Comparative transcriptomic analysis by RNA-seq of Acid ToleranceResponse (ATR) in EHEC O157:H7

Shuangfang Hu a, Xinglong Xiao a, *, Xinwei Wu b, Xingzhou Xia c, Yigang Yu a, **, Hui Wu a

a School of Food Sciences and Engineering, South China University of Technology, Guangzhou City, Guangdong Province 510640, Chinab Department of Microbiology, Guangzhou Center for Disease Control and Prevention, Qide Road No.2, Guangzhou City, Guangdong 510440, Chinac College of Food Science and Technology, Guangdong Ocean University, Zhanjiang City, Guangdong Province 524088, China

a r t i c l e i n f o

Article history:Received 29 August 2016Received in revised form5 December 2016Accepted 15 January 2017Available online 18 January 2017

Keywords:E. coli O157:H7RNA-sequenceATR

Chemical compounds studied in this article:Ethidium bromide (PubChem CID: 14710)Hydrochloric acid (PubChem CID: 313)Agarose (PubChem CID: 11966311)

a b s t r a c t

ATR in EHEC O157:H7 and the cross-protection effects thus induced lead to the uncertainty of food safety.This study aimed to identify factors that are associated with the ATR in EHEC O157:H7 under stomachacidity using RNA-seq. In total, 223 DEGs in E. coli O157:H7 and 110 DEGs in E. coli after acid treatmentwere identified, including 118 upregulated and 105 downregulated in EHEC O157:H7, and 89 upregulatedand 21 downregulated in E. coli ATCC 25922. According to our results, when facing the ATR environment,protein Asr regulated the whole acid resistance process. In E. coli ATCC 25922, cadA and cadB mediate thelysine decarboxylation consumed the extra protons inside and keep the neutral pH value in cytoplasm toprotect the E. coli from death. In contrast, oxidation-reduction mediated by ahpC, which was activated bythe depression of oxyR, and hydrogenation mediated by mhpA consumed the protons inside keeping theneutral pH value to protect the EHEC O157:H7 from harm induced by a low pH environment. In addition,EHEC O157:H7 expressed more anti-oxidation enzymes to repair damage caused by acid stress, whichenhanced its resistance to acid compared to E. coli ATCC 25922.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

The serotype EHEC O157:H7 is a foodborne pathogen that has alow infectious dose (10-102 cfu), which is an important cause ofhemolytic uremic syndrome (Phillips, 1999). EHEC O157:H7 cantolerate harsh pH 2 environment of the stomach (Foster, 2004) andacid fruit juice (Lee, Kim, & Kang, 2015; Sanz-Puig, Pina-Perez,Martinez-Lopez, & Rodrigo, 2016; Tomadoni, Cassania, Moreiraa,& Ponce, 2015). Acid adaptation of EHEC O157:H7 increases survivalin acidic foods (Leyer, Wang, & Johnson, 1995). Due to its strongtolerance to low pH and the cross-protection effect induced(Gabriel, 2012), EHEC O157:H7may form a huge food safety hazard.

The acid resistancemechanism of E. coli has beenwidely studiedin the past decades, and can be summarized with the following 3

aspects. First, it consumes protons by decarboxylose or hydroge-nase. The amino acid decarboxylase-dependent AR systems (AR2and AR3) are thought to consume protons that leak into the cellduring acid stress by decarboxylation of arginine or glutamate(Richard & Foster, 2004). In addition, hydrogenase in E. coli hasbeen suggested to decrease cytoplasmic acid stress and contributeto its acid resistance systems by consuming protons (Noguchi,Riggins, Eldahan, Kitko, & Slonczewski, 2010). Second, it regulatesacid response factors. H-NS (Krin, Danchin, & Soutourina, 2010),gene asr (acid shock RNA) (Olesen and Jespersen, 2010; Seputieneet al., 2003), sORF (called iroK) (Warnecke, Lynch, Lipscomb, &Gill, 2012) and RpoS (Castanie-Cornet et al., 1999) are required forgrowth at acidity environment. Third, it prevents cells from damagesuch as proteins aggregation and DNA degradation. HdeA and HdeBhelp prevent periplasmic-protein aggregation and the solubiliza-tion of several model substrate proteins at acidic pH (Smith &Fratamico, 2012). Acetylation of the transcription factor RcsB pre-vents DNA binding and increases acid stress susceptibility(Castano-Cerezo et al., 2014).

