Biology subjects

Fulde, M.

Publications and source records attributed to Fulde, M..

7 recordsLinked to original sources

bioRxiv2026

Defining a New Standard: Human Platelet Lysate Supports Proliferation and Differentiation of Primary Respiratory Epithelial Cells

Air-liquid interface (ALI) cultures consisting of well-differentiated primary respiratory epithelial cells (PRECs) provide a versatile in vitro model for pharmacological studies and to investigate host-pathogen interactions. Proliferation and differentiation of PRECs require complex media containing several growth factors, hormones, and nutrients. Usually, some of these essential components are provided by the addition of fetal calf serum (FCS). However, several disadvantages of FCS and, most importantly, ethical concerns regarding the method of serum collection have encouraged researchers to find alternatives. Human platelet lysate (hPL) has emerged as a promising alternative to FCS for supporting cell expansion in vitro. In the present study, we investigated the effects of different concentrations of hPL on the proliferation of porcine PRECs and their subsequent differentiation under ALI conditions. Cell morphology was assessed by phase-contrast microscopy, while cell proliferation was evaluated using the ClickTech EdU Cell Proliferation Kit and visualization of proliferating cells by fluorescence microscopy. Differentiation under ALI conditions was monitored by immunofluorescence staining of ciliated cells and the establishment of an intact epithelial barrier was confirmed by measuring transepithelial electrical resistance (TEER). We found that 5% hPL supported efficient cell growth and the subsequent formation of a functional, well-differentiated airway epithelium comparable to or even better than 10% FCS. Thus, hPL offers a reproducible, ethically sound, and scalable alternative to FCS for complex cell culture models in respiratory research, drug development, and host-pathogen interaction studies. LO_SCPLOWAYC_SCPLOW SO_SCPLOWUMMARYC_SCPLOWRespiratory epithelial cells from the lungs of slaughtered animals, such as pigs, can be used for cell culture models to study respiratory diseases and drug development. Air-liquid interface (ALI) cultures closely mimic the natural environment of the airways by exposing the cells to air, making them a valuable alternative to animal experiments. To grow and mature properly, these cells require nutrients and growth factors that are commonly supplied by serum from unborn calves (FCS). However, for ethical and scientific reasons, the use of FCS should be avoided. Therefore, we evaluated whether human platelet lysate (hPL) derived from expired blood donations could replace FCS in ALI cultures. We found that adding 5% hPL to the medium supported efficient cell growth and the development of a well-differentiated airway epithelium. This approach enables the use of an improved and ethically superior model of the (porcine) respiratory tract in accordance with the 3Rs principle.

cell biology↗

bioRxiv2026

Cost and benefits of gene amplification-mediated antibiotic resistance

Bacteria evolve antibiotic resistance through various mechanisms, including horizontal gene transfer, sequence-altering mutations, and tandem duplications/amplifications in genomic regions that increase the dosage of resistance determinants. This phenomenon, known as gene duplication-amplification (GDA), is a transient and reversible genetic mechanism that allows bacteria to rapidly adapt to antibiotic stress. Due to the unstable nature of amplification, the quantitative relationship between gene copy number, resistance levels, and the associated physiological costs remains poorly defined. Here, we constructed strains with fixed GDA copy numbers in a clinical Enterobacter cloacae strain. Using growth curve and time-kill assays, we show that ceftazidime (CAZ) resistance scales with GDA copy number, but the benefit under selection comes with a growth cost in the absence of antibiotic. Notably, we find that while GDA provides a survival advantage under CAZ pressure, it simultaneously increases susceptibility to rapid killing at supra-MIC concentrations. Our analysis further illustrates that survival is jointly shaped by gene dosage, antibiotic concentration, and exposure time. Together, these results clarify that GDA-mediated resistance is highly context dependent, shaped by trade-offs between resistance benefits and physiological costs.

microbiology↗

bioRxiv2025

Discovery of a new fibronectin-binding surface protein of Streptococcus canis with serum opacification activity through Transposon Directed Insertion-Site Sequencing

