Biology subjects

Matysik, A.

Publications and source records attributed to Matysik, A..

9 recordsLinked to original sources

bioRxiv2024

Toxin-glycolipid interactions measured by imaging surface plasmon resonance on artificial membranes predicts diffusion behavior and lipid dependence of binding to cells

Membrane-protein interactions mediate cellular invasion by toxins, and are thought to involve organized plasma membrane lipid domains, often containing glycolipids, other sphingolipids, and/or cholesterol. Here, we characterize an isolated glycolipid-interacting domain of the tetanus toxin heavy chain (Hc) as a fluorescently labelled peptide, TeNT46, and describe its membrane dynamics and binding characteristics on artificial bilayers and cellular membranes. We show that this novel ganglioside-interacting probe TeNT46 retains the glycolipid binding preferences of the parent toxin, using imaging-SPR (iSPR) on a micro-patterned hybrid bilayer surface. On live cell membranes, using fluorescence correlation spectroscopic (FCS) diffusion measurements to compare TeNT46 to the well-studied GM1-binding toxin CTxB, we find that both probes display ordered domain-binding characteristics, but distinct cholesterol and sphingolipid dependencies. Strikingly, the contrasting lipid requirements of TeNT46 from those of CTxB in cells are predicted by their iSPR binding preferences on hybrid synthetic membranes. Based on the combined findings from iSPR and FCS, we propose a model for toxin-membrane interaction whereby a unique lipid constellation determines optimum binding for each probe independently of lateral confinement, which is more generally imposed by cholesterol. Our resulting understanding of the specific lipid requirements of these toxin targets and their dynamics in cell membranes could be important for the future design of preventive membrane-based nano-decoys and cell-delivery tools.

cell biology↗

bioRxiv2023

The HtrA chaperone monitors sortase-assembled pilus biogenesis in Enterococcus faecalis

Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the {Delta}srtA{Delta}htrA background partially restores the observed defects of the {Delta}srtA{Delta}htrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal tolerance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations. Author summaryTo explore the role of the HtrA chaperone/protease in E. faecalis off-pathway pilus clearance, we deleted htrA in an E. faecalis OG1RF {Delta}srtA strain known to retain polymerized pili on the cell membrane. Cells in the {Delta}srtA{Delta}htrA background are hyper-piliated, possess altered morphology, and are more susceptible to cell envelope-targeting antibiotics as compared to the parent OG1RF strain. RNA sequencing of the {Delta}srtA{Delta}htrA strain revealed transcriptional changes reminiscent of a membrane stress response. This response was pilus-dependent and contained several members of the CroR regulon. Inactivation of the response regulator CroR in the {Delta}srtA{Delta}htrA background restored (at least partially) piliation and cell morphology but not antibiotic susceptibility, linking CroR for the first time to pilus biogenesis and endogenous cell envelope stress.

microbiology↗

bioRxiv2021

The composition and function of Enterococcus faecalismembrane vesicles

Membrane vesicles (MVs) contribute to various biological processes in bacteria, including virulence factor delivery, host immune evasion, and cross-species communication. MVs are frequently being discharged from the surface of both Gram-negative and Gram-positive bacteria during growth. In some Gram-positive bacteria, genes affecting MV biogenesis have been identified, but the mechanism of MV formation is unknown. In Enterococcus faecalis, a causative agent of life-threatening bacteraemia and endocarditis, neither mechanisms of MV formation nor their role in virulence has been examined. Since MVs of many bacterial species are implicated in host-pathogen interactions, biofilm formation, horizontal gene transfer, and virulence factor secretion in other species, we sought to identify, describe, and functionally characterize MVs from E. faecalis. Here we show that E. faecalis releases MVs that possess unique lipid and protein profiles, distinct from the intact cell membrane, and are enriched in lipoproteins. MVs of E. faecalis are specifically enriched in unsaturated lipids that might provide membrane flexibility to enable MV formation, providing the first insights into the mechanism of MV formation in this Gram-positive organism.

microbiology↗

bioRxiv2019

Cellular chaining influences biofilm formation and structure in Group A Streptococcus

Group A Streptococcal (GAS) biofilm formation is an important pathological feature contributing to the antibiotic tolerance and progression of various GAS infections. Although a number of bacterial factors have been described to promote in vitro GAS biofilm formation, the relevance of in vitro biofilms to host-associated biofilms requires further understanding. In this study, we demonstrate how constituents of the host environment, such as lysozyme and NaCl, can modulate GAS bacterial chain length and, in turn, shape GAS biofilm morphology and structure. Disruption of GAS chains with lysozyme results in biofilms that are more stable. Based on confocal microscopy, we attribute the increase in biofilm stability to a dense and compact three-dimensional structure produced by de-chained cells. To show that changes in biofilm stability and structure are due to the shortening of bacterial chains and not specific to the activity of lysozyme, we demonstrate that augmented chaining induced by NaCl or deletion of the autolysin gene mur1.2 produced defects in biofilm formation characterized by a loose biofilm architecture. We conclude that GAS biofilm formation can be directly influenced by host and environmental factors through the modulation of bacterial chain lengths, potentially contributing to persistence and colonization within the host. Further studies of in vitro biofilm models incorporating physiological constituents such as lysozyme may uncover new insights into the physiology of in vivo GAS biofilms.

