Deletion of Salmonella enterica serovar typhimurium sipC gene

Maryam Safarpour Dehkordi，Abbas Doosti，Asghar Arshi

Biotechnology Research Center，Islamic Azad University Shahrekord Branch，Shahrekord，Iran

Deletion of Salmonella enterica serovar typhimurium sipC gene

Maryam Safarpour Dehkordi，Abbas Doosti*，Asghar Arshi

Biotechnology Research Center，Islamic Azad University Shahrekord Branch，Shahrekord，Iran

ARTICLE INFO

Article history:

in revised form 6 Jul，

2nd revised form 13 Jul 2015

Accepted 18 Aug 2015

Available online 17 Oct 2015

Salmonella enterica serovar

typhimurium

sipC gene

TA cloning

Gene construct

pET-32 vector

Objective:To construct a novel plasmid as Salmonella enterica serovar typhimurium（S. typhimurium）sipC gene knockouts candidate.

Methods:In this research，5′upstream and 3′downstream regions of S.typhimurium sipC gene and kanamycin gene were PCR amplified.Each of these DNA fragment was cloned into pGEM T-easy vector.The construct was confirmed by PCR and restriction digest.

Results:PCR amplified 320，206 and 835 bp DNA fragments were subcloned into pET-32 vector resulting with a plasmid called pET-32-sipC up-kan-sip C down.

Conclusions:The new plasmid（pET-32-sipC up-kan-sip C down）is useful for genetic engineering and for future manipulation of S.typhimurium sipC gene.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2015.09.003

1.Introduction

Salmonella is a member of the Enterobacteriaceae family，a large group of Gram-negative，non-spore-forming bacilli and facultative anaerobes［1］.Salmonella enterica（S.enterica）causes nearly 99%of Salmonella infections in animals and humans［2］.The S.enterica includes about 2600 diverse serotypes which consist of two species，Salmonella bongori and S.enterica and are divided into six subspecies:salamae，enterica，diarizonae，arizonae，indicaandhoutenae［1］. S.enterica serovar typhimurium（S.typhimurium）is a widely distributed food-borne pathogen and one of the most popular causes of bacterial food-borne disease in humans and animals and deaths globally［3，4］.Salmonellosis is considered one of the most significant food-borne zoonoses that is viewed in two kinds of different diseases，enteric fever which can be typhoid or paratyphoid and gastroenteritis which is non-typhoidal［5，6］. Bacterial pathogen in human causes 21 million cases of typhoid fever and 200000 deaths per year，mainly in Southern Asia，Africa and South America［7］.The discovery of a newvaccine for salmonellosis is a challenge for the scientists. Several vaccines have undergone clinical tests.The need of an efficacious vaccine against salmonellosis providing strong humoralaswellascellularimmunitystillpersists［8］. S.enterica serovars have been classified based on reactivity of antigen to somatic lipopolysaccharide，flagellar and capsular antigens.From a clinical aspect，these may be broadly grouped on the basis of host range and disease representation［9］.The Salmonella pathogenicity islands（SPIs）1 and 2 are two important virulence determinants of S.enterica.They encode type III secretion systems（T3SS），which carry the effector proteins and enable the injection of effector proteins directly into the cytosol of eukaryotic cells.These effectors finally manipulate the cellular functions of the infected host and comfort the development of the infection［10］.At present，21 SPIs have been recognized in Salmonella.Many of the identified SPI-encoded genes have only putative functions with no obvious role in Salmonella pathogenesis.

The SPI-1 locus is a 40 kb chromosomal island，which carries among all the others genes needed for the biosynthesis of a functional T3SS，a number of some effector and regulatory proteins and their chaperones［1］.The T3SS allows the secreted proteins to pass through the bacterial outer and inner membranes and a translocon creates a pore in the host cell membrane［11］. Location and function of these proteins in this system are shown below.Inner membrane proteins:InvA，SpaP，Q，R，S；associated inner membrane proteins:InvA，E；outer membrane proteins:InvG，PrgK，PrgH；chaperone:SicA；putative chaperone:InvI；secreted proteins involved in secretion:InvJ，SpaO；secreted proteins with an effector function（target proteins）:SipA，SipB，Salmonella invasion protein C（SipC），SipD，SptP［12］.

