MCB Rising Star
Featured here are publications from MCB graduate students and postdoctoral fellows. If you would like to have an article featured here, please submit it here.
Advisor: Dr. Cezar Khursigara
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956531/pdf/zbc504.pdf and https://www.jove.com/t/61799/semi-quantitative-analysis-peptidoglycan-liquid-chromatography-mass
Peptidoglycan (PG) is an important component of bacterial cell walls that participates in numerous cellular processes including cellular division and host pathogen interactions. Due to the importance of PG to the growth of bacteria, PG is often the target of antimicrobials. Understanding the changes to the PG can provide insights into the mechanisms behind bacterial resistance to these PG-targeted antimicrobials. The basic structure of PG is composed of a repeating β-1,4-linked disaccharide of N-acetyl glucosamine and N-acetyl muramic acid which is appended with a short five amino acid peptide. The peptide sidechains can be crosslinked between adjacent polysaccharide strands producing a mesh-like structural component of the bacterial cell membrane. The synthesis of the PG is highly conserved, especially in Gram-negative bacteria. However, once produced PG can undergo considerable maturation and/or modification generating considerable composition variation. Within two publications (JBC and JOVE) we described the application of new bioinformatic methodology to examine the composition of PG at a level of detail not seen with older methodology. With the JBC publication, we used this new methodology to demonstrate the compositional changes to the PG between two different growth morphologies, namely free-swimming planktonic and stationary biofilm within the Gram-negative bacterium Pseudomonas aeruginosa. The Jove article provides a visual reference to performing this new bioinformatic methodology. The ultimate findings of the JBC paper revealed the composition of PG in P. aeruginosa that was eight times more complex than seen in that bacteria before. It also highlighted several PG modifications that could be important to each growth morphology. These PG modifications found could provide insights into the mechanisms behind the increased antimicrobial resistance that is seen in the biofilm growth morphology. Erin Anderson Figure
This work was supported by Canadian Institutes of Health Research Operating Grants to C.M.K. and A.J.C. and an Alexander Graham Bell Canada Graduate Scholarships–Doctoral Program of the Natural Sciences and Engineering Research Council of Canada to E.M.A.
Kristen Van Gelder
Advisor: Dr. Tariq Akhtar
Dolichol is an essential molecule to the process of protein N-glycosylation. It serves as an anchor in the endoplasmic reticulum (ER) membrane, where N-glycans, composed of 14 sugar molecules, are assembled. This glycan is then transferred to proteins entering the ER via the secretory pathway and undergo further modifications. Historically, it is the accumulation of dolichol that has been considered the ‘rate-limiting’ step in N-glycosylation. Therefore, this study aimed to enhance dolichol accumulation by manipulating the enzymes involved in its biosynthesis using a transient expression platform in Nicotiana benthamiana. Co-expression of a Solanum lycopersicum (tomato) cis-prenyltransferase (CPT) and its partner protein, CPT binding protein (CPTBP), that catalyze the biosynthesis of polyprenol, the dolichol precursor, led to a 400-fold increase in the levels of polyprenols but resulted in only modest increases in dolichol accumulation. However, when combined with a newly characterized tomato polyprenol reductase, dolichol biosynthesis was enhanced by approximately 20-fold. Furthermore, we showed that the accumulation of dolichol alleviated some bottleneck in protein N-glycosylation. Taken together these results indicate that to effectively enhance the accumulation of dolichol, coordinated synthesis and reduction of polyprenol to dolichol, is strictly required. Kristen Van Gelder Figure
We would like to thank Michael Mucci and Leane Illman (University of Guelph) for help with plant growth and maintenance, Dr. Michaela Strüder-Kypke (University of Guelph) for her technical expertise with confocal microscopy, Dr. Sameer Al-Abud-Wahid (University of Guelph) for his expertise and assistance with NMR, Drs. Dyanne Brewer and Armen Charchoglyan (University of Guelph) for their assistance with MS, and Dr. Heather Roshon (University of Waterloo) for providing Lemna gibba cultures. We would also like to thank Dr. Liliana Surmacz (Polish Academy of Science) for kindly sending the Δdfg10 yeast strain, Dr. Jonathon Krieger of Bioinformatics Solutions Inc. for operating the mass spectrometer and Dr. Nichollas Scott for helpful discussions and suggestions of software platforms. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) [Grant no. RGPIN-2014-05628] to Tariq Akhtar and NSERC postgraduate scholarship-doctoral to Kristen Van Gelder.
