protein is defective at transcription activation due to loss of binding to most DNA binding sites in activated promoters but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R, and N142D are defective for interaction with RNAP alpha but retain functional DNA binding activity. Using X-ray crystallography, we show that the wild-type SmcR dimer and the three RNAP-interaction mutants exhibit two conformations of the helix-turn-helix DNA binding domain. Conversely, the SmcR N55I X-ray crystal structure is limited to only one conformation and is restricted in recognition of single base-pair variations in DNA binding site sequences. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNA polymerase and a multi-conformational DNA binding domain that permits recognition of variable DNA sites. Thus, the LuxR/HapR/SmcR proteins balance specificity for quorum-sensing targets and diversity to accommodate binding at hundreds of sites genome-wide. SignificanceThe cell-cell communication system called quorum sensing controls expression of genes required for virulence in Vibrio bacteria species, including the potent human pathogen Vibrio vulnificus. The master transcriptional regulator of quorum-sensing genes in vibrios belongs to the LuxR/HapR/SmcR family. These regulators directly activate and repress transcription of >100 genes via binding to degenerate sites in promoter regions. We used X-ray crystallography to determine the structure of mutant SmcR proteins. Our experiments reveal that SmcR recognizes diverse sequences via a DNA binding domain that samples multiple conformations to accommodate variations in palindromic DNA sequences. Significantly, the DNA binding domain of SmcR is completely conserved in LuxR/HapR/SmcR family proteins, suggesting that this mechanism is representative of quorum-sensing regulation in other vibrios.

biochemistry↗

bioRxiv2020

Species-specific quorum sensing represses the chitobiose utilization locus in Vibrio cholerae

The marine facultative pathogen Vibrio cholerae forms complex multicellular communities on the chitinous shells of crustacean zooplankton in its aquatic reservoir. V. cholerae-chitin interactions are critical for the growth, evolution, and waterborne transmission of cholera. This is due, in part, to chitin-induced changes in gene expression in this pathogen. Here, we sought to identify factors that influence chitin-induced expression of one locus, the chitobiose utilization operon (chb), which is required for the uptake and catabolism of the chitin disaccharide. Through a series of genetic screens, we identified that the master regulator of quorum sensing, HapR, is a direct repressor of the chb operon. We also found that the levels of HapR in V. cholerae are regulated by the ClpAP protease. Furthermore, we show that the canonical quorum sensing cascade in V. cholerae regulates chb expression in a HapR-dependent manner. Through this analysis we found that signaling via the species-specific autoinducer CAI-1, but not the inter-species autoinducer AI-2, influences chb expression. This phenomenon of species-specific regulation may enhance the fitness of this pathogen in its environmental niche. ImportanceIn nature, bacteria live in multicellular and multispecies communities. Microbial species can sense the density and composition of their community through chemical cues using a process called quorum sensing (QS). The marine pathogen Vibrio cholerae is found in communities on the chitinous shells of crustaceans in its aquatic reservoir. V. cholerae interactions with chitin are critical for the survival, evolution, and waterborne transmission of this pathogen. Here, we show that V. cholerae uses QS to regulate the expression of one locus required for V. cholerae-chitin interactions.

microbiology↗

bioRxiv2017

Bioinformatics Core Competencies for Undergraduate Life Sciences Education

Bioinformatics is becoming increasingly central to research in the life sciences. However, despite its importance, bioinformatics skills and knowledge are not well integrated in undergraduate biology education. This curricular gap prevents biology students from harnessing the full potential of their education, limiting their career opportunities and slowing genomic research innovation. To advance the integration of bioinformatics into life sciences education, a framework of core bioinformatics competencies is needed. To that end, we here report the results of a survey of life sciences faculty in the United States about teaching bioinformatics to undergraduate life scientists. Responses were received from 1,260 faculty representing institutions in all fifty states with a combined capacity to educate hundreds of thousands of students every year. Results indicate strong, widespread agreement that bioinformatics knowledge and skills are critical for undergraduate life scientists, as well as considerable agreement about which skills are necessary. Perceptions of the importance of some skills varied with the respondents degree of training, time since degree earned, and/or the Carnegie classification of the respondents institution. To assess which skills are currently being taught, we analyzed syllabi of courses with bioinformatics content submitted by survey respondents. Finally, we used the survey results, the analysis of syllabi, and our collective research and teaching expertise to develop a set of bioinformatics core competencies for undergraduate life sciences students. These core competencies are intended to serve as a guide for institutions as they work to integrate bioinformatics into their life sciences curricula.\n\nSignificance StatementBioinformatics, an interdisciplinary field that uses techniques from computer science and mathematics to store, manage, and analyze biological data, is becoming increasingly central to modern biology research. Given the widespread use of bioinformatics and its impacts on societal problem-solving (e.g., in healthcare, agriculture, and natural resources management), there is a growing need for the integration of bioinformatics competencies into undergraduate life sciences education. Here, we present a set of bioinformatics core competencies for undergraduate life scientists developed using the results of a large national survey and the expertise of our working group of bioinformaticians and educators. We also present results from the survey on the importance of bioinformatics skills and the current state of integration of bioinformatics into biology education.

bioinformatics↗