Biology subjects

Bakker, S. E.

Publications and source records attributed to Bakker, S. E..

5 recordsLinked to original sources

bioRxiv2026

Biofilm-deficient mutants of Pseudomonas aeruginosa have wild-type levels of antibiotic tolerance in a model of cystic fibrosis lung infection

1Opportunistic, biofilm-forming pathogens such as Pseudomonas aeruginosa can employ an array of strategies to reduce the impact of antibiotics on their survival. The biofilm matrix can prevent antibiotics from reaching bacteria embedded within it; general changes in metabolic activity alter susceptibility to specific drugs dependent on the target; changes in the membrane and the expression of channel or pump proteins embedded within it affect drug uptake and efflux; and production of antibiotic-degrading enzymes can remove the threat. In this study, we report that biofilm-deficient mutants of two well-studied lab strains of P. aeruginosa (PA14 and PAO1) have wild-type (WT) levels of tolerance to colistin and meropenem when allowed to establish mature populations in an ex vivo pig lung model of cystic fibrosis lung infection. This result was unexpected, given previous work in our lab that suggested physical protection of cells from colistin by the biofilm matrix was important for tolerance. The biofilm defects in the mutants were confirmed using electron and light microscopy, and cryo scanning electron microscopy was used to visualise the hydrated biofilm matrix in the WT. Using RNA sequencing of the PA14 WT and an isogenic mutant lacking the pel polysaccharide, we were able to identify a small number of differences in the responses of the two genotypes to the lung environment and to exposure to sub-bactericidal colistin in the lung model. Notably, there was differential upregulation of the MexXY-OprM and MexEF-OprN multidrug efflux pumps. Hence, mature biofilm structure is not essential for tolerance, as other mechanisms may be sufficient in this environment. However, the relative roles of biofilm matrix versus cellular changes in physiology in conferring antibiotic tolerance in this environment remain to be fully elucidated.

microbiology↗

bioRxiv2025

In vitro multi-assay cytotoxicity assessment of iron oxide nanoparticles on neural cells and addressing spectrophotometric interference

Iron-rich nanoparticulate matter air pollution exposure is implicated in the pathophysiology of Alzheimers disease. The use of colorimetric cell viability assays to ascertain the potential cytotoxicity of iron oxide nanoparticles have highlighted conflicting results in various in vitro models, because of spectrophotometric interference. Attempts to resolve this confounding factor to reveal accurate measurements have been insufficient. To address this interference issue, we tested three control methods (readout correction, plate transfer, and above-plate reading) on in vitro cytotoxicity measurements via Neutral Red Uptake (NRU), Resazurin, and MTT assays, of iron oxide and magnetite nanoparticulate exposure on SK-N-SH and IMR-32 human neural cell lines. We observed varying degrees of interference based on cell line, nanoparticle type, exposure time, assay type, and the readout method (colorimetric or fluorometric). The readout correction method did not remove interference completely, the plate transfer method tended to cause overestimation of cell viability beyond the original interference, and fluorescence readings from above the plate were less sensitive than the conventional bottom reading. The Resazurin assay with fluorometric readout presented no interference in most conditions investigated in this work, therefore presents advantages over both colorimetric assays and interference control methods. It is recommended to forego colorimetric assays in future studies of nanoparticle cytotoxicity as they are more susceptible to interference.

pharmacology and toxicology↗

bioRxiv2024

Structure and physiological investigation of arginylated actin

Actin is an evolutionarily conserved cytoskeletal protein with crucial roles in cell polarity, division, migration, and muscle contraction. Actin function is regulated in part by posttranslational modifications. One such modification in non-muscle cells is arginylation, in which an arginine residue is added to the N-terminus of {beta}-actin. What is the structure of arginylated {beta}-actin (R-{beta}-actin), are its interactions with other proteins altered and what phenotypes result when R-{beta}-actin is the sole actin isoform present in the cell? Here we report the 4.2 [A] structure of ADP-bound human R-{beta}-actin filaments, the overall structure of which is nearly identical to the filaments made of non-arginylated actin. In vitro functional assays using isoform-pure actins with defined post-translational modifications reveal that the interaction between myosin-II and actin is altered upon actin arginylation, due to frequent detachment of myosin-II from R-actin filaments. In vivo, we find that replacement of the only actin gene in Schizosaccharomyces pombe with a synthetic gene encoding R-Sp-actin reduces Arp2/3-based actin patches while thickening the formin-induced actin. Furthermore, consistent with altered interactions between myosin-II and R-actin filaments, the assembly and constriction of cytokinetic actomyosin ring are perturbed in the R-Sp-actin cells. Thus, despite the overall structural similarity of arginylated and non-arginylated actin filaments, actin arginylation affects actin filament assortment into distinct subcellular structures and its interaction with myosin II.

biochemistry↗

bioRxiv2024

A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia

Defined as a pneumonia occurring after more than 48 hours of mechanical ventilation via an endotracheal tube, ventilator-associated pneumonia results from biofilm formation on the indwelling tube, seeding the patients lower airways with pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. Currently there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates biofilm formation of these pathogens in a host-mimicking environment, and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilator airway mucus (SVAM) medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-SEM were employed to characterise the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication, or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved biofilm eradication of all pathogens. Our in vitro endotracheal tube (IVETT) model informs on fundamental microbiology in the ventilator-associated pneumonia context, and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants. ImportanceThe incidence of ventilator-associated pneumonia in mechanically ventilated patients is between 5-40%, increasing to 50-80% in patients suffering from coronavirus disease 2019 (COVID-19). The mortality rate of ventilator-associated pneumonia patients can reach 45%. Treatment of the endotracheal tube biofilms that cause ventilator-associated pneumonia is extremely challenging, with causative organisms able to persist in endotracheal tube biofilm despite appropriate antimicrobial treatment in 56% of ventilator-associated pneumonia patients. Flawed antimicrobial susceptibility testing often means that ventilator-associated pneumonia pathogens are insufficiently treated, resulting in patients experiencing ventilator-associated pneumonia recurrence. Here we present an in vitro endotracheal tube biofilm model that recapitulates key aspects of endotracheal tube biofilms, including dense biofilm growth and elevated antimicrobial tolerance. Thus our biofilm model can be used as a ventilated airway simulating environment, aiding the development of anti-ventilator-associated pneumonia therapies and antimicrobial endotracheal tubes that can one day improve the clinical outcomes of mechanically ventilated patients.

microbiology↗

bioRxiv2021

Helical Ordering of Envelope Associated Proteins and Glycoproteins in Respiratory Syncytial Virus Filamentous Virions

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Treatments for RSV disease are however limited and efforts to produce an effective vaccine have so far been unsuccessful. Understanding RSV virion structure is an important prerequisite for developing interventions to treat or prevent infection but has been challenging because of the fragility of virions propagated in cell culture. Here we show, using cryogenic electron microscopy (cryoEM) and cryogenic electron tomography (cryoET) of RSV particles cultivated directly on transmission electron microscopy (TEM) grids, that there is extensive helical symmetry in RSV filamentous virions. We have calculated a 16 [A] resolution three-dimensional reconstruction of the viral envelope, targeting the matrix protein (M) that forms an endoskeleton below the viral membrane. These data define a helical lattice of M proteins, showing how M is oriented relative to the viral envelope and that helical ordering of viral glycoproteins that stud the viral envelope is coordinated by the M layer. Moreover, the helically ordered viral glycoproteins in RSV filamentous virions cluster in pairs, which may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV filamentous virions. Overall, the structural data obtained provides molecular insight into the organization of the virion and the mechanism of its assembly.

microbiology↗