Invasion is initiated by pathogen binding to the host cell surface［13］.After invasion and entering the lumen of the small intestine，environmental conditions enable T3SS-1 genes to be expressed and subsequently the secretion system to be assembled at the bacterial membrane［14］.Cell entry is determined by rearrangements of the actin cytoskeleton，which is partly interceded by SipC，a component of the bacterial translocon，at the place of bacteria-host-cell contact［1］.SipC includes two membrane-spanning areas with C-and N-terminal domains front of the host-cell cytoplasm.This topological assembly of this effector protein is a reason to the actin nucleation and the translocation processes.Remarkably，both of these processes are primarily dependent on the C-terminus of SipC.SipC can localize actin polymerization at bacterial attachment site and employ actin directly［15］.Kanamycinisinactivatedwiththeaminophosphotransferases by transferring theγ-phosphate to the OH group in the 3′position of the pseudosaccharide.The kan gene was clonedand transformed in Escherichia coli（E.coli）cells［16］.

The purpose of this study was to generate a plasmid that carries kanamycin resistance gene replacement of S.typhimurium native sipC gene with Up-Kan-Down fragment，using homologous recombination technique.

2.Materials and methods

2.1.Bacterial complex，plasmids construction and media

In this study，the protocol and informed consent forms were approved by the Islamic Azad University，Shahrekord Branch，Shahrekord，Iran with 17621105 grant number.Virulent S. Typhimurium（ATCC-13311）collected from Microbiology Laboratory of Islamic Azad University of Shahrekord Branch was preserved as frozen glycerol stocks and cultured into Luria-Bertani（LB）broth until the log growth phase（optical density 600=0.9）was reached.The kan gene was isolated from the pET-28 vector.For cloning，preservation of DNA fragment and subcloning，pGEM T-easy vector using TA cloning kit（Promega，U.S）and pET-32 vector with E.coli strain TOP10F′were used，respectively.Bacterial cultures were grown at 37°C in LB broth and LB agar plates.

2.2.Extraction of genomic DNA from S.typhimurium

DNA was isolated from colonies of bacteria using DNA extraction kit（DNP™ Kit，CinnaGen，Iran）according to the manufacturer's protocol.The quality of DNA was checked on 1% agarose gel electrophoresis and quantified by spectrophotometric mensuration at 260 nm，according to Sambrook and Russell［17］.

2.3.Amplification of flanking regions of sipC gene and kan gene

Oligonucleotide primers were designed for amplification of flanking regions of sipC gene of S.typhimurium.The sequences oftheseprimerswereup-sipC-F:5′-ATGTCTAGA CCCTAAATAAAGTGGCG-3′and up-sipC-R:5′ATTAG ATCTCTCCCTTTATTTGGCAG-3′（accessionnumber: CP008928.1）containing Xba I and Bgl II restriction sites and down-sipC-F:5′-ATTGAGCTCTGACCACTGAAAGCCAC-3′anddown-sipC-R:5′-ACACTCGAGTAATACCCAGACTT TCCG-3′（accession number:CP007360.1）containing Sac I and Xho I restriction sites.Primers were used for amplification of up and down region of sipC gene，respectively.Moreover，kan-F: 5′-ATAAGATCTATGAGCCATATTCAGCGTG-3′and kan-R:5′-ATAGAGCTCTTAGAAAAATTCATCCAG-3′primers containing Bgl II and Sac I restriction sites were designed for amplification of kan gene and pET-28 vector was used as template.Underlined sequences indicate restriction sites.

Three collections of PCR programs were performed singly in high volume for amplification of kan gene and up and down regions of sipC gene.PCR amplification programs were carried out in a total volumes of 25μL in 0.2μL tubes containing 1μL of template DNA，1μmol/L of each primer，2.5μmol/L of 10×PCR buffer，2μmol/L MgCl2，200μmol/ L deoxy-ribonucleoside triphosphate and 1 unit of Taq DNA polymerase（Fermentas，Germany）.For the optimal amplification of flanking regions of sipC gene and kan gene，an initial denaturation step was performed at 95°C for 5 min，then amplified for 32 cycles of denaturation at 94°C for 1 min，annealing at 62°C for 1 min and extension at 72°C for 1 min.Lastly，a final extension phase was programmed for 5 min at 72°C and amplified samples were held at 10°C.

2.4.Analysis of PCR products

The amplified products（10μL）were analysed by electrophoresis in 1%agarose gel in tetrabromoethane［tris-base 10.8 g 89 mmol/L，boric acid 5.5 g 2 mmol/L，ethylene diamine tetraacetic acid 4 mL of 0.5 mol/L ethylene diamine tetraacetic acid（pH 8.0）buffer］.Constant voltage of 80 V was used for products differentiation.The 100 bp DNA ladder（Fermentas，Germany）was used as a molecular weight marker. After electrophoresis，the gel was stained with ethidium bromide and images were taken in UVIdoc gel documentation systems（UK）.