Advisor: Dr. Georgina Cox
Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands
Laurenne E. Petrie, Allison C. Leonard, Julia Murphy, Georgina Cox
Staphylococcus aureus is a human pathogen that causes a wide range of infections. The ability of the organism to adhere to our skin and mucosa is key for colonization and the establishment of infection. Our research describes the development of a novel whole cell-based assay that allows for the sensitive detection of S. aureus adhesion to immobilized host ligands. Using this assay, we performed the first genome wide screen identifying the genetic determinants of S. aureus adhesion to three clinically relevant human ligands. From our results we created the S. aureus Genetic Adhesion Network outlining non-essential genes involved in adhesion to each of the host ligands profiled. Here we identified known adhesion-related genetic loci, which validated our assay, as well as a number of genes that have never before been associated with S. aureus adhesion. The Genetic Adhesion Network and our novel adhesion assay are valuable tools that can be used for the development novel therapeutics aimed at preventing S. aureus host cell adhesion during infection.
I would like to acknowledge Maritza Vatta, Dr. David Heinrichs, and Dr. Mariya Goncheva for their assistance and contribution to our publication. This research was funded through the New Frontiers in Research Fund-Exploration Grant (NFRFE-2018-01058), a Medical Research Grant from the J.P. Bickell Foundation, and the Canada Foundation for Innovation (JELF 37730).
Advisor: Dr. Baozhong Meng
Seasonal dynamics and tissue distribution of two major viruses associated with grapevine Leafroll under cool climate condition
Mehdi Shabanian, Huogen Xiao, Baozhong Meng
I am Mehdi Shabanian, a PhD candidate in the Department of Molecular and Cellular Biology, with Dr. Baozhong Meng as my advisor. I joined Dr. Meng’s research program in June 2015. Research on grapevine viruses and viral diseases in Canada has commenced only recently. In my recent publication the focus was on the seasonal dynamics and tissue distribution of major pathogenic viruses associated with grapevine leafroll disease (GLD). This disease complex is caused by five virus species, namely Grapevine leafroll-associated virus (GLRaV-1 to 4 and GLRaV-7). This project is important not only for the understanding of the biology and pathogenesis of grapevine viruses but also for their timely and reliable diagnosis. This paper has been published in the European Journal of Plant Pathology (Shabanian et al. 2020). In this publication we provide answers to some important questions about the biology of GLRaV-2 and GLRaV-3 under cool climate condition, including the best time window and tissues for sampling, the temporal dynamic of each virus over the growing season, and the best diagnostic method. Below I will provide a brief overview of this research project as part of my PhD program. Grapevines have played a major role in human history, religion, and economics worldwide. Most grapes come from the common grapevine, Vitis vinifera, which was first cultivated approximately 6000 to 8000 years ago. Archaeological evidence suggests that the first grape and wine production originated between the Black Sea region and Iran; through the influence of the Roman Empire it spread to Europe. The Canadian grape and wine industry evolved over a century ago. However, it was not until the 1970s when the industry transitioned from local, small-scale wineries to a large-scale commercial production of premium wines. The industry is a significant driver of the Canadian economy as it provides 37,382 full-time jobs, contributes to business revenue, and income totalling $9.04 billion in economic impact. Ontario is the largest producer of grape and wine in Canada, with the majority of the vineyards located in the Niagara Peninsula, Prince Edward County, and the Essex Pelee Island Coast. Viral diseases are responsible for severe economic losses to the grape and wine industry worldwide. At the present, more than 80 distinct virus species have been identified in grapevines which are responsible for decreased yields, poor quality of grapes, and shortened lifespan of vineyards. GLD is one of the most widespread and destructive disease affecting grape and wine industries leading to