Biology subjects

Goldhawk, D. E.

Publications and source records attributed to Goldhawk, D. E..

3 recordsLinked to original sources

bioRxiv2023

Cellular distribution and motion of essential magnetosome proteins expressed in mammalian cells

Magnetosomes are organelle-like structures within magnetotactic bacteria that store iron biominerals in membrane-bound vesicles. In the bacteria, formation of these structures is highly regulated by approximately 30 genes which are conserved throughout different species. To compartmentalize iron in mammalian cells for magnetic resonance imaging using gene-based contrast, we are introducing key magnetosome proteins. We have previously expressed essential magnetosome genes mamI and mamL as fluorescent fusion proteins in the human melanoma cell line MDA-MB-435 and confirmed their co-localization and interaction. Here we investigate the expression of magnetosome genes mamB and mamE in MDA-MB-435 cells, using confocal fluorescence microscopy to observe expression patterns and to analyze particle mobility. Custom software was developed to characterize fluorescent particle trajectories. In mammalian cells, essential magnetosome proteins displayed different diffusive behaviours. However, all magnetosome proteins travelled at similar velocities when undergoing directed motion, suggesting that MamL, MamL+MamI, MamB, and MamE interact with similar mammalian mobile elements. These results confirm that localization and interaction of essential magnetosome proteins is tenable in the mammalian intracellular compartment.

synthetic biology↗

bioRxiv2023

Essential magnetosome proteins MamI and MamL from magnetotactic bacteria interact in mammalian cells

To detect cellular activities deep within the body using magnetic resonance platforms, magnetosomes are the ideal model of genetically-encoded nanoparticles. These membrane-bound iron biominerals produced by magnetotactic bacteria are highly regulated by approximately 30 genes; however, only a few magnetosome genes are essential and may constitute the root structure upon which biominerals form. To test this, essential magnetosome genes mamI and mamL were expressed as fluorescent fusion proteins in mammalian cells. Localization and potential protein-protein interaction(s) were investigated using confocal microscopy and fluorescence correlation spectroscopy (FCS). Enhanced green fluorescent protein (EGFP)-MamI and the red fluorescent Tomato-MamL displayed distinct intracellular localization, with net-like and punctate fluorescence, respectively. Remarkably, co-expression revealed co-localization of both fluorescent fusion proteins in the same punctate pattern. An interaction between MamI and MamL was confirmed by co-immunoprecipitation. In addition, changes in EGFP-MamI distribution were accompanied by acquisition of intracellular mobility which all Tomato-MamL structures displayed. Truncation of the MamL C-terminal cationic peptide partially disrupted MamI-MamL colocalization but not mobility. Analysis of extracts from these cells by FCS was consistent with an interaction between fluorescent fusion proteins, including an increase in particle radius. Co-localization and interaction of MamI and MamL demonstrate that these essential magnetosome proteins may have a role in assembly of the magnetosome in any cell type.

synthetic biology↗

bioRxiv2022

Bacterial association with metals enables in vivo tracking of microbiota using magnetic resonance imaging

Bacteria constitute a significant part of the biomass of the human microbiota, but their interactions are complex and difficult to replicate outside the host. Exploiting the superior resolution of magnetic resonance imaging (MRI) to examine signal parameters of selected human isolates may allow tracking of their dispersion throughout the body. We investigated longitudinal and transverse MRI relaxation rates and found significant differences between several bacterial strains. Common commensal strains of lactobacilli display notably high MRI relaxation rates, partially explained by outstanding cellular manganese content, while other species contain more iron than manganese. Lactobacillus crispatus show particularly high values, 4-fold greater than any other species; over 10-fold greater signal than relevant tissue background; and a linear relationship between relaxation rate and fraction of live cells. Different bacterial strains have detectable, repeatable MRI relaxation rates that in future may enable tracking of their persistence in the human body for enhanced molecular imaging. IMPORTANCE To understand how spatial and temporal distribution of microbiota impact human health, dynamic tools for monitoring microbiota landscapes inside the host are needed. Particularly when considering the complexity of the gastrointestinal tract and the microbiota that dwell within, tools for monitoring deep segments of the gut non-invasively are required. Medical imaging provides solutions that enable the study of microorganisms in their preferred niche regardless of health status. To bootstrap this technology, we investigated the magnetic resonance imaging (MRI) properties of bacterial isolates and showed that outstanding signal detection is an inherent property of several strains. Among these, we showed that bacteria relying on manganese metabolism have an MRI characteristic that is distinct from mammalian cells. Our findings will lead to direct and safe imaging of bacteria; influence how we monitor both infection and gut health; and help direct the use of antibiotics to curtail the growing threat of antibiotic resistance.

microbiology↗