Biology subjects

Fowler, V. G.

Publications and source records attributed to Fowler, V. G..

11 recordsLinked to original sources

bioRxiv2026

Loss of GdpP function in Staphylococcus aureus confers β-lactam-specific antibiotic tolerance and promotes invasive infection

The emergence of antibiotic tolerance in Staphylococcus aureus reduces antibiotic efficacy by allowing bacterial survival despite prolonged antibiotic exposure, the molecular basis of which remains poorly understood. Moreover, the phenotypic indistinguishability of tolerant isolates in antimicrobial susceptibility testing impedes effective diagnosis and therapy. Increased concentration of the second-messenger, cyclic-di-AMP (CDA), has recently been implicated in tolerance to {beta}-lactams as well as other cell-wall-reactive antibiotics. Using the ScanLag assay, Tolerance-Disk test, and traditional methodologies and employing isogenic mutagenized strains, we demonstrate that loss of GdpP function, a phosphodiesterase that hydrolyzes CDA, confers tolerance specifically to {beta}-lactam antibiotics independent of their class. The extent of {beta}-lactam tolerance correlated directly with the intracellular CDA concentration and inversely with the inhibition of bacterial cell-wall synthesis. {Delta}gdpP mutants caused higher mortality than wild-type strains in the Galleria mellonella infection model upon {beta}-lactam treatment, suggesting GdpP-mediated tolerance could lead to {beta}-lactam treatment failure. Large-scale within-host evolution analysis demonstrated that MRSA and MSSA strains isolated from patients acquire GdpP loss-of-function mutations during invasive infections but not during nasal carriage. Overall, this study highlights the clinical relevance of gdpP mutations, frequently selected in persistent S. aureus infections, as key mediators that could promote treatment failure due to {beta}-lactam tolerance.

microbiology↗

bioRxiv2025

Dissecting the epigenome dynamics in human immune cells upon viral and chemical exposure by multimodal single-cell profiling

Environmental and pathogen exposure can lead to profound remodeling of the gene-regulatory landscape across human immune cell populations. Here, we present a single-nucleus chromatin accessibility atlas of human immune cells comprising 271,299 cells and 319,420 candidate regulatory elements from individuals exposed to HIV-1, COVID-19, Influenza virus, organophosphates, as well as healthy controls. Our longitudinal HIV cohort reveals epigenetic signatures of T cell exhaustion manifested in changes in the accessibility at binding sites for the FOXP family transcription factors. Upon severe exposure to SARS-Cov2 we identified changes in chromatin accessibility manifesting in a regulatory switch in cytokine networks characterized by the downregulation of the NF-{kappa}B motif family, alongside concordant changes in AP-1 and IRF factor networks in CD14+ classical monocytes. By integrating single-cell profiles of DNA methylation from matched samples, we created a multimodal epigenome atlas of human immune cell states using the accessibility-derived candidate regulatory elements. Both modalities exhibit complementary epigenetic signatures at transcription factor binding sites associated with cell state, as exemplified in the process of memory formation in T-cells, where BATF, AP-1, and ETS motifs exhibit significant epigenetic covariance across both epigenomic layers. Finally, by linking potentially regulatory DNA methylation signatures to changes in chromatin accessibility in monocytes, we observe that severe COVID-19 involves selective, multiomics remodeling of epigenetic profiles at TF binding sites manifested in concordant DNA methylation and accessibility dynamics at inflammation-associated regulatory TFs.

bioinformatics↗

bioRxiv2025

The innate immune protein calprotectin incapacitates the bactericidal activity of β-lactam antibiotics

