Biology subjects

Lewis, J. P.

Publications and source records attributed to Lewis, J. P..

12 recordsLinked to original sources

bioRxiv2026

Nitrate-Reducing Commensals Reshape Oral Biofilm Ecology and Reveal Hcp as a Critical Determinant of Porphyromonas gingivalis Persistence

Dietary nitrate (NO-) supplementation is emerging as a promising strategy for suppressing oral pathobionts through microbial generation of reactive nitrogen species (RNS), including nitrite (NO2-) and nitric oxide (NO). However, the mechanisms that enable periodontal pathogens to survive nitrate-derived nitrosative stress within polymicrobial communities remain poorly understood. Previously, we identified the hybrid cluster protein (Hcp) as a major nitrosative stress defense factor in Porphyromonas gingivalis demonstrating [~]170-fold induction of hcp expression following nitrite exposure and as a requirement for survival at physiologically relevant nitrite concentrations. Here we investigated the role of Hcp in promoting P. gingivalis persistence within nitrate-reducing biofilms. Using human ex vivo plaque biofilms, we found that Hcp is essential for P. gingivalis survival under both basal and nitrate-supplemented conditions. In a defined nine-species biofilm model, nitrate reduction suppressed wild-type P. gingivalis, whereas deletion of hcp ({Delta}hcp) resulted in complete population clearance. Metatranscriptomics revealed that nitrate-induced hcp expression was not restricted to P. gingivalis but was part of a coordinated nitrosative stress response shared among oral anaerobes, including Prevotella intermedia, Fusobacterium nucleatum, and Veillonella atypica. Moreover, nitrate reduction disrupted a previously synergistic interaction between Veillonella spp. and P. gingivalis, converting a supportive relationship into an inhibitory microenvironment that constrained pathogen survival. Collectively, these findings identify Hcp-mediated nitrosative stress resistance as a major determinant of fitness within nitrate-reducing biofilms and reveal RNS as key ecological force shaping interactions between commensal nitrate reducers and periodontal pathogens. These results provide a mechanistic framework linking dietary nitrate metabolism to oral microbiome homeostasis.

microbiology↗

bioRxiv2025

Identification and characterization of Porphyromonas gingivalis TonB

Porphyromonas gingivalis is one of the major bacterial pathogens responsible for the initiation and progression of periodontal disease. The bacterium codes for multiple TonB-dependent receptors required for acquisition of nutrients such as heme and vitamin B12, although the identity of a TonB energy transducer has yet to be identified. Here we identify a potential TonB protein encoded by PG0785, generate a deficient strain, and characterize its biological significance. Bioinformatics analysis reveals that the PG0785 has unique features confined to the Cytophaga-Flavobacterium-Bacteroides (CFB) group of bacteria but shares similarities in the C-terminal domain (CTD) to well characterized proteins from Helicobacter pylori and Escherichia coli. Similarity at a protein level, as well as the genomic locus organization, was identified with the TonB proteins characterized in other Bacteroidota species. Loss of PG0785 led to significant alteration of the proteome: proteins mediating gene regulation, protein translation, protection against reactive radical species (oxygen and nitrogen), and glycosylation were up-regulated and polysaccharide export, efflux protein, and lipoproteins were downregulated. Furthermore, phenotypic studies showed that a mutant lacking PG0785 cannot accumulate heme on its surface and is deficient in gingipain protease activity. In addition, reduction of the capsular layer was detected in the TonB-deficient strain. Thus, while the mutant interacted and invaded eukaryotic cells at much higher levels than the wild type, it had significantly reduced ability to survive with host cells. ImportanceThe TonB system is indispensable for energy transduction in Gram-negative bacteria and is mainly associated with nutrient uptake. Although well investigated in Proteobacteria, the knowledge regarding the system in the CFB group of bacteria is lagging. In contrast to Proteobacteria, the intestinal Bacteroidota have multiple TonBs and their novel involvement in sugar utilization has been demonstrated. This study shows that the oral Bacteroidota, Porphyromonas gingivalis, also has TonB but unexpectedly, it is not involved in iron homeostasis or nutrient acquisition but rather in processes involved in maturation of proteases and cell surface remodeling. As maintenance of outer membrane integrity is mainly associated with Tol motor our study shows possible crosstalk between the energy transducers. Also, this study further demonstrates the functional versatility of the TonB system depending on the environmental niche and the metabolic requirements of the bacteria.

microbiology↗

bioRxiv2025

Nested Connections: Local Phage and Broad Plasmid Sharing in the Honey Bee Mobilome

