Biology subjects

Kreth, J.

Publications and source records attributed to Kreth, J..

9 recordsLinked to original sources

bioRxiv2026

Paired oral clinical specimens reveal the underlying ecology supporting the emergence of inflammophilic microbiome communities

BackgroundInflammatory oral diseases are associated with reproducible shifts from commensal-dominated microbiota toward pathobiont-enriched communities, yet the ecological mechanisms underlying the emergence of inflammophiles remain poorly understood. This study aims to investigate if host-derived inflammatory environments act as selective pressures that restructure microbial metabolism and community organization during disease progression. MethodsWe performed 16S rRNA gene sequencing of patient-matched pediatric dental plaque and odontogenic abscess specimens to capture microbial community transitions across an inflammatory ecological gradient. Community ecology modeling and inferred metagenomic analyses were used to identify taxa and functional programs associated with commensal and inflammophilic states. ResultsPatient-matched comparisons revealed a reproducible ecological selection gradient linking inflammatory environments to expansion of metabolically specialized inflammophiles and depletion of carbohydrate-utilizing commensals. Commensal-dominated plaque communities exhibited anabolic, carbohydrate-centered metabolic capacity, whereas abscess microbiota were enriched for catabolic metabolism, amino acid fermentation, and antimicrobial resistance, consistent with adaptation to inflammation-driven nutrient landscapes and immune pressure. ConclusionsThese findings support a model in which host inflammation drives ecological restructuring of the oral microbiome toward metabolically adapted inflammophilic communities. Defining the metabolic requirements and selective pressures governing these transitions provides a framework for microbiome-directed therapeutic strategies aimed at restoring ecological stability during inflammatory dysbiosis.

bioinformatics↗

bioRxiv2025

Comparative Characterization Reveals Conserved and Divergent Ecological Traits of Oral Corynebacteria

Corynebacteria are abundant members of the oral microbiome and increasingly recognized as key structural organizers of supragingival biofilms. Despite their prevalence, the ecological roles and phenotypic traits of many oral corynebacterial species remain poorly defined. Here, we isolated and characterized two new strains, Corynebacterium durum JJ2 and Corynebacterium argentoratense MB1, and compared them with reference strains Corynebacterium durum JJ1 and Corynebacterium matruchotii ATCC14266. Phenotypic assays revealed that C. durum strains displayed robust aggregation, thick biofilm formation, and extensive extracellular polymeric substance (EPS) networks, whereas C. argentoratense and C. matruchotii formed thinner biofilms with minimal EPS production. All four strains secreted extracellular membrane vesicles (EMVs) capable of inducing chain elongation in Streptococcus sanguinis, underscoring a conserved interspecies signaling function. Genomic analysis demonstrated close relatedness between C. durum and C. matruchotii, while C. argentoratense was more distantly related, with a reduced genome, fewer metabolic pathways, and absence of nitrate reductase genes, consistent with its inability to grow under anaerobic conditions. These findings suggest that C. argentoratense may represent a less specialized or transient inhabitant of the oral cavity, whereas C. durum and C. matruchotii are well adapted to the oral niche. Together, this study expands our understanding of phenotypic diversity, metabolic capacity, and interspecies interactions among oral corynebacteria, highlighting their potential importance as biofilm organizers and contributors to oral microbial ecology. ImportanceOral Corynebacteria contribute to the structural and ecological stability of supragingival communities. Yet, their species-level functions remain poorly defined. By isolating and characterizing new strains of Corynebacterium durum and Corynebacterium argentoratense, and comparing them with reference strains including C. matruchotii, we provide new insight into their phenotypic diversity, metabolic capacity, and ecological roles. Our results demonstrate that C. durum strains form robust biofilms enriched in extracellular polymeric substances, while C. argentoratense produces thinner biofilms and lacks the genomic features required for anaerobic growth, suggesting a less specialized or transient role in the oral cavity. Importantly, we show that extracellular membrane vesicles secreted by all tested strains promote chain elongation in Streptococcus sanguinis, highlighting a conserved mechanism of interspecies communication. These findings advance our understanding of how oral corynebacteria contribute to biofilm organization and microbial homeostasis, and position them as critical but understudied players in oral microbial ecology.