{beta}-lactam antibiotics are widely used to treat bacterial infections, yet treatment failures frequently occur even without resistance. Here, we show that the innate immune protein calprotectin (CP), released by neutrophils and abundant at infection sites, induces tolerance to {beta}-lactam antibiotics in Staphylococcus aureus. CP is a potent zinc chelator and was found to inhibit the activity of S. aureus autolysins, zinc-dependent enzymes essential for bacterial lysis following {beta}-lactam-mediated inhibition of cell wall synthesis. This protection was independent of bacterial growth or metabolism and was specific to {beta}-lactam antibiotics. Mechanistically, CP inactivated the amidase activity of Atl, the major S. aureus autolysin, through zinc sequestration. In vivo, oxacillin was significantly more effective in CP-deficient mice, demonstrating that CP reduces {beta}-lactam efficacy during infection. These findings reveal a host-derived mechanism of antibiotic tolerance and suggest that zinc availability at infection sites may directly influence {beta}-lactam treatment outcomes.

microbiology↗

bioRxiv2024

A host-directed adjuvant resuscitates and sensitizes intracellular bacterial persisters to antibiotics

There are two major problems in the field of antimicrobial chemotherapy-antibiotic resistance and antibiotic tolerance. In the case of antibiotic tolerance, antibiotics fail to kill the bacteria as their phenotypic state affords them protection from the bactericidal activity of the antibiotic. Antibiotic tolerance can affect an entire bacterial population, or a subset of cells known as persister cells. Interaction with the host induces the formation of persister cells in numerous pathogens, with reactive oxygen and nitrogen species production being heavily implicated in the collapse of bacterial energy levels and entrance into an antibiotic tolerant state. Here, we developed a high-throughput screen to identify energy modulators for intracellular Staphylococcus aureus. The identified compound, KL1, increases intracellular bacterial energy and sensitizes the persister population to antibiotics, without causing cytotoxicity or bacterial outgrowth. We demonstrate that KL1 exhibits adjuvant activity in a murine model of S. aureus bacteremia and intracellular infection of Salmonella Typhimurium. Transcriptomic analysis and further studies on its mechanism of action reveal that KL1 modulates host immune response genes and suppresses the production of reactive species in host macrophages, alleviating one of the major stressors that induce antibiotic tolerance. Our findings highlight the potential to target intracellular persister cells by stimulating their metabolism and encourage larger efforts to address antibiotic tolerance at the host-pathogen interface, particularly within the intracellular milieu.

microbiology↗

bioRxiv2024

An engineered prodrug selectively suppresses β-lactam resistant bacteria in a mixed microbial setting

The rise of {beta}-lactam resistance necessitates new strategies to combat bacterial infections. We purposefully engineered the {beta}-lactam prodrug AcephPT to exploit {beta}-lactamase activity to selectively suppress resistant bacteria producing extended-spectrum-{beta}-lactamases (ESBLs). Selective targeting of resistant bacteria requires avoiding interaction with penicillin-binding proteins, the conventional targets of {beta}-lactam antibiotics, while maintaining recognition by ESBLs to activate AcephPT only in resistant cells. Computational approaches provide a rationale for structural modifications to the prodrug to achieve this biased activity. We show AcephPT selectively suppresses gram-negative ESBL-producing bacteria in clonal populations and in mixed microbial cultures, with effective selectivity for both lab strains and clinical isolates expressing ESBLs. Time-course NMR experiments confirm hydrolytic activation of AcephPT exclusively by ESBL-producing bacteria. In mixed microbial cultures, AcephPT suppresses proliferation of ESBL-producing strains while sustaining growth of {beta}-lactamase-non-producing bacteria, highlighting its potential to combat {beta}-lactam resistance while promoting antimicrobial stewardship. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/606422v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@cd7f9aorg.highwire.dtl.DTLVardef@1747bc6org.highwire.dtl.DTLVardef@6a3419org.highwire.dtl.DTLVardef@825aa6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Human Immune Cell Epigenomic Signatures in Response to Infectious Diseases and Chemical Exposures