For the acid resistance of E. coli O157:H7, low pH restored theDNA-binding activity of heat-denatured Dps low pH (2.2 and 3.6),but not at higher pH (>pH4.6) (Choi, Baumler, & Kaspar, 2000).

* Corresponding author. Research Center of Food Safety and Detection, School ofFood Sciences and Engineering, South China University of Technology, 381WushanRoad, Tianhe District, Guangzhou City, Guangdong Province 510640, China.** Corresponding author. Research Center of Food Safety and Detection, School ofFood Sciences and Engineering, South China University of Technology, 381WushanRoad, Tianhe District, Guangzhou City, Guangdong Province 510640, China.

E-mail addresses: fexxl@scut.edu.cn (X. Xiao), harebell_hu@foxmail.com (Y. Yu).

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LWT - Food Science and Technology 79 (2017) 300e308

Jeong, Hung, Baumler, Byrd, and Kaspar (2008) found that acidstress leads to the damage of chromosomal DNA, which wasaccentuated in dps and recAmutants. Metabolism-related proteins,including phosphoglycerate kinase (PGK), GadAB, adenine phos-phoribosyltransferase (APRT), dihydrodipicolinate synthase(DHDPS) and translation related proteins (e.g., elongation factor Tu,and elongation factor G), protein folding (e.g., alkyl hydroperoxidereductase), and membrane proteins (e.g., ompA precursor andompR) upregulated when responsed to mild pH such as pH 4.5 byHCl or to pH 5.5 by lactic acid (Huang, Tsai,& Pan, 2007). Accordingto microarray result, King, Lucchini, Hinton, and Gobius (2010)raised an assumption that enterohemorrhagic E. coli strain pos-sesses additional molecular mechanisms contributing to acidresistance that are absent in K-12.

However, it is still unclear about what plays a critical role inO157:H7 acid resistance to extremely acid environments after mildpH adaption. The ATR (acid tolerance response) system we discusshere is focused on research in enteric bacteria (Foster & Moreno,1999). Specifically, bacteria has the ability to survive extreme lowpH (pH 3.0 to 4.0) such as stomach acidity if first adapted tomild pH(pH 5.5 to 6.0) which could be implied as a mixture of meat stuffand stomach gastric juice (Foster, 1991).

The purpose of our study was to describe the major tran-scriptomic features of ATR in E. coli O157:H7 by comparing themRNA differentiate between E. coli O157:H7 and common non-toxic E.coli under stomach acidity after mild pH adaption, usinghigh-throughput RNA-sequencing (RNA-seq). RNA-seq has beenused for transcriptome analysis as an alternative to other tran-scriptomic technologies such as microarrays, due to advantagesthat include a large dynamic range and high technical reproduc-ibility (Wang et al., 2015). This work provided new physiologicalinsights into the ATR mechanism of E. coli O157:H7 and contributesa comprehensive illustration of mechanism in E. coli O157:H7responding to the acid stress environment.

2. Method and materials

2.1. Growth conditions of strains and acid treatment

E. coli O157:H7 NCTC 12900, was obtained from the NationalType Collection Center (London, United Kingdom). E. coli ATCC25922 was obtained from the American Type Culture Collection(Manassas, USA). Bacteria cells from the stationary phase wereharvested by centrifugation at 4000!g for 3 min, washed twice insterile water, and then resuspended in Luria-Bertani (LB) broth toproduce a final concentration of 6 log CFU/ml. Specifically for theacid stressed group, the LB broth was regulated by HCl to obtain apH of 5.5 and the bacteria were grown in an acid LB broth micro-cosm and maintained at 37 "C for 1 h during the acid adaptionprocedure. Subsequently, the LB broth was regulated by HCl toobtain a pH of 3.0 and the cells were grown in an acid LB brothmicrocosm and maintained at 37 "C for another hour. For the un-stressed control group without acid stress, bacteria at the concen-tration of 6 log CFU/ml was incubated in neutral LB broth at 37 "Cfor 2 h.