Honey bees rely on bacterial symbionts for their nutritional needs and for protection against invading pathogens. Genetic diversity among strains within the colony has the potential to impact symbiont function and subsequently the benefits that honey bees receive from them. Mobilized genes vectored by mobile genetic elements (MGEs) like phages, transposons, conjugative elements and plasmids, are known to rapidly alter bacterial phenotypes. We identified phages and plasmids in genomes of the symbiont Bombella apis between two colonies, with the goal of understanding which MGEs contribute to strain diversification as well as MGE distribution across colonies and between microbial species. Interestingly, we found some B. apis strains carry plasmids while all harbor a diversity of integrated phages, with only one phage clusters conserved across all. Identified B. apis phages are not found outside of the Bombella and Saccaribacter species, suggesting some host specificity for these MGEs. Of the five plasmids discovered, two appear to be phage-plasmids with high similarity to phages found in previously sequenced B. apis genomes. Interestingly, three plasmids in B. apis shared significant average nucleotide identity with known plasmids from acetic acid bacteria isolated from flowers, plants, and fermented foods. This result suggests that B. apis has acquired MGEs, either vertically or horizontally, from plant- and fermented-food associated AABs. Overall, our findings suggest that MGE content varies between colonies and has the potential to shape genetic and phenotypic variation between strains.

microbiology↗

bioRxiv2024

Full-length structure and heme binding in the transcriptional regulator HcpR

HcpR is a CRP-family transcriptional regulator found in many Gram-negative anaerobic bacteria. In the perio-pathogen Porphyromonas gingivalis, HcpR is crucial for the response to reactive nitrogen species such as nitric oxide (NO). Binding of NO to the heme group of HcpR leads to transcription of the redox enzyme Hcp. However, the molecular mechanisms of heme binding to HcpR remain unknown. In this study we present the 2.3[A] structure of the P. gingivalis HcpR. Interdomain interactions present in the structure help to form a hydrophobic pocket in the N-terminal sensing domain. A comparison analysis with other CRP-family members reveals that the molecular mechanisms of HcpR-mediated regulation may be distinct from other family members. Using docking studies, we identify a putative heme binding site in the sensing domain. In vitro complementation and mutagenesis studies verify Met68 as an important residue in activation of HcpR. Finally, heme binding studies with purified forms of recombinant HcpR support Met68 and His149 residues as important for proper heme coordination in HcpR.

microbiology↗

bioRxiv2024

Targeting of SUMOylation leads to cBAF complex stabilization and disruption of the SS18::SSX transcriptome in Synovial Sarcoma

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein. and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complex and amplified presence of an SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) that drives an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program and SSs are sensitive to the small molecule SAE1/2 inhibitor, TAK-981. Mechanistically, TAK-981 de-SUMOylates the cBAF subunit SMARCE1, stabilizing and restoring cBAF on chromatin, shifting away from SS18::SSX-ncBAF-driven transcription, associated with DNA damage and cell death and resulting in tumor inhibition across both human and mouse SS tumor models. TAK-981 synergized with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX-ncBAF transcriptome, identifying a therapeutic vulnerability in SS, positioning the in-clinic TAK-981 to treat SS.

cancer biology↗

bioRxiv2023

Whole Genome Sequencing Based Analysis of Inflammation Biomarkers in the Trans-Omics for Precision Medicine (TOPMed) Consortium

Inflammation biomarkers can provide valuable insight into the role of inflammatory processes in many diseases and conditions. Sequencing based analyses of such biomarkers can also serve as an exemplar of the genetic architecture of quantitative traits. To evaluate the biological insight, which can be provided by a multi-ancestry, whole-genome based association study, we performed a comprehensive analysis of 21 inflammation biomarkers from up to 38,465 individuals with whole-genome sequencing from the Trans-Omics for Precision Medicine (TOPMed) program. We identified 22 distinct single-variant associations across 6 traits - E-selectin, intercellular adhesion molecule 1, interleukin-6, lipoprotein-associated phospholipase A2 activity and mass, and P-selectin - that remained significant after conditioning on previously identified associations for these inflammatory biomarkers. We further expanded upon known biomarker associations by pairing the single-variant analysis with a rare variant set-based analysis that further identified 19 significant rare variant set-based associations with 5 traits. These signals were distinct from both significant single variant association signals within TOPMed and genetic signals observed in prior studies, demonstrating the complementary value of performing both single and rare variant analyses when analyzing quantitative traits. We also confirm several previously reported signals from semi-quantitative proteomics platforms. Many of these signals demonstrate the extensive allelic heterogeneity and ancestry-differentiated variant-trait associations common for inflammation biomarkers, a characteristic we hypothesize will be increasingly observed with well-powered, large-scale analyses of complex traits.

