Speeding towardstranscriptomics

Kristin Sæbø Pettersen

Norwegian Veterinary Institute

"Speeding towards –omics ... - chemical and microbiological food analysis"

AOAC Europe - NMKL - NordVal International symposium

4 th of June, 2019

Overview

• Transcriptomics in a food safety omics perspective

• L. monocytogenes – a case study using transcriptomics to analyse the effect of visible light• Applied transcriptomics on other foodborne pathogenic

bacteriae

• Take home messages

Incidence and cost of foodborne illness

Cook et al 2018

Foodomics and Food Safety:Where we are

Foodomic analyses of food from the

production to the consumption(Andielkovic et al 2017)

Use of omics methods for theadvancement of food quality and food safety

Word cloud (graphical representation of word frequency) showing various state-of-the-art “omics” technologies

(Cook et al 2018)

Foodomics: A novel approach for food microbiology

Fig. 1. (A) Foodolomics-related papers published between 2001-2016 (based on ISIS web of science). (Xu et al 2017)

The central biological dogma

Cook et al 2018

DNA is transcribed to mRNA and mRNA is translated to protein. New technologies (e.g., “omics” technologies) have been developed to study changes in DNA, mRNA, and protein under varying physiological and environmental conditions (Cook et al 2018).

Each of the different omics data types provide a different layer and can confirm assumptions made at prior levels.

Transcriptomics • Transcriptomics is the study of all RNA in one cell or a

population of cells • precise measurement of transcription, • full picture of the extent and complexity of transcriptomes.

• Two techniques in transcriptomics: • Microarrays

• can't be used for unknown RNA characterization since microarray was designed for known sequences

• RNA-sequencing (RNA-Seq). • can be used for qualitative and quantitative analysis on any RNA type,

including messenger RNAs (mRNAs), microRNAs, small interfering RNAs, and long noncoding RNAs.

• Foodomics: transcriptomic analysis reveals potentially “food safety targets” in complex physiological pathways• Ex; find synergies of hurdle technology effects to benefit food safety

Xu et al, 2017

Transcriptomics technologies

(Lowe et al 2017)

RNA sequencing

RNA microarray

Expressed

sequence tag

Serial/cap analysis

of gene expression

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2004 2006 2008 2010 2012 2014 2016 2018 2020

Popularity

Year

Microarray:…RNA-Seq:…

Source: Google trends, Girum Tessema

Google search topics trend “Microarray” and “RNA-Seq»

(Lowe et al, 2017)

(Lowe et al, 2017)

Comparison of contemporary methods

(Lowe et al, 2017)

Sequencing technology platforms commonly used for RNA-Seq.

(Lowe et al, 2017)

Transcriptomics on L. monocytogenes: a case study

(USDA Economic Research Service,Cook et al, 2018)

Cost of various foodborne pathogens

Visible light –why should L. monocytogenes care?

http://www.nature.com/ng/journal/v48/n3/full/ng.3515.html

https://ing.dk/sites/ing/files/topillustration/2017/09/fordampningenergi1.jpg

http://www.faculty.virginia.edu/rwoclass/astr1230/im/human-eye-crossec.jpg

Effect of visible light

• Reactive Oxygen Species (ROS) produced in illuminated bacteria - phototoxic effect

• Production of antioxidants – light sensitivity

• NB! Low amounts of ROS that has been found to induce cell growth.

• Intense blue light, preferably at 415nm, is better than red light for bacteria killing.

Lubart et al 2011

Initially experiment

https://www.slideshare.net/VaniJhaveri/vision-in-animals

What are the deeper mechanisms? Which genes are activated? (Transcriptomics….)

http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/105292327/44350_36a.jpg

https://www.ncbi.nlm.nih.gov/Structure/pdb/2BKP

https://images.finncdn.no/dynamic/1280w/2017/8/vertical-5/30/6/103/375/_1447457110.jpg

https://www.spaniaidag.no/wp-content/uploads/2016/08/paella3-700x350.jpg

2 409 significantregulatedgenes (ca 80 %)

Listeria monocytogenes

How do we find which genes the bacterium expresswhen exposed to visible light?