2.2. RNA extraction, library construction, and sequencing

The total RNA of each sample was isolated using the RNeasyProtect Bacteria kits (QIAGEN, USA), as per the manufacturer's in-structions. The total RNA was treated with RNase-free DNase I(Takara Bio, Japan) for 30 min at 37 "C to remove residual DNA.mRNA was enriched using a MICROBExpress Bacterial mRNAEnrichment Kit (Ambion, USA) to remove rRNA. RNA quality wasverified using an Agilent 2100 Bio-analyzer (Agilent Technologies,s

Santa Clara, CA) and was also checked by RNase free agarose gelelectrophoresis. All mRNA was broken into short fragments byadding fragmentation buffer.

Taking these short fragments as the template, first-strand cDNAwas generated with random hexamer-primed reverse transcriptionbyusing the SuperScript VILO cDNASynthesis Kit (Life Technologies,USA), followed by the synthesis of the second-strand cDNA usingRNase H and DNA polymerase I. RNaseH and RNaseA removed theresidual RNA. The cDNA fragments were purified using an AMPureXP Beads Kit (BioCanal, China). These purified fragments were thenwashed with EB buffer for end reparation poly (A) addition andligated to sequencing adapters. Following agarose gel electropho-resis and extraction of cDNA from gels, the cDNA fragments werepurified using an AMPure XP beads Kit (BioCanal, China) andenrichedbyPCR to construct thefinal cDNA library. The cDNA librarywas sequenced on the Illumina sequencing platform (IlluminaHiSeq™ 2000) using the paired-end technology from Gene DenovoCo. (Guangzhou, China). All samples were sequenced twice.

2.3. Reads alignment and normalization of gene expression levels

A Perl program was written to select clean reads by removinglow quality sequences (there were more than 50% bases withquality lower than 20 in one sequence), reads with more than 5% Nbases (bases unknown), and reads containing adaptor sequences.Sequencing reads were mapped to reference sequence by theSOAPaligner/soap2 (Li et al., 2009), a tool designed for shortsequence alignment. All expression data statistics and visualizationwas conducted with the R package (http://www.r-project.org/).

2.4. Differentially expressed genes (DEGs) and function enrichmentanalyses

After the expression level of each gene was calculated, differ-ential expression analysis was conducted using edgeR (Robinson,McCarthy, & Smyth, 2010). The false discovery rate (FDR) wasused to determine the threshold of the p value inmultiple tests, andfor the analysis, a threshold of the FDR# 0.01 and an absolute valueof log2 Ratio $ 1.5 were used to judge the significance of the geneexpression differences.

The differentially expressed genes were used for GO and KEGGenrichment analyses according to a method similar to thatdescribed by Zhang (Zhang et al., 2013). Both GO terms and KEGGpathways with a Q-value # 0.05 are significantly enriched in DEGs.

2.5. Quantitative real-time PCR (qRT-PCR) validations

Several differentially expressed genes were selected for qPCRanalysis to verify if gene expression was consistent between theRNA-seq and qRT-PCR. Total RNA was isolated using TRIzol reagentaccording to the manufacturer's protocol, and the RNAwas used forcDNA synthesis using reverse transcriptase. The rssA gene of E. coliwas used as an internal gene (Peng, Stephan, Hummerjohann, &Tasara, 2014). The 7500 Fast Real-time PCR System (Applied Bio-system, Foster, USA) was used for thermocycling and to recordchanges in fluorescence. Quantitation of each transcript wasrepeated using total RNA as the starting materials and each qPCRwas performed in triplicate. The primers are listed in Table 1.

3. Results

3.1. Data processing and analysis

Using the Illumina sequencing platform, raw reads were pro-duced for stressed and unstressed samples, respectively, and after

S. Hu et al. / LWT - Food Science and Technology 79 (2017) 300e308 301

strict quality control and data filtration, clean reads were harvested,with an average length of 100 bp (data not shown). In the followingfigures and tables in this paper, O157 or EHEC O157:H7 is short forE. coli O157:H7 NCTC 12900 and E. coli is short for non-toxic E. coliATCC 25922. After removing contaminated and low-quality se-quences, all reads were mapped onto the published transcriptome,which contains 5449 Unigenes. We mapped 4926 known genes,and no new gene was found. In total, the mapped genes were 4798,3731, 4788 and 3727 for O157-A, E. coli-A, O157eC, and E. coli-C,respectively. Unigenes represented by at least one mapped readwere accepted for subsequent analyses. Approximately 99.48%,99.47%, 99.57%, and 98.69% of the total gene had coverage between80 and 100% for O157-A, E. coli-A, O157-C and E. coli-C respectively(data not shown). These basic data of RNA sequencing presentedthat the data were confidential for further analysis.