microbiology↗

bioRxiv2023

The prevalence of Fusobacterium nucleatum subspecies in the oral cavity stratifies by local health status

The ubiquitous inflammophilic pathobiont Fusobacterium nucleatum is widely recognized for its strong association with a variety of human dysbiotic diseases such as periodontitis and oral/extraoral abscesses, as well as multiple types of cancer. F. nucleatum is currently subdivided into four subspecies: F. nucleatum subspecies nucleatum (Fn. nucleatum), animalis (Fn. animalis), polymorphum (Fn. polymorphum), and vincentii/fusiforme (Fn. vincentii). Although these subspecies have been historically considered as functionally interchangeable in the oral cavity, direct clinical evidence is largely lacking for this assertion. Consequently, we assembled a collection of oral clinical specimens to determine whether F. nucleatum subspecies prevalence in the oral cavity stratifies by local oral health status. Patient-matched clinical specimens of both disease-free dental plaque and odontogenic abscess were analyzed with newly developed culture-dependent and culture-independent approaches using 44 and 60 oral biofilm/tooth abscess paired specimens, respectively. Most oral cavities were found to simultaneously harbor multiple F. nucleatum subspecies, with a greater diversity present within dental plaque compared to abscesses. In dental plaque, Fn. polymorphum is clearly the dominant organism, but this changes dramatically within odontogenic abscesses where Fn. animalis is heavily favored over all other fusobacteria. Surprisingly, the most commonly studied F. nucleatum subspecies, Fn. nucleatum, is only a minor constituent in the oral cavity. To gain further insights into the genetic basis for these phenotypes, we subsequently performed pangenome, phylogenetic, and functional enrichment analyses of oral fusobacterial genomes using the Anvio platform, which revealed significant genotypic distinctions among F. nucleatum subspecies. Accordingly, our results strongly support a taxonomic reassignment of each F. nucleatum subspecies into distinct Fusobacterium species. Of these, Fn. animalis should be considered as the most clinically relevant at sites of active inflammation, despite being among the least characterized oral fusobacteria.

microbiology↗

bioRxiv2023

Microbiome imaging goes &agrave la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform

The human microbiome is predominantly composed of facultative and obligate anaerobic bacteria that live in hypoxic/anoxic polymicrobial biofilm communities. Given the oxidative sensitivity of large fractions of the human microbiota, green fluorescent protein (GFP) and related genetically-encoded fluorophores only offer limited utility for live cell imaging due the oxygen requirement for chromophore maturation. Consequently, new fluorescent imaging modalities are needed to study polymicrobial interactions and microbiome-host interactions within anaerobic environments. The fluorescence-activating and absorption shifting tag (FAST) is a rapidly developing genetically-encoded fluorescent imaging technology that exhibits tremendous potential to address this need. In the FAST system, fluorescence only occurs when the FAST protein is complexed with one of a suite of cognate small molecule fluorogens. To expand the utility of FAST imaging, we sought to develop a modular platform (Click-FAST) to democratize fluorogen engineering for personalized use cases. Using Click-FAST, investigators can quickly and affordably sample a vast chemical space of compounds, potentially imparting a broad range of desired functionalities to the parental fluorogen. In this work, we demonstrate the utility of the Click-FAST platform using a novel fluorogen, PLBlaze-alkyne, which incorporates the widely available small molecule ethylvanillin as the hydroxybenzylidine head group. Different azido reagents were clicked onto PLBlaze-alkyne and shown to impart useful characteristics to the fluorogen, such as selective bacterial labeling in mixed populations as well as fluorescent signal enhancement. Conjugation of an 80 [A] PEG molecule to PLBlaze-alkyne illustrates the broad size range of functional fluorogen chimeras that can be employed. This PEGylated fluorogen also functions as an exquisitely selective membrane permeability marker capable of outperforming propidium iodide as a fluorescent marker of cell viability.

microbiology↗

bioRxiv2023

Adaptation of prokaryotic toxins for negative selection and cloning-independent markerless mutagenesis (CIMM) in Streptococcus species