Processing rawsequences and alignwith a referencegenome

Sequencing

Countingsequences and statistiscalanalysis

Pathwayfunction analysis

https://www.digikey.com/-/media/Images/Article%20Library/TechZone%20Articles/2017/July/Ultraviolet%20Radiation%20Att

20 min = ca 8 s180 min = ca 60 s

https://www.hegnar.no/var/hegnar/storage/images/8/3/1/2/1842138-1-nor-NO/55254_binary_12390_full_article.jpg

Considerations for experimentaldesign• Include a bioinformatician in early phase of planning

experimental design and budget!

• Snaphsot! Versus fingerprinting….

• Stationary versus exponential growth phase

• Time of storage (RNA quality)

• rRNA – mRNA -> depletion

• Reference level• Global transcriptomics: you assume no difference• Single gene expression: reference gene and control group

• Depth

• Read length

• Single – paired –end reads

Applied transcriptomics on other foodborne pathogenic bacteria

This research provides for the first time valuable insights into gene expression patterns and

potential regulatory mechanisms that may be employed by the highly virulent C. jejuni

IA3902 to colonize and survive within the inhospitable gallbladder environment where it

potentially serves as a chronic nidus of infection for spread of disease between animals and

humans.

Applied transcriptomics on other foodborne pathogenic bacteria

Applied transcriptomics on other foodborne pathogenic bacteria

Transcriptomics analysis on ampicillin-induced and non-ampicillin-induced biofilms were performed by RNA-sequencing, differentially expressed genes identification and annotation, GO functional and KEGG pathway enrichment. The viability and biomass of ampicillin-induced biofilm showed dramatical increase compared to the non-ampicillin-induced biofilm. A total of 530 differentially expressed genes (DEGs) with 167 and 363 genes showing up- and down-regulation, respectively, were obtained. Upon GO functional enrichment, 183, 252, and 21 specific GO terms in biological process, molecular function and cellular component were identified, respectively. Eight KEGG pathways including “Microbial metabolism in diverse environments”, “S. aureus infection”, and “Monobactam biosynthesis” were significantly enriched. In addition, “beta-lactam resistance” pathway was also highly enriched. In ampicillin-induced biofilm, the significant up-regulation of genes encoding multidrug resistance efflux pump AbcA, penicillin binding proteins PBP1, PBP1a/2, and PBP3, and antimicrobial resistance proteins VraF, VraG, Dlt, and Aur indicated the positive response of S. aureus to ampicillin. The up-regulation of genes encoding surface proteins ClfB, IsdA, and SasG and genes (cap5B and cap5C) which promote the adhesion of S. aureus in ampicillin induced biofilm might explain the enhanced biofilm viability and biomass.

Take home message

• Transcriptomics and food safety• Mooving towards transcriptomics

• Not yet at the «applied stage», such as genomics, maybemore suitable for revealing deeper mechanims and new«targets»

• Broad spectrum visible light has a significant impacton several of the foodborne bacterial pathogens• To be included in preventive strategies for contamination?

• Experimental design in the lab when simulating host conditions…..

Acknowledgement

• Funding: Pathogens in the Food Chain, NFR 221663 –F40

• Co-authors: «Shining Light on L. monocytogenes – a transcriptome analysis»

• Arvind YM Sundaram*, Taran Skjerdal **, Yngvild Wasteson ***, Toril Lindbäck ***, Anne Kijewski ***, Marina Aspholm ***

• *Norwegian Sequencing Centre, Department of Medical Genetics, Oslo University Hospital, Norway

• **Norwegian Veterinary Institute, Ullevålsveien 68, N-0450 Oslo, Norway

• ***Norwegian University of Life Sciences, Campus Adamstuen, Ullevålsveien 72, 0454 Oslo, Norway

• Terje Christensen, Statens Strålevern

• Ellen Christensen, Veterinærinstituttet

• Gro Johannessen, Veterinærinstituttet

Food Micro, Berlin, September 2018; Posterprice