3.2. DEGs

DEGs from the collection of mapped Unigenes were screenedusing the criteria of log 2 fold change $1.5 and FDR <0.01; weidentified 110 DEGs after acid treatment in non-toxic E. coli. Ofthose, 89 were upregulated and 21 were downregulated (Fig. 1A).223 DEGs in EHEC O157:H7 after acid treatment were identified. Ofthose, 118 were upregulated and 105 were downregulated (Fig. 1B).The Venn diagram indicated that, within 233 DEGs in EHECO157:H7 and 110 DEGs in non-toxic E. coli, 192 genes were specif-ically altered in EHEC O157:H7, 79 genes specifically were altered innon-toxic E. coli, and only 31 genes appeared differentiallyexpressed both in EHEC O157:H7 and non-toxic E. coli (Fig. 1C).

3.3. GO analysis of DEGs

Gene Ontology is a standardized system for gene functionalclassification, containing 3 domains that are classified by biologicalprocess, cellular components, and molecular functions of geneproducts (Zhenzhen et al., 2014). To determine the function ofdifferentially expressed genes, all of the DEGs in this study wereannotated to terms in the GO database. In EHEC O157:H7, 125 out of192 DEGs were annotated in the GO database, i.e., 55 genes wereannotated in cellular components, 102 genes were annotated inmolecular functions and 102 genes were annotated in biologicalprocesses. In non-toxic E. coli, 54 out of 79 DEGS were annotated in

the GO database, i.e. 35 DEGs were annotated in cellular compo-nents, 38 genes were annotated in molecular functions and 47genes were annotated in biological processes.

There are 3 biological processes (cellular component organiza-tion or biogenesis cellular, locomotion and signaling.) and 2 cellularcomponents (organelle and part organelle) that went through analteration in EHEC O157:H7 compared to non-toxic E. coli (Table 2).These biological processes may be associated with a higher acidtolerance of EHEC O157:H7 compared to other Enterobacteriaceae.

3.4. KEGG analysis of DEGs

KEGG pathway enrichment analysis was also carried out toelucidate the interaction of acid mediated pathways in stress re-sponses. The DEGs in non-toxic E. coliwere matched to 18 differentKEGG pathways. Themost significantly changed pathways includedABC transporters, Taurine and hypotaurine metabolism and LysineDegradation. The DEGs in EHEC O157:H7 were matched to 18different KEGG pathways, including Lysine Degradation Pathways.As for the 79 DEGs in non-toxic E. coli, ten terms from the ProcessOntology with p-value as good or better than 1 were cellularresponse to starvation, extracellular stimulus and nutrient levels,single-organism process and transmembrane transport. In contrast,the most significantly changed pathways in EHEC O157:H7included Peroxisome, Riboflavin metabolism and Amoebiasis.

3.5. qRT-PCR validation

qRT-PCR analysis for 11 genes (7 upregulated and 4 down-regulated) was used to validate the RNA-seq data in this study. Asshown in Fig. 4, qRT-PCR data correlated well with the RNA-seqdata (R2 ¼ 0.8851). Overall, the qRT-PCR data showed patternssimilar to those obtained from RNA-seq for these genes, althoughthe particular values of fold-change were different.

4. Discussion

In this study, combining DEGs identified and functional analysissuch as KEGG and GO annotation (Fig. 2), we focused on thefollowing aspects associated with ATR from a whole insight.

Table 1Primer pairs used for qRT-PCR validation.

Gene name Gene annotation Primer pair Reference

rssA encoding 16 S ribosomalRNA F,50-CTCTTGCCATCGGATGTGCCCA- 30

R,50-CCAGTGTGGCTGGTCATCCTCTCA- 30(Peng et al., 2014)

asnA asparagine synthetase AsnA F,50-CTGGGCGGGAATTAAAGCAACC- 30

R,50-CTCCTGGCTGTGTACGAAGTGG- 30(Zhou et al., 2011)

ispA geranyltranstransferase F,50-GCATTATTAGGTGGTAAGC- 30

R,50-ATGAGTAAGCGTGGATAC- 30(Zhou et al., 2011)

ompF phosphoporin protein E F,50-TTCTGGCAGTGATCGTCCCT 30

R,50-GCCGTAATCGAAAGAACCAAC 30(Monr!as et al., 2014)

