R. Landstorfer et al. BMC Genomics, Audrey Segura

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1 Comparison of strand-specific transcriptomes of enterohemorrhagic Escherichia coli O157:H7 EDL933 under eleven different environmental conditions including radish sprouts and cattle feces R. Landstorfer et al. BMC Genomics, 2014 Audrey Segura Journal Club of Microbiology, 18 th of November

2 Enterohemorrhagic E. coli (EHEC) Worldwide zoonotic foodborne pathogen o Hemorrhagic colitis o Hemolytic uremic syndrome : HUS (kidney failure) EHEC serotype O157:H7: most outbreaks and sporadic cases of HUS Public health problem: no therapy in humans (antibiotics treatment under debate) 2

3 Carriage of EHEC Major reservoir of EHEC strains: gastrointestinal tract of ruminants Fecal shedding Contamination of the environment Outbreak in US, 2006 (Uhlich et al., 2008) Outbreak in Japan, 1996 (Watanabe et al., 1999) Multiple infection sources Large environmental niches of EHEC Outbreak in US, 1996 (CDC, 1997) 3

4 Ecology of EHEC Ecology of this pathogen outside its human host unknown EHEC strains persistent in environment (Semenov et al., 2010) Large spectrum of environmental niches of EHEC (water, plants, animals) (Duffitt et al., 2011 ; Barker et al., 1999) Lack of global approaches to investigate gene regulation of EHEC from a broad environmental spectrum o Classical techniques are not enough sensitive o Many genes are not characterized: annotated as hypothetical genes 4

5 Hypothetical genes in EHEC 1/3 of the genes of EHEC annotated as hypothetical encoding proteins that do not have homology with any other protein Sequencing Genome Transcriptomic Prediction of hypothetical genes No evidence for the expression of these genes Confirmation of the expression of the hypothetical genes 5

6 Aim of the study To identify genes of EHEC involved in environmental and plant persitence with a special focus on hypothetical genes from a large variety of ecological niches Approach used: Strand-specific RNA-seq to EHEC Sequencing the transcriptome of EHEC O157:H7 under several culture conditions 6

7 Strain and culture conditions Strain: E. coli O157:H7 EDL933 Culture conditions: -LB : reference medium, 37 C -LB-15 C -LB-pH9 -LB-pH4 -LB-nitrite ph6: 200 mg/l sodium nitrite -LB-antibiotics: sulfamethoxazole + trimethoprim -LB-solid -M9 minimal medium (MM) -Spinach (extract) -Radish sprouts -Cattle feces RNA extraction when bacteria enter the stationary growth phase Strand-specific RNA-Seq: SOLiD System and Illumina System 7

8 Test the reproducibility of the sequencing process Sequencing of two technical replicates by SOLiD System Comparison of RPKM values of each replicate Coefficient correlation R 2 = 1 Combinaison of technical replicates for further analysis Sequencing of two biological replicates on two different plateforms: SOLiD System and Illumina System Comparison of RPKM values of each replicates for each plateform Coefficient correlation R 2 =

9 Sequencing results: number of reads Genome size: bp Plasmid size: bp (1.7%) 26.1 millions reads mapped to the EHEC genome and the plasmid po157 9

10 Sequencing results: expression of annotated gene Annotated gene in EDL933: 5379 genes Determination of the number of transcribed genes by an estimation of a threshold of backgroung transcription Statistical evaluation to classify genes into «active» or «inactive» 5142 transcribed genes (96%) of which 4877 significantly regulated (log 2 -Fold Change 3 or -1) Number of transcribed genes differs for different conditions 10

11 Sequencing results: in LB-antibiotics and feces conditions LB-antibiotics: lowest number of transcribed genes In feces : low number of transcribed genes compared to sprouts or spinach juice EHEC require more transcribed genes to survive in other conditions 11

12 Sequencing results: hypothetical genes Annotated hypothetical genes in EDL933: 1771 transcribed genes (32.9%) 1559 significantly regulated genes (log 2 -Fold Change 3 or -1) Number of hypothetical genes highly regulated (log 2 -Fold Change 5) Culture conditions Number of hypothetical gene LB-15 C 3 Minimal medium 14 LB-pH9 0 LB-pH4 3 LB-nitrite 1 LB-antibiotics 26 LB-solid 2 Spinach 9 Radish sprouts 9 Cattle feces 13 12