microbiology↗

bioRxiv2019

Streptococcus pyogenes capsule promotes microcolony-independent biofilm formation

Biofilms play an important role in the pathogenesis of Group A Streptococcus (GAS), a gram-positive pathogen responsible for a wide range infections and significant public health impact. Although most GAS serotypes are able to form biofilms, there is large heterogeneity between individual strains in biofilm formation, as measured by standard crystal violet assays. It is generally accepted that biofilm formation includes initial adhesion of bacterial cells to a surface, followed by microcolony formation, biofilm maturation, and extensive production of extracellular matrix that links together proliferating cells and provides a scaffold for the three-dimensional biofilm structure. However, our studies show that for GAS strain JS95, microcolony formation is not an essential step in static biofilm formation, and instead, biofilm can be effectively formed from slow-growing or non-replicating late exponential or early stationary planktonic cells, via sedimentation and fixation of GAS chains into biofilms. In addition, we show that the GAS capsule specifically contributes to the alternative, sedimentation-initiated biofilms. Microcolony-independent, sedimentation biofilms are similar in morphology and 3-D structure to biofilms initiated by actively dividing planktonic bacteria. We conclude that GAS can form biofilms by an alternate, non-canonical mechanism that does not require transition from microcolony formation to biofilm maturation, and which may be obscured by biofilm phenotypes that arise via the classical biofilm maturation processes.\n\nIMPORTANCEThe static biofilm assay is a common tool for easy biomass quantification of biofilm forming bacteria. However, S. pyogenes biofilm formation as measured by the static assay is strain dependent and yields heterogeneous results for different strains of the same serotype. In this study, we show that two independent mechanisms, for which the protective capsule contributes opposing functions, may contribute to static biofilm formation. We propose that separation of these mechanisms for biofilm formation might uncover previously unappreciated biofilm phenotypes that may otherwise be masked in the classic static assay.

microbiology↗

bioRxiv2019

Sortase-assembled pili promote extracellular electron transfer and iron acquisition in Enterococcus faecalis biofilm

Enterococcus faecalis sortase-assembled endocarditis and biofilm-associated pili (Ebp) are a virulence factor implicated in enterococcal biofilm-associated infections and gastrointestinal colonization. We previously showed that E. faecalis biofilm metabolism is influenced by extracellular electron transfer (EET) under iron-rich conditions, raising the question of whether Ebp pili also play a role in EET. Here, we report a novel role of Ebp pili in E. faecalis adhesion to the iron oxides magnetite, goethite and hematite, where the EbpA tip adhesin contributes to this interaction. Adhesion by Ebp pili is conditionally important for EET to iron oxides, as pilus mutants are attenuated in EET under non-static growth conditions. In alignment with the established role of EET in redox homeostasis, we find that EET to ferricyanide supports E. faecalis anaerobic growth on glycerol. Further, in an antibiotic-treated mouse gastrointestinal colonization model, we show that E. faecalis mutants deficient in EET poorly colonize the intestinal niche. Taken together, our findings suggest that Ebp pili can influence E. faecalis metabolic fitness by promoting EET to iron oxides, raising new questions of how Ebp pili shape E. faecalis interactions with environmental ecosystems. Furthermore, the important role of EET in E. faecalis colonization of the dysbiotic gastrointestinal environment highlights the need for further inquiry into how EET contributes to E. faecalis microbial pathogenesis. ImportanceIn this study, we explored the interplay between extracellular electron transfer (EET) and an Enterococcus faecalis biofilm factor, the endocarditis and biofilm-associated pili (Ebp). We demonstrate that Ebp pili have a novel role in adhesion to iron oxides, which consequently promotes EET to iron oxides under non-static conditions. Along with our findings that E. faecalis EET can be coupled to anaerobic cell growth, our results point to a potential ecological role of Ebp pili in natural environments, outside of its established function in adhesion to host ligands. We provide the first evidence of the contribution of EET to E. faecalis colonization of the antibiotic-treated murine intestinal niche, which adds to the limited experimental evidence linking EET and microbial pathogenesis, as well as highlights the need for further studies of EET in bacterial pathogens.

microbiology↗

bioRxiv2018

Apparent oxygen half saturation constant for nitrifiers: genus specific, inherent physiological property, or artefact of colony morphology?