asr acid shock protein F,50-ACTGCGACGACCACCAAAG-30

R,50-TCTGGGCAGGTGCTGCTT-30(Inger Olesen and LeneJespersen, 2010)

rpoS RNA polymerase, sigma S (sigma38) factor F,50-AACGGCGGGCAATTTTTAC-30

R,50-CGCCGCCGGATGATC-30(Inger Olesen and LeneJespersen, 2010)

oxyR Activator, hydrogen peroxide-inducible genes F,50-GAAGCACAGACCCACCAGTT-30 R,50-CAAACAACGGCACTTCAATG-30 (Wang et al., 2009)soxR redox-sensitive transcriptional activator SoxR F,50-GCATTAAAGCGCTGCTAACC-30 R,50-ATTGCCGCTGTTACGGATAC-30 (Wang et al., 2009)ycfR multiple stress resistance protein BhsA F,50-GCGATTTTAAGCTCCATGTC-30 R,50-GCTGTTCCATGGAGGGTATT-30 (Wang et al., 2009)dps DNA starvation/stationary phase

protection protein DpsF,50-TTATGAGTACCGCTAAAT-30

R,50-ATTTATTCGATGTTAGAC-30(Kabir et al., 2004)

gadA glutamate decarboxylase alpha F,50-GAATATCCGCAATCCGC-30

R,50-GGTTGTGGGAACTCATA-30(Kabir et al., 2004)

dnaK molecular chaperone DnaK F,50ATGGGTAAAATAATTGGT-30

R,50GATACGGCCTGCGTCTTT-30(Kabir et al., 2004)

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4.1. Upregulated genes under acid stress

Genes associated with the FeeS system (such as ykgJ, fes, ybfA,grxA, ydeA, and glnA), transportation and membrane (such as ycaD,yceL, yjcB, yohJ, and mokP) and stress resistance (such as ycfR, hdfR,and asr) and were upregulated in both EHEC O157:H7 and non-toxic E. coli. Gene grxA dramatically upregulated 8.01 and 9.71 log2 fold in EHEC O157:H7 and non-toxic E. coli, respectively. Gluta-redoxin (GRX) acted in antioxidant defense by reducing dehy-droascorbate, peroxiredoxins, and methionine sulfoxide reductaseand were shown to bind iron-sulfur clusters (Ye, Nadar, Li, & Rosen,2014). Gene yohJ (UPF0299 family inner membrane protein)expression increased 20.44 and 3.79 in EHEC O157:H7 and non-toxic E. coli, respectively. Gene hdfR (LysR family transcriptionalregulator), an H-NS-dependent flhDC regulator, were upregulated6.62 and 5.38, in EHEC O157:H7 and non-toxic E. coli, respectively.H-NS also positively affected motility via the flhDC master operon(Krin et al., 2010). Protein Asr (acid shock protein) is required forgrowth at moderate acidity (pH 4.5) and its ability to survive sub-sequent transfer to extreme acidity (pH 2.0) (Seputiene et al., 2003).Gene asr expression increased 13.57 and 5.65 in EHEC O157:H7 andnon-toxic E. coli, respectively. The dramatic expression increasinggene asr, yohJ and hdfR may have regulated a strong resistance to

the acid environment. However, the mechanism as to why theexpression of asr was much higher in EHEC O157:H7 compared tonon-toxic E. coli is still unknown.

4.2. Protons consumption

In AR2 and AR3, the glutamate decarboxylase GadA and thearginine decarboxylase AdiA, are pyridoxal phosphate-containingenzymes that replace the a-carboxyl groups of their amino acidsubstrates with a proton that is recruited from the cytoplasm.When facing an ATR environment, the 4.03 log 2 fold upregulationof cadA (lysine decarboxylase) and 5.42 log 2 fold upregulation ofcadB (lysine/cadaverine antiporter) in non-toxic E. colimay play thesame role as GadA and AdiA to keep the appropriate pH value insidethe cytoplasm. The cadBA and speF-potE operons contribute to anincrease in pH of the extracellular medium through excretion ofcadaverine and putrescine and the consumption of a proton, and asupply of carbon dioxide during the decarboxylation reaction inE. coli JM109 (Soksawatmaekhin, Uemura, Fukiwake, Kashiwagi, &Igarashi, 2006). However, gene cadB was found upregulatingslightly (1.35 log 2 fold) in EHEC O157:H7, and the mechanism howEHEC O157:H7 consumed the extra protons by another way.

