Biology subjects

Putzel, G.

Publications and source records attributed to Putzel, G..

8 recordsLinked to original sources

bioRxiv2025

Establishing benchmarks for quantitative mapping of m6A by Nanopore Direct RNA Sequencing

Nanopore direct RNA sequencing (DRS) coupled with Dorado modification-aware basecalling enables mapping of epitranscriptomic modifications including N6-methyladenosine (m6A) at the level of individual RNAs. However, the sensitivity, specificity, and reproducibility of this method remain unclear and have only recently begun to be addressed through systematic benchmarking studies. Here, we aimed to establish a best-practice workflow for DRS-based epitranscriptomic analyses. Specifically, we evaluated multiple Dorado versions and models using RNA isolated from primary cells and unmodified in vitro transcribed RNAs. We further utilized an m6A methyltransferase inhibitor as a specificity control. We established that stringent filtering is necessary to reduce false-positive calls and found that Dorado predictions captured an increasing proportion of GLORI sites detected at high m6A/A proportions. Further, by applying DRS to human primary fibroblasts and HD10.6 neurons, we detected cell type-specific differences in the predicted m6A/A proportions at conserved sites. Our study thus presents the first systematic comparison of Dorado and GLORI from the same input RNA and expands characterization of the m6A epitranscriptome to fibroblasts and neurons.

bioinformatics↗

bioRxiv2025

Gastrointestinal colonization as a source of Staphylococcus aureus in atopic dermatitis

Atopic dermatitis (AD) is a prevalent inflammatory skin disease with complex pathogenesis. Both skin and gut microbiota influence AD, with Staphylococcus aureus, in particular, exacerbating the disease. However, the relationship between S. aureus colonization in the gut and skin, and whether it affects AD, remains unclear. Using a combination of culture-based methods, microbiome analysis, and genome sequencing of S. aureus from multiple body sites of children with and without AD, we found that the gut represents a major reservoir of genetically diverse S. aureus that is transmitted to the skin, including mutants associated with worse disease. We validated this association between S. aureus gastrointestinal colonization and AD in an independent human cohort and demonstrated its direct effect on disease in an infantile AD mouse model, wherein S. aureus gastrointestinal colonization worsened skin inflammation. Overall, this study identifies a previously unrecognized S. aureus reservoir, with implications for microbiota-targeting therapies in AD.

microbiology↗

bioRxiv2024

Collagen binding adhesin restricts Staphylococcus aureus skin infection

Staphylococcus aureus causes approximately 80% of skin and soft tissue infections (SSTIs). Collagen is the most abundant human extracellular matrix protein with critical roles in wound healing, and S. aureus encodes a collagen binding adhesin (Cna). The role of this protein during skin infections is unknown. Here we report that inability to bind collagen results in worsened pathology of intradermal {Delta}cna S. aureus infection. WT/Cna+ S. aureus showed reduced infection severity, aggregate formation, and significantly improved clearance of bacteria. Cna binds to the collagen-like domain of serum C1q protein to reduce its opsonophagocytic functions. We demonstrate that infection of C1qKO mice with WT bacteria show results similar to the {Delta}cna group. Conversely, inability to bind collagen resulted in an amplified inflammatory response caused in part by macrophage and neutrophil small molecule mediators released at the infection site (MMP-9, MMP-12, LTB4), resulting in increased immune cell infiltration and death.

microbiology↗

bioRxiv2024

Sex-dependent gastrointestinal colonization resistance to MRSA is microbiome and Th17 dependent