We report that a single Nitrospira sublineage I OTU performs nitrite oxidation in several full-scale domestic wastewater treatment plants (WWTPs) in the tropics (29-31 {degrees}C). Contrary to the prevailing theory for the relationship between nitrite oxidizing bacteria (NOB) and ammonia oxidizing bacteria (AOB), members of the Nitrospira sublineage I OTU had an apparent half saturation coefficient, Ks(app) lower than that of the full-scale domestic activated sludge cohabitant AOB (0.09 {+/-} 0.02 g O2 m-3 versus 0.3 {+/-} 0.03 g O2 m-3). Paradoxically, NOB may thus thrive under conditions of low oxygen supply. Low dissolved oxygen (DO) conditions could enrich for and high aeration inhibit the NOB in a long-term lab-scale reactor. The relative abundance of Nitrospira gradually decreased with increasing DO until it was washed out. Nitritation was sustained even after the DO was lowered subsequently. Based on 3D-fluorescence in situ hybridization (FISH) image analysis, the morphologies of AOB and NOB microcolonies responded to DO levels in accordance with their apparent oxygen half saturation constant Ks(app). When exposed to the same oxygenation level, NOB formed densely packed spherical clusters with a low surface area-to-volume ratio compared to the Nitrosomonas-like AOB clusters, which maintained a porous and non-spherical morphology. Microcolony morphology is thus a way for AOB and NOB to regulate oxygen exposure and sustain the mutualistic interaction. However, short-term high DO exposure can select for AOB and against NOB in full-scale domestic WWTPs and such population dynamics depend on which specific AOB and NOB species predominate under given environmental conditions.

systems biology↗

bioRxiv2017

Extracellular Electron Transfer Powers Enterococcus faecalis Biofilm Metabolism

Enterococci are important human commensals and significant opportunistic pathogens associated with endocarditis, urinary tract infections, wound and surgical site infections, and medical device associated infections. These infections often become chronic upon the formation of biofilm. The biofilm matrix establishes properties that distinguish this state from free-living bacterial cells and increase tolerance to antimicrobial interventions. The metabolic versatility of the Enterococci is reflected in the diversity and complexity of environments and communities in which they thrive. Understanding metabolic factors governing colonization and persistence in different host niches can reveal factors influencing the transition from commensal to opportunistic pathogen. Here, we report a new form of iron-dependent metabolism for Enterococcus faecalis where, in the absence of heme, respiration components can be utilised for extracellular electron transfer (EET). Iron augments E. faecalis biofilm growth and generates alterations in biofilm matrix, cell spatial distribution, and biofilm matrix properties. We identify the genes involved in iron-augmented biofilm growth and show that it occurs by promoting EET to iron within biofilm.\n\nSignificanceBacterial metabolic versatility is often key in dictating the outcome of host-pathogen interactions, yet determinants of metabolic shifts are difficult to resolve. The bacterial biofilm matrix provides the structural and functional support that distinguishes this state from free-living bacterial cells. Here, we show that the biofilm matrix provides access to resources necessary for metabolism and growth which are otherwise inaccessible in the planktonic state. Our data shows that in the absence of heme, components of Enterococcus faecalis respiration (l-lactate dehydrogenase and acetaldehyde dehydrogenase) may function as initiators of EET through the cytoplasmic membrane quinone pool and utilize matrix-associated iron to carry out EET. The presence of iron resources within the biofilm matrix leads to enhanced biofilm growth.

microbiology↗

bioRxiv2016

Drosophila BEACH domain autophagic adaptor blue cheese shuttles between vesicle populations and is required for an early step in autophagy

Drosophila melanogaster blue cheese (bchs) encodes a large BEACH (Beige and Chediak-Higashi) family protein that is postulated to function as an adaptor protein with roles in vesicle trafficking. Mutation in bchs leads to the accumulation of ubiquitinated aggregates in aged brains, presumably because of a conserved function with its human homologue Autophagy-Linked FYVE (ALFY), which interacts with Atg5 and p62 to promote the clearance of aggregate-prone proteins. In this study, we present pharmacological and genetic evidence using a well-defined larval motorneuron paradigm that in Drosophila bchs mutants, autophagic deficit contributes to neurodegeneration. Specifically, we show that motorneuron death in larvae is accompanied by the accumulation of prominent ubiquitinated aggregates in synaptic termini, and that these are sensitive to autophagy modulating drugs. In primary bchs neurons, early autophagic compartments increase in number and intensity based on Atg5 expression, but fail to progress to Atg8-labelled compartments, indicating non-clearance. A rescuing transgene encoding the longest Bchs BEACH domain isoform not only reverses this defect, but also greatly increases Atg8 compartment number and rescues neuronal death. Although only a small fraction of Bchs colocalizes with these markers under wild-type conditions, the population of Bchs that does associate with autophagosomes shuttles between different locations depending on how autophagy is induced. These observations, together with epistatic relationships between bchs mutant alleles and autophagy-modulating drugs and genetic backgrounds, points to a model whereby BEACH domain isoforms of Bchs participate in the early steps of autophagy by recruiting Atg5 to target substrates for clearance, and that Bchs association with different parts of the autophagy machinery depends upon the type of autophagic stress imposed upon the neuron.

cell biology↗