According to the GO annotations of DEGs in EHEC O157:H7, only

Fig. 1. Global comparison of transcript profiles and DEGs in ATR: (A) Global transcript profiles of E. coli; (B) Global transcript profiles of O157; and (C) Venn diagram for DEGsidentified for each strain as indicated.

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2 terms from the Process Ontology had a p-value better than 1,which indicated that these 2 processes were dramatically differentfrom the control group. The 2 processes were responses to stress(including genes yafO, yaiB, ahpC and so on) and secondary meta-bolic process (including genes mhpA, entC and entE).

The addition of exogenous catalase or other antioxidant factorswere proven to enhance the resistance of Aeromonas hydrophila(Casabianca et al., 2015), E. coli (Zhao, Bi, Hao, & Liao, 2013) andVibrio vulnificus (Abe, Ohashi, Ren, Hayashi, & Endo, 2007) under astressed state. Hydrogenase in E. coli has been suggested todecrease cytoplasmic acid stress and contribute to its acid resis-tance systems (Noguchi et al., 2010). All of these indicated thathydroperoxide reductase or hydrogenase, which consume protons,may help bacteria to prevent the cytoplasm pH value change whenencountering acidity. Gene ahpC which expressed alkyl hydroper-oxide reductase, upregulated 3 log 2 fold compared to the controlsample in EHEC O157:H7. This reaction is a typical 2-cys peroxidereductase; it can directly consume the protons and transfer theperoxide substrate into water or the corresponding alcohol. Theupregulation of ahpC in ATR EHEC O157:H7 may contribute tomaintain the intercellular pH value by consuming the extra pro-tons, and finally help the cells go through adverse conditions.

In addition, the enzyme was expressed by entE transferredphosphorus-containing groups and the enzyme expressed by entCtransferred hydroxyl groups. These 2 reactions associated withsiderophore group nonribosomal peptides accumulated the enter-obactin inside cells, an archetype for microbial iron transport. GenemhpAmediated an oxidation-reduction reaction, specifically, actingon paired donors, with incorporation or reduction of molecularoxygen. During this process, with NADH or NADPH as one donor,and incorporation of one atom of oxygen into the other donor,protons were consumed.

4.3. Acid response regulation factors

RpoS is considered to be a critical switch for the whole regula-tion to resist the adverse environment in E. coli (Christensen-

Dalsgaard, Jorgensen, & Gerdes, 2010; Vanlint, Rutten, Govers,Michiels, & Aertsen, 2013). According to our results, the anti-adapter protein IraP (yaiB), which was an RpoS stabilizer duringPi starvation downregulated 2.2 log 2 fold. The inactivation of oxyRand both oxyR and rpoS genes had little effect on the viability of Pi-starved cells (Moreau, Gerard, Lutz, & Cozzone, 2001). Gene oxyRdownregulated 1.05 log 2 fold based on to our results. Theexpression of the ahpCF genes were enhanced in an OxyR-dependent manner in response to the hydrogen peroxide chal-lenge (Moreau et al., 2001). The expression of ahpCwas increased inthe oxyRmutant background in response to hydrogen peroxide andthe loss of oxyR is suggested to benefit the hydrogen peroxideinducible gene expression (Xie, Peng, Hu, Wang, & Zhang, 2013).Our results revealed that the inactivation of oxyR-stimulated geneahpC upregulated 3 log 2 fold compared to the control sample inEHEC O157:H7, which may play an essential role in the ATR system.This indicated that oxyR instead of rpoS may account for the ATR.

Toxin YafO is one of the mRNA interferases induced by DNAdamage (Christensen-Dalsgaard et al., 2010). The expression of yafOupregulated 1.62 log 2 fold conferred that the DNA damagehappened in cells. DNA binding protein tra, DNA polymerase Vsubunit D umuD and DNA binding transcriptional regulator ydaKwere depressed under the ATR environment, because acid stressleads to the damage of chromosomal DNA (Christensen-Dalsgaardet al., 2010).