2.5.Cloning of sipC gene and plasmid construction

The PCR products were purified using gel extraction kit（Bioneer，Korea）according to manufacture's protocol.All PCR products were cloned into pGEM T-easy vector and the recombinant vectors were transformed by heat shock at 42°C and calcium chloride method into E.coli TOP10F′competent cells in LB culture media（Merck，Germany）.Kanamycin resistance was used for the selection.The presence of flanking regions of sipC gene and kan gene was confirmed by restriction endonucleases analysis.

2.6.Subcloning of the sipC and kan genes

The up-sipC fragment was removed from the pGEM T-easy vector by Xba I-Bgl II double digestion and subcloned in Xba I-Bgl II linearized pET-32 to get pET-32-sipC up.Then，kan fragment was released from the pGEM T-easy vector byBgl II-Sac I double digestion and subcloned into Bgl II-Sac I linearized pET-32-sipC up producing pET-32-sipC up-kan. Finally，pGEM-sipC down double digested with Sac I-Xho I and down fragment of sipC was subcloned into Sac I-Xho I linearized pET-32-sipC up-kan to produce pET-32-sipC upkan-sipCdownrecombinantvector.Competentcellsof E.coli TOP10F′strain were transformed with pET-32-sipC up-kan-sipC down recombinant vector.Flanking regions of sipC gene and kan gene containing restriction point of Xba I，Bgl II，Sac I and Xho I were inserted in polyclonal site in pET-32.E.coli TOP10F′strain competent cells were used for transformation and cultured in LB agar media containing ampicillin.The final construct was confirmed by double digestion by Xba I-Xho I and by PCR using up-sipC-F and down-sipC-R primers.

3.Results

DNA was successfully extracted and PCR amplified products for flanking regions of sipC and kan genes on 1%agarose gel revealed 320 bp，206 bp and 835 bp fragments，respectively（Figure 1）.

In the next step，the up and down regions of sipC gene of S. typhimurium and kan gene were cloned with TA cloning techniqueinpGEMT-easyvectorsuccessfully，separately（Figure 2）.

Plasmid purification and Xba I，Bgl II，Sac I and Xho I restriction endonuclease digestion of pET-32-sipC up-kan-sipC down recombinant plasmid confirmed the correction of up and down regions of sipC gene of S.typhimurium and kan gene cloning（Figure 3）.A 7261 bp large fragment was related to pET-32 vector（5900 bp）and 320，206 and 835 fragments were up and down regions of sipC gene and kan gene bands，respectively（Figure 3）.

4.Discussion

S.enterica is a facultative intracellular pathogen of universal significance and Gram-negative enteric bacterium［18］.While some have a restricted host range，for example the serovars typhi and pullorum are restricted to humans and chickens and most of the S.enterica serovars can infect a broad range of cold and warm-blooded animals and humans.S.enterica infects its hosts by the oral route and causes two types of disease: a gastroenteritis determined by the extension of bacteria in the intestinal tract and typhoid fever that determined by the invasion of the systemic compartment［19，20］.SipC is a Salmonella actin-binding protein that nucleates actin filament formation［12］.This protein was found to enhance the entry of wild-type S. Typhimurium into cultured cells，by interacting with phospholipid membranes and oligomerizing in solution.Research to date on sipC genes and SipC proteins that have been obtained，further study of the molecular properties and performance are discussed.Nuclear actin-related activity displacement factor SipC was placed under study by Chang et al.who reported that the central region of the protein SipC for nuclear actin and the C-terminal amino acid region for transfer factors are required［21，22］.Gene cloning allows scientists to find exclusive genes，cut them out and insert them into the genome of another organism and is the act of making copies of a single gene. Genetic engineering is the process of cloning genes into new organisms to change the protein product.Bacterial plasmids used in gene cloning naturally contain antibiotic resistance genes［23］.He CH et al.［24］constructed a recombinant plasmid based on AAV gene.This gene carried human endothelial nitric oxide synthase.The study by He CH et al. showed that pSNAV-eNOS was successfully constructed with the ability to express human endothelial nitric oxide synthase mRNA in cultured animals and humans cells，which is similar to the results of our study，but the plasmid and restriction enzyme of that research differed from those of our study［24］. The study of cloning of the encoding enterohemorrhagic E.coli Shiga-like toxin subunits to pGEM T-easy vector showed that cloning of this gene was successful and their findings were similar to this study［25］.Another study of Peerayeh SN et al.［26］in Iran，constructed a recombinant vector based on UreB122 gene that carried the urease of Helicobacter pylori.Prokaryote expression vector pET-32a was inserted with UreB122 gene.The recombinant vector was used to transform the competent E.coli DH5α.This study showed that pET-32a-UreB122 was successfully constructed and the expression of recombinant protein was induced by isopropythio-β-D-galctoside at different concentrations［26］. Haghi et al.generated a gene construct based on porA gene［27］.This protein（PorA）is a major member of the outer membrane of Neisseria meningitidis and functions as a cationic protein.porA was cloned into prokaryotic expression vector pET-32a and recombinant vector was transformed into competent Origami B（DE3）cells.Haghi et al.study showed that pET-32a-porA was successfully constructed and recombinant protein was overexpressed，which is similar to our study［27］.A gene construct to vacB gene deletion of Brucella melitensis（B.melitensis）vacB gene that was generated by Iranianresearchersin2012isanimportantgeneof B.melitensis that encoded a RNase R.This construct carries a kanamycin resistance gene replacement in downstream and upstream region of vacB gene of B.melitensis.Results showed that cloning of this gene was successful and their findings are similar to our research［16］.In another study in China，a recombinant vector for deletion of yncD gene in S.enterica was constructed.The yncD gene encodes a putativeTonB-dependenttransportersandwasidentified recently as an in vivo induced antigen［28］.In this study，yncD deletion mutant was successfully constructed in pYG4 vector，which is similar to the results of the present research［28］.The recombinant bacteria have become a useful instrument in various aspects.In this research，we have constructed the new recombinant plasmid carrying a kanamycin resistance gene replacement in up and down regions of sipC gene of S. typhimurium by improving the plasmid of E.coli.