Infective endocarditis is a rare but severe disease in humans and dogs which can be caused by Streptococcus canis. The bacterial factors that mediate endothelial adherence remain poorly defined. To understand how S. canis can adhere to and invade the endocardium, we combined a high-throughput approach called transposon directed insertion-site sequencing (TraDIS) with a physiologically relevant endothelial infection model that incorporates venous-range shear stress to identify S. canis genes required for host cell interaction. A saturated transposon library of clinical strain IMT49926 was screened in a microfluidic infection assay, enabling genome-wide selection of mutants impaired in endothelial adhesion. Comparative analysis of input and non-adherent output pools revealed several candidate genes, including a fibronectin-binding LPXTG-anchored surface protein with high homology to streptococcal serum opacity factors (SOFs). Our findings identified ScSOF as a multifunctional surface protein that plays an important role in the infection potential of S. canis. It facilitates adhesion to endothelial cells, prevents endothelial wound closure, contributes to the streptococcal surface architecture, binds fibronectin, opacifies serum, and inhibits {beta}-haemolytic activity. ScSOF represents a strong candidate for future studies into pathogenesis, immune evasion, and (potentially) vaccine or therapeutic targeting in S. canis infections. Authors summaryStreptococcus canis is a bacterium commonly found in healthy dogs and cats, but it can sometimes cause serious infections in animals and humans, including infective endocarditis, a dangerous infection of the heart lining. To cause this disease, the bacteria must first attach to and invade the cells that line blood vessels. However, very little is known about how S. canis can do this. In this study, we used a genetic screening method that includes all the genes from S. canis to identify which bacterial genes are needed for attachment to human endothelial cells under conditions that mimic real blood flow. We discovered a previously uncharacterized surface protein, which we named ScSOF, that plays several important roles during infection. ScSOF helps the bacteria bind to fibronectin, a major host tissue protein, alters the bacterial surface structure, and causes serum opacification, a known marker of virulence in related streptococci. When ScSOF was removed, the bacteria were much less able to attach to endothelial cells, cause cell damage, or interfere with the healing of endothelial wounds. Our findings show that ScSOF is a key factor that enables S. canis to interact with host tissues and may contribute to heart valve infections.

microbiology↗

bioRxiv2025

Phylodynamics and genome-wide association studies reveal the expansion of modern Streptococcus canis in Germany

Streptoccocus canis is a leading canine pathogen causing 22.4% of canine streptococcal infections. However, knowledge of factors contributing to S. canis expansion is limited. This study uses population genetics to structure a S. canis dataset of 585 isolates and identify genetic markers contributing to the success of each dominant lineage. The dataset is composed of canine, feline and bovine isolates primarily from Germany collected between the years 1996 and 2021. We performed Multi-Locus Sequence Typing (MLST) for an initial analysis and clustered the population with fastBAPS based on the whole genome. Dated phylogenies were inferred with BEAST and Genome-Wide Association Studies (GWAS) were conducted with Scoary. MLST showed that in Germany there are two dominant groups of canine S. canis, ST43 (n= 75) and ST9 (n= 51), which were grouped into BAPS-6 and BAPS-5, respectively. The BAPS-6 cluster emerged as early as 1988 with major expansion starting around 2010. We showed via GWAS that this cluster is associated with a putative Streptococcus anginosus derived integrative and conjugative element carrying a putative cysteine protease. Furthermore, the BAPS-5 cluster emerged around 1786 with a bovine/feline subcluster appearing around 1879. This subcluster is associated with a variant of the streptococcal lac operon, which appears to have resulted from an exchange with Streptococcus dysgalactiae. We present the largest population genetics study to date for S. canis where we show that its expansion is associated with genetic exchange with other streptococcal species leading to increased pathogenic potential (BAPS-6) or host adaptation (BAPS-5). ImportanceStreptococcus canis is a bacterium which poses considerable threat to the health of dogs through superficial infections. Current research could benefit from understanding the reasons for its expansion and spread into other hosts. We looked at the genomes of 585 S. canis isolates collected during a 25-year period and identified which parts of the population were the most successful. With powerful statistical methods, we found that two groups of S. canis were the most effective: a canine group which had become more pathogenic by acquiring DNA from Streptococcus anginosus, and another group which spread from dogs into cats and cows by acquiring genetic material for lactose consumption from Streptococcus dysgalactiae. For the first time, we link the expansion of major S. canis groups to genetic exchange events with closely related streptococcal species to understand S. canis evolution and spread.