4.4. ROS removal

Although, acid stress could lead to diverse harms to cells, EHECO157:H7 turned out to be more capable of repairing damages otherthan non-toxic E. coli. Peroxisomes are an important cellular sourceof different signaling molecules, including ROS (Del Rio and Lopez-Huertas, 2016). Environmental stress could stimulate the accumu-lation of ROS in cells which has the ability to cause antioxidantdamage (Xiang, Hu, Hu, Pan, & Ren, 2015). There are 2 ways toprevent oxidation damage caused by an adverse environment,namely the production of direct scavengers of ROS and indirect cell

Table 2GO annotation of DEGs.

Biological Process Cellular Component Molecular Function

O157 vs E. coli single-organism process;cellular process;metabolic process;localization;response to stimulus;biological regulation;regulation of biological process.

Membrane;membrane part;cell;cell part.

catalytic activity;binding;transporter activity;

O157-A vs O157-C single-organism process;cellular process;metabolic process;localization;response to stimulus;biological regulation;regulation of biological process;

Membrane;membrane part;cell;cell part;

catalytic activity;binding;transporter activity;

cellular component organization or biogenesis;locomotion;multi-organism process;signaling.

macromolecular complex;organelle;organelle part.

molecular transducer activity.

E. coli eA vs E. coli -C single-organism process;cellular process;metabolic process;localization;response to stimulus;biological regulation;regulation of biological process;

Membrane;membrane part;cell;cell part;

catalytic activity;binding;transporter activity;

multi-organism process. Macromolecular complex. enzyme regulator activity;molecular function regulator.

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responses (Apak, €Ozyürek, Güçlü, & Çapano#glu, 2016). Indirect cellresponses include heat shock response, the production of cellsignaling molecules and the synthesis of antioxidants such as

catalase, SOD, glutathione and so on (Apak et al., 2016). WhenVibrio vulnificus faced the low temperature, the expression ofglutathione S-transferase (GSTs) upregulated almost ten folds from

Fig. 2. Transcript profiles of genes enhancing ATR in O157.

S. Hu et al. / LWT - Food Science and Technology 79 (2017) 300e308 305

6 h to 24 h later at 4 "C (Abe et al., 2007). GSTs comprise a family ofisozymes best known for their ability to catalyze the conjugation ofthe reduced form of glutathione (GSH) to xenobiotic substrates forthe purpose of detoxification (Kanai, Takahashi, & Inoue, 2006).GenemetK regulated the synthesis of S-adenosylmethionine whichacted as a precursor during the synthesis of GSH (Koirala et al.,2015). Across the peroxisomal targeting signal (PTS) such as sodA

and sodC, peroxisomal proteins synthesized in the cytoplasm werebonded to receptors. The receptor then directed the complex to theperoxisome. Oxidoreductase ldhA and nirD catalyzed the accumu-lation of NADH. According to our results, genes associated withiron-sulfur clusters and cysteine biosynthesis upregulated in ATR inEHEC O157:H7. That is also consistence with the result of King et al.(2010), which indicated that genes involved in oxidative and iron

Fig. 3. Model for the regulation of ATR in O157.

Fig. 4. qRT-PCR validations.

S. Hu et al. / LWT - Food Science and Technology 79 (2017) 300e308306

and manganese uptake involved with hydrochloric acid at pH 5.5.

5. Conclusion

To conclude, when facing the ATR environment, protein Asrregulated the whole acid resistance process. In non-toxic E. coli,cadA and cadB mediate the lysine decarboxylation consuming theextra protons inside and keep the neutral pH value in cytoplasm toprotect the E. coli from death. In contrast, oxidation-reductionmediated by ahpC, which was activated by the depression ofOxyR, and hydrogenation mediated by mhpA, consumed the pro-tons inside keeping the neutral pH value to protect the EHECO157:H7 from harm induced by a low pH environment. Besides,EHEC O157:H7 expressed more anti-oxidation enzymes to repairdamage caused by acid stress, which enhanced its resistance to acidcompared to non-toxic E. coli (Fig. 3).

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China [No.31101279; and No. 31271867]; the Scienceand Technology Program Foundation of Guangdong Province [No.2013B021100005; No. 2013A090100014; and No.2015A030401025]; the Natural Science Fund of Guangdong [No.2016A030313449]; and the Fundamental Research Funds for theCentral Universities [2015ZZ123]. We would like to thankGuangzhou Gene Denovo Biotechnology Co., Ltd., China, for itstechnical assistance. We also thank LetPub (www.letpub.com) forits linguistic assistance during the preparation of this manuscript.

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