The epigenomic landscape of human immune cells is dynamically shaped by both genetic factors and environmental exposures. However, the relative contributions of these elements are still not fully understood. In this study, we employed single-nucleus methylation sequencing and ATAC-seq to systematically explore how pathogen and chemical exposures, along with genetic variation, influence the immune cell epigenome. We identified distinct exposure-associated differentially methylated regions (eDMRs) corresponding to each exposure, revealing how environmental factors remodel the methylome, alter immune cell states, and affect transcription factor binding. Furthermore, we observed a significant correlation between changes in DNA methylation and chromatin accessibility, underscoring the coordinated response of the epigenome. We also uncovered genotype-associated DMRs (gDMRs), demonstrating that while eDMRs are enriched in regulatory regions, gDMRs are preferentially located in gene body marks, suggesting that exposures and genetic factors exert differential regulatory control. Notably, disease-associated SNPs were frequently colocalized with meQTLs, providing new cell-type-specific insights into the genetic basis of disease. Our findings underscore the intricate interplay between genetic and environmental factors in sculpting the immune cell epigenome, offering a deeper understanding of how immune cell function is regulated in health and disease.

genomics↗

bioRxiv2022

Cytokine-expression patterns reveal coordinated immunological programs associated with persistent MRSA bacteremia

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a common, life-threatening infection that imposes up to 30% mortality even when appropriate therapy is used. Despite in vitro efficacy, antibiotics often fail to resolve the infection in vivo, resulting in persistent MRSA bacteremia. Recently, several genetic, epigenetic, and proteomic correlates of persistent outcomes have been identified. However, the extent to which single variables or composite patterns operate as independent predictors of outcome or reflect shared underlying mechanisms of persistence is unknown. To explore this question, we employed a tensor-based integration of host transcriptional and proteomic data across a well-characterized cohort of patients with persistent and resolving MRSA bacteremia outcomes. Tensor-based data integration yielded high correlative accuracy with persistence and revealed immunologic signatures shared across both the transcriptomic and proteomic datasets. We find that elevated proliferation of mature granulocytes associates with resolving bacteremia outcomes. In contrast, patients with persistent bacteremia heterogeneously exhibit correlates of granulocyte dysfunction or immature granulocyte proliferation. Collectively, these results suggest that transcriptional and proteomic correlates of persistent versus resolving bacteremia outcomes are complex and may not be disclosed by conventional modeling. However, a tensor-based integration approach can help to reveal consensus molecular mechanisms in an interpretable manner. Significance StatementWhile antibacterial therapies effectively resolve MRSA in vitro, these treatments often fail to clear MRSA bacteremia in vivo, suggesting that host-pathogen interactions are essential to persistent MRSA bacteremia. Recent studies have identified genetic, transcriptomic, and proteomic determinants of MRSA persistence. These determinants independently, however, provide insufficient mechanistic insight and it is unclear if they indicate unique or overlapping persistence mechanisms. Here, we use tensor-based decomposition to jointly analyze cytokine and transcriptomic measurements from patients with MRSA bacteremia. Results indicate that persistence mechanisms integrated across biological modalities reflect diverging mechanisms of persistent bacteremia. Ultimately, these results may help to identify future therapeutic targets for treating persistent MRSA bacteremia.

immunology↗

bioRxiv2022

All Staphylococcus aureus bacteraemia strains have the potential to cause infective endocarditis: results of GWAS and experimental animal studies