genetics↗

bioRxiv2022

Iron deficiency modulates metabolic landscape of Bacteroidetes promoting its resilience during inflammation

Bacteria have to persist in low iron conditions in order to adapt to hosts nutritional immunity. Since the knowledge of iron stimulon of Bacteroidetes is sparse, we examined oral (Porphyromonas gingivalis and Prevotella intermedia) and gut (Bacteroides thataiotaomicron) representatives for their ability to adapt to iron deplete and iron replete conditions. Our transcriptomics and comparative genomics analysis shows that many iron-regulated mechanisms are conserved within the phylum. Those include genes upregulated in low iron: fldA (flavodoxin), hmu (hemin uptake operon) and loci encoding ABC transporters. Downregulated were: frd (ferredoxin), rbr (rubrerythrin), sdh (succinate dehydrogenase/fumarate reductase), vor (oxoglutarate oxidoreductase/dehydrogenase), and pfor (pyruvate:ferredoxin/ flavodoxin oxidoreductase). Some genus-specific mechanisms, such as the B. thetaiotaomicrons sus coding for carbohydrate metabolism and the xusABC coding for xenosiderophore utilization, were also identified. While all bacteria tested in our study had the nrfAH operon coding for nitrite reduction and were able to reduce nitrite levels present in culture media, the expression of the operon was iron dependent only in B. thetaiotaomicron. It is noteworthy that we identified a significant overlap between regulated genes found in our study and the B. thetaiotaomicron colitis study (Zhu et al; Cell Host Microbe 27: 376-388). Many of those commonly regulated genes were also iron regulated in the oral bacterial genera. Overall, this work points to iron being the master regulator enabling bacterial persistence in the host and paves the way for more generalized investigation of the molecular mechanisms of iron homeostasis in Bacteroidetes.

microbiology↗

bioRxiv2022

Ferroportin Depletes Iron Needed for Cell Cycle Progression in Head and Neck Squamous Cell Carcinoma

Ferroportin (FPN), the only identified eukaryotic iron efflux channel, plays an important role in iron homeostasis and is down regulated in many cancers. To determine if iron related pathways are important for HNSCC progression and proliferation, we utilize a model of FPN over-expression to simulate iron depletion and probe associated molecular pathways. HNSCC cells are sensitive to iron chelation and ferroptosis, but a non-transformed normal oral keratinocyte (NOK) cell line is not. We found that FPN expression inhibits HNSCC cell proliferation and colony formation but NOK cells are unaffected. Inhibition of cell proliferation is rescued by the addition hepcidin. Decreases in proliferation are due to the disruption of iron homeostasis via loss of labile iron caused by elevated FPN levels. This in turn protects HNSCC cells from ferroptotic cell death. Expression of FPN induces DNA damage, activates p21 and reduces mRNA levels of cyclin proteins thereby inhibiting cell cycle progression of HNSCC cells, arresting cells in S-phase. Induction of FPN severely inhibits Edu incorporation and increases {beta}-galactosidase activity, indicating cells have entered senescence. Finally, in an oral orthotopic mouse xenograft model, FPN induction yields a decrease of tumor growth. Our results indicate that iron plays a role in HNSCC cell proliferation and sustained growth and ferroptosis iron based therapeutic strategies may have potential therapeutic benefit.

cancer biology↗

bioRxiv2021

Clonal hematopoiesis is driven by aberrant activation of TCL1A

A diverse set of driver genes, such as regulators of DNA methylation, RNA splicing, and chromatin remodeling, have been associated with pre-malignant clonal expansion of hematopoietic stem cells (HSCs). The factors mediating expansion of these mutant clones remain largely unknown, partially due to a paucity of large cohorts with longitudinal blood sampling. To circumvent this limitation, we developed and validated a method to infer clonal expansion rate from single timepoint data called PACER (passenger-approximated clonal expansion rate). Applying PACER to 5,071 persons with clonal hematopoiesis accurately recapitulated the known fitness effects due to different driver mutations. A genome-wide association study of PACER revealed that a common inherited polymorphism in the TCL1A promoter was associated with slower clonal expansion. Those carrying two copies of this protective allele had up to 80% reduced odds of having driver mutations in TET2, ASXL1, SF3B1, SRSF2, and JAK2, but not DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 by CRISPR editing led to aberrant expression of TCL1A and expansion of HSCs in vitro. These effects were abrogated in HSCs from donors carrying the protective TCL1A allele. Our results indicate that the fitness advantage of multiple common driver genes in clonal hematopoiesis is mediated through TCL1A activation. PACER is an approach that can be widely applied to uncover genetic and environmental determinants of pre-malignant clonal expansion in blood and other tissues.