13 Transcription of genes in cattle feces Regulated known genes in feces: - Metabolic enzymes (glycogen metabolism) - Macromolecule-protection - Associated to cellular or membrane stress Hypothetical up-regulated genes in feces (13): - ycdt: involved in biofilm formation - Membrane protein involved in the metabolism of glucuronate (Ramos et al., 2005), also active during human infection (John et al., 2005) Several regulated genes allowing the survival of EHEC in cattle feces Most are associated to cellular or membrane stress In the colon of ruminant: EHEC under environmental stress? 13

14 Gene expression on radish sprouts (1) Very well growth of EHEC on radish sprouts Sprouts incoculated with 4x10 2 CFU/g plant Regulated genes associated to sugar degradation: fructose, trehalose, arabinose EHEC able to use sugars secreted by plants as carbon sources Regulated genes associated to the response to stress - azor: bacterial azoreductase environmental role unknown (Feng et al., 2012) Azo dyes (class of colorant carcinogenic) Severe environmental problem AZOREDUCTASE NADPH NADP + Reduction of azo dyes (destruction) High induction = Detoxification of plant metabolites directed against bacteria? 14

15 Gene expression on radish sprouts (2) Regulated genes associated to adhesion on the plant surface -csggfedba: curli fiber genes Major factor for the formation of biofilm (Brombacher et al., 2006) Associated to adhesion to plants (Saldana et al., 2011) -bsss: regulatory gene for biofilm formation (Domka et al., 2006) -9 hypothetical genes only regulated on sprouts: biofilm growth and formation (Schambri et al., 2003 ; Hancock et al., 2010) Formation of biofilms on plant surfaces using curli Radish sprouts = Suitable habitat for EHEC survival and proliferation 15

16 Transcriptional pattern of regulated genes Up-regulated genes colored in blue Down-regulated genes colored in red Compared to LB-condition LB-based experiments clustering together except LB-antibiotics: - No growth, cell elongation - Block of DNA synthesis - Genes down-regulated LB-antibiotics condition: outer group Spinach medium and MM : more related regulational pattern Low nutrient content Cattle feces: outer group 16

17 Gene expression in the presence of antibiotics Condition with lowest number of regulated genes Highly up-regulated genes: most originate from prophages -CP-933V -BP-933W Encode the shiga-toxin Condition with highest number of hypothetical regulated genes (26) 16/26 antibiotic-induced hypotheticals are encoded by prophages Hypothetical genes Product References Z0314 and Z0316 High similarities to phage tail fiber of CP-933H This study Z1434 High similarities to phage tail fiber of CP-933W John et al., 2005 Z3371 Unknown protein of the prophage CP-933V This study Antibiototics = SOS-response = phage replication = clinical complications 17

18 Conclusions and discussion (1) Distinguishing weakly transcribed genes from background transcription Classification of genes into «active» or «inactive» by statistical evaluation Discovery of a unique set of «active» genes for each culture condition Several hypothetical genes found to be «active» = new candidates for a detailed functional description (new roles for genes) Adaptation of EHEC to different environmental niches 18

19 Conclusions and discussion (2) EHEC ON CATTLE FECES: Growth and survival of EHEC under environmental stress In this study: Method does not reflect what s happen in environment No confirmation about genes involved in environment persistence in cattle feces EHEC ON RADISH SPROUTS: Growth and survival of EHEC (use plant-specific carbon sources, form biofilm, induce stress response) azor: detoxification role in nature? Test natural plant substances for azor induction Test the behavior of EHEC and other pathogens in nature (ΔazoR mutant) In this study: Mono-association of EHEC and sprouts No confirmation whether plants serve as a natural reservoir of EHEC 19

20 EHEC as a «vegetarian» EHEC able to survive and proliferate in plants (Hou et al., 2013) Possible «vegetarian» life style of EHEC? Some genes induced in EHEC on radish sprouts are found in other plant pathogens To scan the EHEC genome for homologous genes from other plant bacteria which are known to be induced in the respective niche To see if EHEC can develop in this niche and which genes are induced 20