Biology subjects

Yeo, B. K. Y.

Publications and source records attributed to Yeo, B. K. Y..

3 recordsLinked to original sources

bioRxiv2021

N-glycosylation on Oryza Sativa Root Germin-like Protein 1 is conserved but not required for stability or activity

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251{+/-}70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

biochemistry↗

bioRxiv2020

Pseudomonas aeruginosa isolates co-incubated with Acanthamoeba castellanii exhibit phenotypes similar to chronic cystic fibrosis isolates

The opportunistic pathogen, Pseudomonas aeruginosa, is ubiquitous in the environment, and in humans is capable of causing acute and chronic infections. P. aeruginosa, when co-incubated with the bacterivorous amoeba, Acanthamoeba castellanii, for extended periods, produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans, was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early stage amoeba-adapted and non-adapted counterparts. Late-stage amoeba-adapted P. aeruginosa lost competitive fitness compared to non-adapted counterparts when grown in nutrient rich media. However, non-adapted P. aeruginosa were rapidly cleared by amoeba predation, whereas late-stage amoeba-adapted isolates remained in higher numbers 24 h after ingestion by amoeba. In addition, there was reduced uptake by macrophage of amoeba-adapted isolates and reduced uptake by human neutrophils as well as increased survival in the presence of neutrophils. Our findings indicate that the selection imposed by amoeba on P. aeruginosa resulted in reduced virulence over time. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types. Author SummaryPseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans and also causes chronic infections in immune compromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by the bacterial predators, protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here we examined the effect of long-term predation on the genotype and phenotypes expressed by P. aeruginosa. We show that long-term co-incubation with protozoa resulted in mutations in the bacteria that made them less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Thus, predation by protozoa and long term colonization of the human host may represent similar environments that select for similar losses in gene functions.

microbiology↗

bioRxiv2018

Proteomics reveals profound metabolic changes in the alcohol use disorder brain

Changes in brain metabolism are a hallmark of Alcohol Use Disorder (AUD). Determining how AUD changes the brain proteome is critical for understanding the effects of alcohol consumption on biochemical processes in the brain. We used data-independent acquisition mass spectrometry proteomics to study differences in the abundance of proteins associated with AUD in pre-frontal lobe and motor cortex from autopsy brain. AUD had a substantial effect on the overall brain proteome exceeding the inherent differences between brain regions. Proteins associated with glycolysis, trafficking, the cytoskeleton, and excitotoxicity were altered in abundance in AUD. We observed extensive changes in the abundance of key metabolic enzymes, consistent with a switch from glucose to acetate utilization in the AUD brain. We propose that metabolic adaptations allowing efficient acetate utilization contribute to ethanol dependence in AUD.

biochemistry↗