In conclusion our results showed that sipC gene was cloned and subcloned in E.coli successfully.The recombinant gene construct has became a useful tool in various aspects on basic knowledge and applied science.In this research，we have constructed a novel recombinant gene construct that carries a kanamycin resistance gene replacement of S.typhimurium native sipC gene with sipC up-kan-sipC down fragment using homologous recombination technique.The new recombinant construct（pET-32-sipC up-kan-sipC dwon）in this study can be useful for genetics engineering.According to the results of the present study，gene construct that was produced can be used for sipC gene deletion and then manipulated strain can be used for engineering attenuated vaccine against S.typhimurium in future researches.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

The authors would like to thank to the staff of the Biotechnology Research Center of Islamic Azad University of Shahrekord Branch in Southwest Iran.

［1］Velge P，Wiedemann A，Rosselin M，Abed N，Boumart Z，Chauss´e AM，et al.Multiplicity of Salmonella entry mechanisms，a new paradigm for Salmonella pathogenesis.Microbiologyopen 2012；1（3）:243-58.

［2］McClelland M，Sanderson KE，Spieth J，Clifton SW，Latreille P，Courtney L，et al.Complete genome sequence of Salmonella enterica serovar Typhimurium LT2.Nature 2001；413（6858）: 852-6.

［3］Paião FG，Arisitides LGA，Murate LS，Vilas-Bˆoas GT，Vilas-Boas LA，Shimokomaki M.Detection of Salmonella spp，Salmonella Enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay.Braz J Microbiol 2013；44（1）:37-41.

［4］Srikanth CV，Mercado-Lubo R，Hallstrom K，McCormick BA. Salmonella effector proteins and host-cell responses.Cell Mol Life Sci 2011；68（22）:3687-97.

［5］Wasyl D，Sandvang D，Skov MN，Baggesen DL.Epidemiological characteristics of Salmonella Typhimurium isolated from animals and feed in Poland.Epidemiol Infect 2006；134（1）: 179-85.

［6］Zhang XL，Jeza VT，Pan Q.Salmonella typhi:from a human pathogen to a vaccine vector.Cell Mol Immunol 2008；5（2）:91-7.

［7］Roumagnac P，Weill FX，Dolecek C，Baker S，Brisse S，Chinh NT，et al.Evolutionary history of Salmonella typhi.Science 2006；314（5803）:1301-4.

［8］Marathe SA，Lahiri A，Negi VD，Chakravortty D.Typhoid fever& vaccine development:a partially answered question.Indian J Med Res 2012；135:161-9.

［9］Pezoa D，Blondel CJ，Silva CA，Yang HJ，Andrews-Polymenis H，Santiviago CA，et al.Only one of the two type VI secretion systems encoded in the Salmonella enterica serotype Dublin genome is involved in colonization of the avian and murine hosts.Vet Res 2014；45:2.