considerable economic loss about $25,000–$40,000 per hectare based on a 30% infection rate. The first surveys of grapevine viruses in Canada were conducted in 1994 and 2001 with no subsequent large-scale studies until 2013 despite the rapid growth of the national grapevine industry. Since 2013, there has seen a sudden outbreak of viral infections throughout Ontario, raising widespread and serious concerns among grape growers, nurseries, and wineries. In response to this urgent need from the grape/wine industry, a preliminary survey using PCR-based methods with primers targeting 17 different viruses was conducted by our laboratory in collaboration with OMAFRA and the grape and wine industry. This study showed that GLRaV-2 and GLRaV-3 are the most prevalent in Ontario vineyards (Xiao et al. 2018). Unfortunately, very limited information was available about their molecular and cellular biology. Little research has been done to understand the changes in titre and distribution of these viruses during growing season. To have a better understanding of the biology of grapevine leafroll viruses, I have conducted a two-year research project. Besides the observation of symptom progression I employed highly sensitive molecular based method (RT-qPCR) as well as the serological method, Western blotting, which detects the virus based on the viral capsid protein (CP). Vines from two premium wine grape cultivars were chosen and labelled: Chardonnay representing white-berried grapes and Cabernet Franc representing red-berried grapes. On the 20th day of each month from May to October leaf sampling was done from leaves on the top of each branch, and in particular months beside the leaves, petioles, fruits and also cane was collected and photographs were taken to record symptoms progression. After sampling, all samples were ground in liquid nitrogen and stored in freezer for later use in different tests. Comparison of the photographs from infected and healthy grapevine at different time points has shown that in both white-berried (Chardonnay) and red-berried (Cabernet Franc) cultivars, symptoms of GLD were first noticeable in late July where discoloration appeared on the margins of basal leaves. For instance, in Cabernet Franc, the main symptoms first appeared as patchy reddening of interveinal regions of leaf lamina and downward rolling; later in October reddening was seen on the entire leaf blades except primary and secondary veins of virtually all the leaves on the infected vine (Figure 1). Based on the findings of this study, we demonstrated that, for both viruses, RNA levels remained low in leaves from May to July, increased rapidly by late August, and peaked in September or October depending on the virus. We also found that the distribution of viral RNA among different tissues varies depending on the growth stage of the vine. For example, in June, young berries contained the highest levels of viral RNA, followed by petiole and leaves. In contrast, in September, cambial scrapings contain the highest level of viral RNA, followed by petioles and leaves. The results from Western blotting are in agreement with those from RT-qPCR for both viruses. For instance, while GLRaV-3 CP was weakly detectable in samples from May and June, higher amounts of CP were detected from July through October. This publication provides key information not only for the timely and reliable detection of both viruses in terms of the best time window and tissue type for sampling, but also it has enabled us to better understand the basic biology of both viruses in terms of their seasonal titer variation and tissue distribution. We have proposed the following guidelines for the best time window and tissue type for sampling and testing in order to detect both viruses under cool climate conditions. If sampling is required early in the season, young berries are the best source. From July until harvest, leaves would serve as the best tissue. During dormancy, cambial scrapings will be a rich source of viruses. To our knowledge, this is the first report on the seasonal dynamics and tissue distribution of GLRaV-2 and GLRaV-3 in wine grapes in Canada.
This project was funded by the Engage (project # EGP469921 and EGP485347) and Discovery (project RGPIN-2014-05306) grant programs of the Natural Sciences and Engineering Research Council of Canada.