The Streptococcus mutans genetic system offers a variety of strategies to rapidly engineer targeted chromosomal mutations. Previously, we reported the first S. mutans negative selection system that functions in a wild-type background. This system utilizes induced sensitivity to the toxic amino acid analog p-chlorophenylalanine (4-CP) as a negative selection mechanism, and was developed for counterselection-based cloning-independent markerless mutagenesis (CIMM). While we have employed this system extensively for our ongoing genetic studies, we have encountered a couple limitations with the system, mainly its narrow host range and the requirement for selection on a toxic substrate. Here, we report the development of a new negative selection system that addresses both limitations, while still retaining the utility of the previous 4-CP-based markerless mutagenesis system. We placed a variety of toxin-encoding genes under the control of the xylose-inducible Xyl-S expression cassette and found the Fst-sm and ParE toxins to be suitable candidates for inducible negative selection. We combined the inducible toxins with an antibiotic resistance gene to create several different counterselection cassettes. The most broadly useful of these contained a wild-type fst-sm open reading frame transcriptionally fused to a point mutant form of the Xyl-S expression system, which we subsequently named as IFDC4. IFDC4 was shown to exhibit exceptionally low background resistance, with 3 - 4 log reductions in cell number observed when plating on xylose-supplemented media. IFDC4 also functioned similarly in multiple strains of S. mutans as well as with S. gordonii and S. sanguinis. We performed CIMM with IFDC4 and successfully engineered a variety of different types of markerless mutations in all three species. The counterselection strategy described here provides a template approach that should be adaptable for the creation of similar counterselection systems in many other bacteria.

genetics↗

bioRxiv2022

Identification of novel PGRP3 protein -protein interactions using yeast two hybrid system

BackgroundPeptidoglycan recognition protein-3 (PGRP3) is a pattern recognition receptor that binds peptidoglycan to elicit an immune response. How PGRP3 mediates these immunomodulatory effects remains unknown. Identifying the proteins that interact with PGRP3 will help begin to address this important knowledge gap. Therefore, in this study our objective was to identify and validate novel PGRP3-protein interactions. MethodsPGRP3-protein interactions were identified using a yeast two-hybrid system. PGRP3 cloned into a pGBKT7 DNA-BD vector and transformed into a Y2HGold yeast strain served as the bait. A normalized universal human cDNA library cloned in a pGADT7 AD vector and pre-transformed into an Y187 yeast strain served as the prey. Y2HGold and Y187 yeast strains were mated and diploids were plated on selective media. True positive clones were sent in for DNA sequencing and corresponding proteins were identified through NCBI Blast. Two proteins were selected for validation. His-tagged PGRP3 expressing 293T cells were then co-transfected with expression vectors containing the identified proteins tagged with c-Myc. Lysates from co-transfected cells were subjected to anti-His and anti-Myc co-immunoprecipitation and analyzed with western blots. ResultsFour unique proteins-- MBNL3, RBP5, ATXN2, and GPATCH8--were identified from our yeast two-hybrid screen. Co-transfection and co-immunoprecipitation assays successfully confirmed MBNL3 and RBP5 as positive interactors with PGRP3. ConclusionsBased on the known functions of MBNL3, RBP5, ATXN2, and GPATCH8, we hypothesize that PGRP3 may be involved in RNA processing, endocytic trafficking, and PPAR{gamma} pathways related to innate immunity.

immunology↗

bioRxiv2022

Development of the First Tractable Genetic System for Parvimonas micra, a Ubiquitous Pathobiont in Human Dysbiotic Disease