［10］Dieye Y，Ameiss K，Mellata M，Curtiss R.The Salmonella pathogenicity island（SPI）1 contributes more than SPI2 to the colonizationofthechickenbySalmonellaentericaserovar typhimurium.BMC Microbiol 2009；9:3.

［11］FookesM，SchroederGN，LangridgeGC，BlondelCJ，Mammina C，Connor TR，et al.Salmonella bongori provides insights into the evolution of the salmonellae.PLoS Pathog 2011；7（8）:e1002191.

［12］Boonyom R，Karavolos MH，Bulmer DM，Khan CM.Salmonella pathogenicity island 1（SPI-1）type III secretion of SopD involves N-and C-terminal signals and direct binding to the InvC ATPase. Microbiology 2010；156（Pt 6）:1805-14.

［13］Sheahan KL，Isberg RR.Identification of mammalian proteins that collaborate with type III secretion system function:involvement of a chemokine receptor in supporting translocon activity.MBio 2015；6（1）:e02023-14.

［14］Yang Y，Qi SH.A new feature selection method for computational prediction of type III secreted effectors.Int J Data Min Bioinform 2014；10（4）:440-54.

［15］Myeni SK，Zhou DG.The C terminus of SipC binds and bundles F-actin to promote Salmonella invasion.J Biol Chem 2010；285（18）: 13357-63.

［16］Yazdani RA，Doosti A，Dehkordi PG.Construction of a novel recombinant vector as Brucella melitensis vacB gene knockout candidate.Afr J Microbiol Res 2012；6（4）:802-8.

［17］Sambrook J，Russel DW.Molecular cloning:a laboratory manual. 3rd ed.New York:Cold Spring Harbour Laboratory；2001.

［18］Stevens MP，Humphrey TJ，Maskell DJ.Molecular insights into farm animal and zoonotic Salmonella infections.Philos Trans R Soc Lond B Biol Sci 2009；364（1530）:2709-23.

［19］Tauxe RV，Pavia AT.Salmonellosis:nontyphoidal.In:Evans AS，Brachman PS，editors.Bacterial infections of humans，epidemiology and control.3rd ed.New York:Plenum Medical Book Co.；1998，p.223-42.

［20］Parry CM，Hien TT，Dougan G，White NJ，Farrar JJ.Typhoid fever.N Engl J Med 2002；374:1770-82.

［21］Gong H，Vu GP，Bai Y，Yang E，Liu F，Lu S.Differential expression of Salmonella type III secretion system factors InvJ，PrgJ，SipC，SipD，SopA and SopB in cultures and in mice. Microbiology 2010；156（Pt 1）:116-27.

［22］Chang J，Chen J，Zhou D.Delineation and characterization of the actin nucleation and effector translocation activities of Salmonella SipC.Mol Microbiol 2005；55（5）:1379-89.

［23］Dyck MK，Lacroix D，Pothier F，Sirard MA.Making recombinant proteinsinanimals-differentsystems，differentapplications. Trends Biotechnol 2003；21（9）:394-9.

［24］He CX，Miao CY，Yao JH，Chen HM，Lu DR，Su DF，et al. Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene.Acta Pharmacol Sin 2003；24（7）:637-40.

［25］Bentancor LV，Bilen M，Brando RJ，Ramos MV，Ferreira LC，Ghiringhelli PD，et al.A DNA vaccine encoding the enterohemorragic Escherichia coli Shiga-like toxin 2 A2and B subunits confers protective immunity to Shiga toxin challenge in the murine model.Clin Vaccine Immunol 2009；16（5）:712-8.

［26］Peerayeh SN，Farshchian M，Sadeghizadeh M，Atoofi J.Cloning and expression of Helicobacter pylori UreB122（a segment of the B-subunit of urease gene）.Iran J Clin Infect Dis 2011；6: 161-4.

［27］Haghi F，Peerayeh SN，Siadat SD，Montajabiniat M.Cloning，expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B.J Infect Dev Ctries 2011；5（12）:856-62.

［28］Xiong K，Chen Z，Xiang G，Wang J，Rao X，Hu F，et al.Deletion of yncD gene in Salmonella enterica ssp.enterica serovar Typhi leads to attenuation in mouse model.FEMS Microbiol Lett 2012；328（1）:70-7.

15 Jun 2015

Abbas Doosti，Shahrekord Biotechnology Research Center，Islamic Azad University Shahrekord Branch，Shahrekord，Iran.

Tel/Fax:+98 381 3361001

E-mail:bioshk@yahoo.com

Peer review under responsibility of Hainan Medical University.