microbiology↗

bioRxiv2023

Mucosal washes are useful for sampling intestinal mucus-associated microbiota despite low biomass

Exploring the dynamic relationship between mucus-associated microbiota and host health is pivotal, yet prevalent studies using stool samples may not accurately represent these bacteria. Here, we explored mucus-associated microbiota in the gastrointestinal tract of mice and the terminal ileum in humans, using three different sample types: mucosal washes, scraping, and intestinal content in mice and biopsies and mucosal washes in humans. We employed DNA quantification and 16S rRNA sequencing to assess how comparable the information yielded from different sample types, evaluating findings relative to expectations from state-of-the-art and under controlled benchmarks. Mucosal washes in mice exhibited higher bacterial DNA and lower host DNA contamination than scraping samples. Similarly, in humans, washes surpassed biopsies in bacterial yield. Despite variations in read counts, microbiota diversity and composition remained remarkably similar between methods in both species, faithfully reflecting expected genotypic and phenotypic differences. We conclude that washes reduce host contamination without inducing substantial compositional bias when sampling mucosal microbiota. Our findings emphasize mucosal washes as alternatives to biopsies in humans and scrapings in mice, providing insights for improving result transferability across hosts. Our research underscores the importance of considering the mucus-associated microbiota to track host-microbiome interactions closer to their actual interface surface.

systems biology↗

bioRxiv2022

Gene amplifications cause high-level resistance against albicidin in Gram-negative bacteria

Antibiotic resistance is a continuously increasing concern for public health care. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against the antibiotic albicidin. We show that this mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of the GyrI-like transcription regulator STM3175 (YgiV) which binds albicidin. X-ray crystallography and molecular docking studies reveal a conserved binding motif that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in Gram-negative bacteria and our experiments confirm that STM3175 homologs can convey resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

microbiology↗

bioRxiv2019

Staphylococcus aureus uses the bacilliredoxin (BrxAB)/bacillithiol disulfide reductase (YpdA) redox pathway to defend against oxidative stress under infections

Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections. The low molecular weight thiol bacillithiol (BSH) is an important defense mechanism of S. aureus for detoxification of ROS and HOCl stress to maintain the reduced state of the cytoplasm. Under HOCl stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolations, which are reduced by bacilliredoxins (BrxA and BrxB). The NADPH-dependent flavin disulfide reductase YpdA is phylogenetically associated with the BSH synthesis and BrxA/B enzymes and was proposed to function as BSSB reductase. Here, we investigated the role of the bacilliredoxin BrxAB/BSH/YpdA pathway in S. aureus COL under oxidative stress and macrophage infection conditions in vivo and in biochemical assays in vitro. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in the S. aureus COL ypdA deletion mutant under control and NaOCl stress. Monitoring the BSH redox potential (EBSH) using the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reduced EBSH upon recovery from oxidative stress. In addition, the ypdA mutant was impaired in H2O2 detoxification as measured with the novel H2O2-specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H2O2 stress in vitro and inside murine J-774A.1 macrophages in infection assays in vivo. Finally, NADPH-coupled electron transfer assays provide evidence for the function of YpdA in BSSB reduction, which depends on the conserved Cys14 residue. YpdA acts together with BrxA and BSH in de-bacillithiolation of S-bacilithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis in S. aureus during recovery from oxidative stress and under infections.

microbiology↗