genetics↗

bioRxiv2020

find-tfbs: a tool to identify functional non-coding variants associated with complex human traits using open chromatin maps and phased whole-genome sequences

MotivationWhole-genome DNA sequencing (WGS) enables the discovery of non-coding variants, but tools are lacking to prioritize the subset that functionally impacts human phenotypes. DNA sequence variants that disrupt or create transcription factor binding sites (TFBS) can modulate gene expression. find-tfbs efficiently scans phased WGS in large cohorts to identify and count TFBSs in regulatory sequences. This information can then be used in association testing to find putatively functional non-coding variants associated with complex human diseases or traits. ResultsWe applied find-tfbs to discover functional non-coding variants associated with hematological traits in the NHLBI Trans-Omics for Precision Medicine (TOPMed) WGS dataset (Nmax=44,709). We identified >2000 associations at P<1x10-9, implicating specific blood cell-types, transcription factors and causal genes. The vast majority of these associations are captured by variants identified in large genome-wide association studies (GWAS) for blood-cell traits. find-tfbs is computationally efficient and robust, allowing for the rapid identification of non-coding variants associated with multiple human phenotypes in very large sample size. Availabilityhttps://github.com/Helkafen/find-tfbs and https://github.com/Helkafen/find-tfbs-demo Contactssebastian.meric.de.bellefon@umontreal.ca and guillaume.lettre@umontreal.ca Supplementary informationSupplementary data are available.

genomics↗

bioRxiv2019

Genome Sequencing Unveils a New Regulatory Landscape of Platelet Reactivity

Exaggerated platelet aggregation at the site of vascular injury is the underlying pathophysiology of thrombotic diseases. Here, we conduct the largest whole genome sequencing (WGS) effort to uncover the genetic determinants of platelet aggregation. Leveraging 3,855 NHLBI Trans-Omics for Precision Medicine (TOPMed) individuals deeply phenotyped for platelet aggregation, we identify 18 loci using single-variant approaches. This includes the novel RGS18 locus encoding a myeloerythroid lineage-specific regulator of G-protein signaling that co-localizes with eQTL signatures for RGS18 expression in platelets. A gene-based approach focusing on deleterious coding variants identifies the SVEP1 gene, previously shown to be associated with coronary artery disease, as a novel determinant of platelet aggregation. Finally, in an integrative approach leveraging epigenetic data on megakaryocytes, we find strong association between rare variants mapping to a super enhancer region for PEAR1. This is a novel finding implicating the importance of rare variants with regulatory potential in a previously documented GWAS-identified locus.

genomics↗

bioRxiv2018

Efficient variant set mixed model association tests for continuous and binary traits in large-scale whole genome sequencing studies

With advances in Whole Genome Sequencing (WGS) technology, more advanced statistical methods for testing genetic association with rare variants are being developed. Methods in which variants are grouped for analysis are also known as variant-set, gene-based, and aggregate unit tests. The burden test and Sequence Kernel Association Test (SKAT) are two widely used variant-set tests, which were originally developed for samples of unrelated individuals and later have been extended to family data with known pedigree structures. However, computationally-efficient and powerful variant-set tests are needed to make analyses tractable in large-scale WGS studies with complex study samples. In this paper, we propose the variant-Set Mixed Model Association Tests (SMMAT) for continuous and binary traits using the generalized linear mixed model framework. These tests can be applied to large-scale WGS studies involving samples with population structure and relatedness, such as in the National Heart, Lung, and Blood Institutes Trans-Omics for Precision Medicine (TOPMed) program. SMMAT tests share the same null model for different variant sets, and a virtue of this null model, which includes covariates only, is that it needs to be only fit once for all tests in each genome-wide analysis. Simulation studies show that all the proposed SMMAT tests correctly control type I error rates for both continuous and binary traits in the presence of population structure and relatedness. We also illustrate our tests in a real data example of analysis of plasma fibrinogen levels in the TOPMed program (n = 23,763), using the Analysis Commons, a cloud-based computing platform.

genetics↗