Biology subjects

Brown, I. R.

Publications and source records attributed to Brown, I. R..

6 recordsLinked to original sources

bioRxiv2026

From Magnetosomes to Vesicles: LemA Proteins as Drivers of Bacterial Membrane Reorganization

The cellular machineries that remodel bacterial membranes during organelle and vesicle formation remain poorly defined. Herein, we report MamQ as a key determinant of the lipid invaginations that enclose the magnetosome. MamQ belongs to the broadly conserved LemA protein family, whose members localize to either the inner or outer membrane. Heterologous overproduction of 13 different LemA proteins in Escherichia coli induced extensive membrane restructuring and vesiculation, indicating a conserved membrane-remodelling activity. In Pseudomonas aeruginosa, overproduction of the inner membrane protein LemA1 (PaLemA1) promoted cytoplasmic membrane vesicle (CMV) formation, whereas the periplasmic-located outer membrane-anchored LemA2 (PaLemA2) drove the formation of bacterial outer-membrane vesicles (OMVs). Conversely, deletion of the corresponding genes reduced extracellular vesicle production. Interactome analyses identified 256 protein partners of PaLemA1 and PaLemA2, including multiple components of the cellular divisome, such as CpoB, FtsE, FtsI, FtsK, FtsN, FtsW, MinE, ParB, ZapG, ZipA. Bacterial two-hybrid assays confirmed interactions between both LemA proteins and the cell division regulator CpoB, whose perturbation markedly altered vesicle formation. Additional interaction partners included PspA, an ESCRT-III-like protein implicated in membrane remodelling. Proteins involved in cell wall metabolism, membrane turnover, lipopolysaccharide (LPS) biogenesis, protein export and maturation, energy metabolism, and stress-responsive gene regulation were also enriched, suggesting a supporting role for PaLemA proteins in coordinating membrane-associated processes. Consistent with this, LemA deletion mutants displayed altered vesicle composition and global changes in gene expression. Furthermore, PalemA2 expression was induced in stationary phase and under envelope stress via the extracytoplasmic sigma factor AlgU, itself an interaction partner of the LemA proteins. Together, our findings indicate the LemA family act as a conserved membrane-remodelling system that, in conjunction with PspA, CpoB and divisome components, organises membrane-localized protein complexes required for bacterial organelle biogenesis and vesicle formation while executing a controlled, protein-machinery-driven mechanism that operates independently of passive chemical blebbing (such as hydrophobic PQS intercalation) or lytic membrane fragmentation.

microbiology↗

bioRxiv2022

Next generation protein-based materials capture and preserve projectiles from supersonic impacts

Extreme energy dissipating materials are essential for a range of applications. The military and police force require ballistic armour to ensure the safety of their personnel, while the aerospace industry requires materials that enable the capture, preservation and study of hypervelocity projectiles. However, current industry standards display at least one inherent limitation. To resolve these limitations we have turned to nature, utilising proteins that have evolved over millennia to enable effective energy dissipation. Specifically, a recombinant form of the mechanosensitive protein talin was incorporated into a monomeric unit and crosslinked, resulting in the production of the first reported example of a talin shock absorbing material (TSAM). When subjected to 1.5 km/s supersonic shots, TSAMs were shown not only to absorb the impact, but to capture/preserve the projectile, making TSAMs the first reported protein material to achieve this.

synthetic biology↗

bioRxiv2022

Exceptional yield vesicle packaged recombinant protein production from E. coli.

We describe a novel system that exports diverse recombinant proteins in extracellular vesicles from E. coli. The vesicles not only compartmentalise toxic, insoluble and disulphide bond containing proteins in a soluble and functional form (e.g. DNaseI, nanobodies and IgG-fusions), but the continued release of the inducible vesicle packaged proteins into the media supports continuous isolation of protein from active culture within a micro-environment allowing stable long-term storage. This technology results in unprecedented yields of vesicle packaged functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine.

molecular biology↗

bioRxiv2020

Localization and Functional Characterization of the Alternative Oxidase in Naegleria

The Alternative oxidase (AOX) is a protein involved in maintaining the Krebs cycle in instances where the respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have disperse distribution and are found in plants, fungi and a few protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates, and include the pathogenic species of N. fowleri, the so-called brain eating amoeba. Using a multidisciplinary approach, we aimed to understand the evolution, localization and function of AOX and the role that plays in Naeglerias biology. Our analyses suggest that the protein was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using a combination of cellular and biochemical techniques, we also functionally characterize N. gruberis AOX in its mitochondria and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism and evolution of Naegleria and other free-living relatives.

microbiology↗

bioRxiv2019

A novel exosome biogenesis mechanism: multivesicular structures budding and rupturing at the plasma membrane

Exosomes are small vesicles secreted by the cells, which mediate intercellular signalling and systemic physiological processes. Exosomes are known to originate from the intraluminal vesicles of the multivesicular endosome that fuses with the plasma membrane. We found that the non-small cell lung cancer (NSCLC) cell lines, HCC15 and A549, secreted exosomes with typical morphology and protein contents. Unexpectedly, transmission electron microscopy images indicated that the cells formed multivesicular structures that protruded from the plasma membrane and ruptured to release the exosomes. There were smooth multivesicular structures surrounded by an ordinary looking membrane, multivesicular structures coated by an electron dense layer with regular spacing pattern, and intermediate forms that combined elements of both. Electron microscopy images suggested that exosomes are release from these structures by burst events and not by the conventional fusion process. The molecular details of this novel mechanism for membrane association, deformation and fusion is to be unveiled in the future.

cell biology↗

bioRxiv2017

A Cell Culture Platform For Cryptosporidium That Enables Long-Term Cultivation And New Tools For The Systematic Investigation Of Its Biology

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking. Currently, Cryptosporidium oocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains (Moredun, Iowa) produce sufficient infectious oocysts to infect subsequent cultures. Oocyst identity was confirmed by specific staining (Crypt-a-glo, Vicia Villosa lectin, Sporo-glo), PCR-based amplification of Cryptosporidium-specific genes, lipidomics fingerprinting, and atomic force microscopy (AFM). Antibody-stained oocysts produced unstained oocysts confirming production of novel oocysts. Infected cultures could be cryoconserved and continued to produce infectious oocysts after resuscitation. Transmission electron microscopy identified all key Cryptosporidium life cycle stages. Infected cultures produced thick-walled (primarily involved in Cryptosporidium transmission between organisms) and thin-walled oocysts (important for Cryptosporidium propagation within a host/tissue) as indicated by DAPI staining (only thin-walled oocysts are permeable to DAPI staining, thus allowing visualisation of sporozoites) and AFM. In conclusion, we present a novel, easy-to-handle cell culture system that enables the propagation, cryopreservation and detailed investigation of Cryptosporidium at a laboratory scale. Its availability will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.

microbiology↗