Advisor: Dr. Rod Merrill
Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora
Olivier Tremblay, Zachary Thow, A. Rod Merrill
Mono-ADP-ribosyltransferase toxins (mARTs) are virulence factors secreted by pathogenic bacteria that disrupt vital host cell processes in diseases like cholera, whooping cough, and diphtheria by binding NAD+ and transferring ADP-ribose to a target macromolecule. Dating back to 2008, the Merrill lab has used a computer-based approach to mine burgeoning bacterial genomic databases to discover new members of the mART family. The ongoing discovery of mARTs helps to understand how bacterial pathogens cause disease by identifying new potential targets for therapeutic development. Further characterization of mART target substrates along with toxin/enzyme structural information about active-site architecture provides the basis for the design of small inhibitory anti-virulence compounds as an alternative to conventional antibiotics. Our recent publication showcases the discovery of Vorin, a new mART produced by the plant pathogen Erwinia amylovora, the causative agent of fire blight, a destructive disease affecting economically important crops such as apples, pears, raspberries and blackberries. We show that Vorin causes a growth-defective phenotype in both S. cerevisiae and E. coli which can be reversed by the direct binding of a cognate antitoxin, VorinI. An investigation of surrounding genes indicates that the toxic effect of Vorin is neutralized by VorinI before secretion to either a eukaryotic or bacterial host, which is a novel secretion strategy for mART toxins. Preliminary mass spectrometry experiments show that Vorin may impair growth in S. cerevisiae by suppressing the initiation of autophagy, causing an accumulation of macromolecular waste leading to cell death, which would represent a novel virulence mechanism for mART toxins. Future work will focus on determining the biological substrate of Vorin and solving the three-dimensional structure of the Vorin-VorinI complex by X-ray crystallography. A sound knowledge of the toxin-antitoxin structural interaction may facilitate inhibitor design for this and other mART toxins involved in bacterial pathogenesis.
This work was supported by the Natural Sciences and Engineering Research Council of Canada, Discovery, DND and Strategic Grants (to A.R.M.). We would like to acknowledge the help of Dr. Jennifer Geddes-McAlister and her research group with sample preparation for LC-MS/MS and with data analysis, and thank them for sharing with us their technical expertise in quantitative proteomics.
Dr. You Wang
Advisor: Dr. Michael Emes; Co-advisor: Dr. Ian Tetlow
AKINβ1, a subunit of SnRK1, regulates organic acid metabolism and acts as a global modulator of genes involved in carbon, lipid, and nitrogen metabolism
You Wang, Liping Wang, Barry J Micallef, Ian J Tetlow, Robert T Mullen, Regina Feil, John E Lunn, Michael J Emes
The sucrose non-fermenting-1-related protein kinase 1 (SnRK1), is a highly conserved heterotrimeric protein kinase in plants and an orthologue of the mammalian AMP-activated protein kinase. While the roles of the α and γ subunits have been studied in detail, little is known about the function of the β subunit, AKINβ1. The effects of altered expression of AKINβ1 on carbohydrate metabolism and gene expression in leaves were investigated in an Arabidopsis T-DNA insertion mutant. The contents of key intermediates in the tricarboxylic acid cycle of the mutant leaves, as well as respiration, were markedly reduced throughout the diurnal cycle. Using RNASeq to compare with the wild type, many genes were differentially expressed in leaves of the akinβ1 mutant in response to light but to a far lesser extent in darkness. Particular isoforms of multigene families involved in malate and lipid metabolism and nitrate uptake exhibited very substantial decreases in expression, showing the importance of the trimer as a global regulator of metabolism. Studies of its subcellular localization revealed that AKINβ1 can be N-myristoylated and localizes to the Golgi. Partitioning of AKINβ1 between the Golgi, nucleus and cytoplasm provides a mechanism to regulate metabolism and gene expression in Arabidopsis.
I thank Dr Jaideep Mathur (Department of Molecular and Cellular Biology, University of Guelph, Canada) for providing the binary vector for subcellular localization studies. This work was supported by NSERC Discovery grant 435781 and by the Max Planck Society (RF and JEL).