Gastrointestinal (GI) colonization by methicillin-resistant Staphylococcus aureus (MRSA) is associated with a high risk of transmission and invasive disease in vulnerable populations. The immune and microbial factors that permit GI colonization remain unknown. Male sex is correlated with enhanced Staphylococcus aureus nasal carriage, skin and soft tissue infections, and bacterial sepsis. Here, we established a mouse model of sexual dimorphism during GI colonization by MRSA. Our results show that in contrast to male mice that were susceptible to persistent colonization, female mice rapidly cleared MRSA from the GI tract following oral inoculation in a manner dependent on the gut microbiota. This colonization resistance displayed by female mice was mediated by an increase in IL-17A+ CD4+ T cells (Th17) and dependent on neutrophils. Ovariectomy of female mice increased MRSA burden, but gonadal female mice that have the Y chromosome retained enhanced Th17 responses and colonization resistance. Our study reveals a novel intersection between sex and gut microbiota underlying colonization resistance against a major widespread pathogen.

immunology↗

bioRxiv2024

Prophage-encoded methyltransferase drives adaptation of community-acquired methicillin-resistant Staphylococcus aureus

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the {Phi}11 prophage (m{Phi}11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage m{Phi}11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an m{Phi}11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPAs role in biofilm formation. Collectively, these data reveal a novel mechanism--epigenetic regulation of staphylococcal gene expression--by which phage can regulate virulence to drive adaptive leaps by S. aureus. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/589803v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b97d6forg.highwire.dtl.DTLVardef@1da30f1org.highwire.dtl.DTLVardef@1c34311org.highwire.dtl.DTLVardef@6834df_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Identification of a proteolysis regulator for an essential enzyme in Mycobacterium tuberculosis

In Mycobacterium tuberculosis proteins that are post-translationally modified with Pup, a prokaryotic ubiquitin-like protein, can be degraded by proteasomes. While pupylation is reversible, mechanisms regulating substrate specificity have not been identified. Here, we identify the first depupylation regulators: CoaX, a pseudokinase, and pantothenate, an essential, central metabolite. In a {Delta}coaX mutant, pantothenate synthesis enzymes were more abundant, including PanB, a substrate of the Pup-proteasome system. Media supplementation with pantothenate decreased PanB levels in a coaX and Pup-proteasome-dependent manner. In vitro, CoaX accelerated depupylation of Pup[~]PanB, while addition of pantothenate inhibited this reaction. Collectively, we propose CoaX contributes to proteasomal degradation of PanB by modulating depupylation of Pup[~]PanB in response to pantothenate levels. One Sentence SummaryA pseudo-pantothenate kinase regulates proteasomal degradation of a pantothenate synthesis enzyme in M. tuberculosis.

microbiology↗

bioRxiv2023

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr increased both respiration and fermentation but decreased ATP levels and growth, suggesting that {Delta}agr cells assume a hyperactive metabolic state in response to reduced metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of {Delta}agr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected {Delta}agr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived "memory" of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Nox2-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

microbiology↗

bioRxiv2022

An integrated strain-level analytic pipeline utilizing longitudinal metagenomic data

The development of sequencing technology and analytic tools have advanced our insights into the complexity of microbiome. Since different strains within species may display great phenotypic variability, studying within-species variations enhances the understanding of microbial biological processes. However, most existing methods for strain-level analysis do not allow for the simultaneous interrogation of strain proportions and genome-wide variants in longitudinal metagenomic samples. In this study, we introduce LongStrain, an integrated pipeline for the analysis of metagenomic data from individuals with longitudinal or repeated samples. Our algorithm improves the efficiency and accuracy of strain identification by jointly modeling the strain proportion and genomic variants in combined multiple samples within individuals. With simulation studies of a microbial community and single species, we show that LongStrain is superior to three extensively used methods in variant calling and proportion estimation. Furthermore, we illustrate the potential applications of LongStrain in the real data analysis of The Environmental Determinants of Diabetes in the Young (TEDDY) study and a gastric intestinal metaplasia microbiome study. We investigate the association between the dynamic change of strain proportions and early life events, such as birth delivery mode, antibiotic treatment, and weaning. By joint analysis of phylogeny and strain transition, we also identify a subspecies clade of Bifidobacterium longum which is significantly correlated with breastfeeding.

bioinformatics↗