Abstract and KeywordsO_ST_ABSAimsC_ST_ABSInfective endocarditis (IE) complicates 10-20% of Staphylococcus aureus bacteraemia (SAB). We aimed to determine whether IE strains of S. aureus are genotypically different or behave differently in experimental endocarditis models as compared to non-IE SAB strains. Methods and ResultsWe conducted a genome wide association study (GWAS) of 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients, and tested a subset of strains in two experimental animal models of IE, one studying the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically damaged rabbit valves. The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences and k-mers analyses in GWASs. In the inflammation-induced IE model in mice no difference was observed between IE and non-IE SAB strains both in adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding vegetation size and CFU. ConclusionS. aureus isolates from SAB patients with and without IE were indistinguishable, by GWAS and by two in vivo models of IE. Thus, S. aureus strain variation is not the primary determinant of IE. Pending the possible identification of host factors predisposing to IE, all strains of S. aureus must be considered in patients as capable of causing this common, lethal infection once they have accessed the bloodstream. Translational PerspectiveStaphylococcus aureus endocarditis (IE) is a deadly complication of S. aureus bacteraemia (SAB). Beyond well-identified host related IE risk factors, whether bacterial features may influence the occurrence of IE in the course of bacteraemia remain elusive. We analysed the genomes of 924 S. aureus strains from IE and non-IE SAB and compared some in two in vivo IE models. We demonstrated that the propensity of S. aureus to cause IE in the course of bacteraemia does not depend on the intrinsic genetic or virulence factors of S. aureus. These findings are of importance for the management of S. aureus bacteraemia.

microbiology↗

bioRxiv2021

Macrophage-produced peroxynitrite induces antibiotic tolerance and supersedes intrinsic mechanisms of persister formation

Staphylococcus aureus is a leading human pathogen that frequently causes chronic and relapsing infections. Antibiotic tolerant persister cells contribute to frequent antibiotic failure in patients. Macrophages represent an important niche during S. aureus bacteremia and recent work has identified a role for oxidative burst in the formation of antibiotic tolerant S. aureus. We find that host-derived peroxynitrite, the reaction product of superoxide and nitric oxide, is the main mediator of antibiotic tolerance in macrophages. Using a collection of S. aureus clinical isolates, we find that, despite significant variation in persister formation in pure culture, all strains were similarly enriched for antibiotic tolerance following internalization by activated macrophages. Our findings suggest that host interaction strongly induces antibiotic tolerance and may negate bacterial mechanisms of persister formation, established in pure culture. These findings emphasize the importance of studying antibiotic tolerance in the context of bacterial interaction with the host suggest that modulation of the host response may represent a viable therapeutic strategy to sensitize S. aureus to antibiotics.

microbiology↗

bioRxiv2020

Chiral evasion and stereospecific antifolate resistance in Staphylococcus aureus

Antimicrobial resistance is a health care crisis. The resistance-conferring mutation F98Y in Staphylococcus aureus dihydrofolate reductase (SaDHFR) reduces effectiveness of antifolates, e.g., trimethoprim (TMP). Although propargyl-linked antifolates (PLAs) are much more resilient than TMP towards F98Y, this substitution still vitiates their inhibition potency. Surprisingly, differences in the enantiomeric configuration at the stereogenic center of PLAs influence the isomeric state of NADPH cofactor. Is resistance correlated with chiral evasion? A mechanism of action underpinning this coupling is unknown. To understand the molecular basis of F98Y-mediated resistance and how PLAs inhibition drives NADPH isomeric states, we used OSPREY to analyze a comprehensive suite of structural, biophysical, biochemical, and computational data. We present a model showing how F98Y SaDHFR exploits a different anomeric configuration of NADPH to evade certain PLAs inhibition, while other PLAs remain resilient to resistance. Our model should enable general design of inhibitors that are resilient to chiral evasion.

biochemistry↗

bioRxiv2018

Identification of a Chimeric M4 Protein and Novel Emm Pattern in Currently Circulating Strains of Emm4 Group A Streptococcus

Group A Streptococcus (GAS) is classified by sequence of the gene encoding the M protein (emm) and the patterns into which emm types are grouped. We discovered a novel emm pattern in emm4 GAS, historically considered pattern E, arising from a fusion event between emm and the adjacent enn gene. We identified the emm/enn fusion event in 51/52 emm4 GAS strains isolated by national surveillance in 2015. GAS isolates with an emm/enn fusion event completely replaced pattern E emm4 strains over a 4-year span in Houston (2013-2017). The novel emm/enn gene fusion and new emm pattern has potential vaccine implications.

genomics↗