Parvimonas micra is a Gram-positive obligate anaerobe and a typical member of the human microbiome. P. micra is among the most highly enriched species at numerous sites of mucosal dysbiotic disease and is closely associated with the development of multiple types of malignant tumors. Despite its strong association with disease, surprisingly little is known about P. micra pathobiology, which is directly attributable to its longstanding genetic intractability. To address this problem, we directly isolated a collection of P. micra strains from odontogenic abscess clinical specimens and then screened these isolates for natural competence. Amazingly, all of the P. micra clinical isolates exhibited various levels of natural competence, including the reference strain ATCC 33270. By exploiting this ability, we were able to employ cloning-independent methodologies to engineer and complement a variety of targeted chromosomal genetic mutations directly within low passage clinical isolates. To create the first P. micra genetic system, we employed renilla-based bioluminescence for highly sensitive reporter studies. This reporter system was then applied for the development of the novel Theo+ theophylline-inducible riboswitch for tunable gene expression studies over a broad dynamic range. Finally, we demonstrate the feasibility of generating Mariner-based Tn-seq libraries for forward genetic screening in P. micra. With the availability of a highly efficient transformation protocol and the current suite of genetic tools, P. micra should now be considered as a fully genetically tractable organism suitable for molecular genetic research. The methods presented here provide a clear path to investigate the understudied role of P. micra in polymicrobial infections and tumorigenesis.

microbiology↗

bioRxiv2021

MYCN mediates cysteine addiction and sensitizes to ferroptosis

Aberrant expression of MYC family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYC induces massive lipid peroxidation upon depletion of cysteine, the rate-limiting amino acid for glutathione biosynthesis and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and irondependent type of cell death. In MYCN-amplified childhood neuroblastoma, MYCN mediates resistance to ferroptosis by activating transsulfuration of methionine to cysteine. MYCN may contribute to spontaneous tumor regression in low-risk neuroblastomas by promoting ferroptosis in cells with epigenetically silenced cystathionine-beta-synthase, the rate-limiting enzyme for transsulfuration. We identified enzymes and antiporter proteins crucial to ferroptotic escape, providing multiple previously unknown sites that may be acted on therapeutically.

cancer biology↗

bioRxiv2020

Competition and Caries on Enamel of a Dual-species Biofilm Model of Streptococcus mutans and Streptococcus sanguinis.

Imbalances within the dental biofilm trigger dental caries, currently considered a dysbiosis and the most prevalent non-communicable disease. There is still a gap in knowledge about the dynamics of enamel colonization by bacteria from the dental biofilm in caries. The aim, therefore, was to test whether the sequence of enamel colonization by a typically commensal and a cariogenic species modifies biofilms cariogenicity. Dual-species biofilms of Streptococcus mutans (Sm) and Streptococcus sanguinis (Ss) on saliva-coated enamel slabs were inoculated in different sequences: Sm followed by Ss (Sm-Ss), Ss followed by Sm (Ss-Sm), Sm and Ss inoculated at the same time (Sm=Ss) and the single-species controls Sm followed by Sm (Sm-Sm) and Ss followed by Ss (Ss-Ss). Biofilms were exposed to 10% sucrose, 3x/day for 5 days and the slabs/biofilms were retrieved to assess demineralization, viable cells, biomass, proteins, polysaccharides and H2O2 production. When compared with Sm-Sm, primary inoculation with Ss reduced demineralization (p<0.05). Both Ss-Sm and Sm=Ss sequences showed reduction in biomass, protein and polysaccharide content (p<0.05). The highest S. sanguinis viable cells and H2O2 production and the lowest acidogenicity were observed when Ss colonized enamel before Sm (p<0.05). Initial enamel adherence with commensal biofilms seems to induce more intense competition against more typically cariogenic species, reducing cariogenicity. ImportanceThe concept of caries as an ecological disease implies the understanding of the intricate relationships among the populating microorganisms. Under frequent sugars exposure, some the bacteria from the oral biofilm develop pathogenic traits that lead to oral imbalances, known as dysbiosis. Depending on which microorganism colonizes the dental surface first, different competition strategies may be developed. Since the study of the interactions in the entire dental biofilm is not an easy task, in this article we model the interplay among these microorganisms using a caries-inducing (S. mutans) and a health-associated species (S. sanguinis). Initial enamel adherence with S. sanguinis seems to induce more intense competition against more typically caries-inducing species. Besides continuous exposure with sugars, early colonization of the enamel by highly cariogenic species, like S. mutans, appears to be needed to develop caries lesions, as well. Promoting early colonization by health-associated bacteria, such as S. sanguinis, could help maintaining oral health, delaying dysbiosis.

microbiology↗