articulo yersinia base abstract.docx

8/16/2019 articulo Yersinia Base Abstract.docx

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UNIVERSIDAD TÉCNICA DE MACHALAUNIDAD ACADÉMICA DE CIENCIAS QUÍMICAS Y DE LA SALUD

ESCUELA DE CIENCIAS MÉDICASCÁTEDRA DE MEDICINA TROPICAL

Yersinia Base: a genomic resource and analysis platform for comparative

analysis of YersiniaShi Yang Tan , Avirup Dutta , Nicholas S Jakubovics , Mia Yang Ang , Cheuk Chuen

Siow , Naresh VR Mutha , a!e" e#"ari , $ei Yee $ee , %uat Jah$ong , an" Siew $oh Choo &

Author in'or!ation ► Article notes ► Cop#right an" (icense in'or!ation ►

Abstract

Background

Yersinia is a genus of Gram-negative rod shaped bacteria belonging to theEnterobacteriaceae family. The majority ofYersinia species are environmental andnon-disease causing in mammals and can be isolated from many locations such asfresh water and soil. Some species can also be isolated from sources like patient sbody or even dog faeces three of the Yersinia species are known to cause diseasein human! the plague bacillus Y. pestis and the enteropathogens Y.enterocolitica and Y. pseudotuberculosis . The most notorious and virulent speciesin this genus" Y. pestis " the causative agent of plague" is a recently emergedlineage from Y. pseudotuberculosis #lague" which has caused three pandemics inhuman history $ is mainly a %oonotic infection caused byY. pestis " a rodentpathogen that can be transmitted to humans through the bite of an infected flea.&nfection can lead to acute febrile lymphadenitis also called bubonic plague or maycause septicemia" pharyngeal" meningeal and fatal pneumonia plague The 'orld(ealth )rgani%ation *'()+ has categori%ed this disease as re-emergent in someparts of the world as plague reappeared in ,alawi" ,o%ambi ue" and &ndia in

//0" in 1lgeria in 2334" and in 5ibya in 233/ $. The potential re-emergence of plague is a significant worldwide public health ha%ard. The problem has becomeeven more severe with the emergence of multi-drug resistant strains and thepotential use of plague as a bioweapon

Yersinia enterocolitica " the other notorious human pathogen of thegenus Yersinia is a %oonotic agent that causes gastrointestinal disease in humans.This species is also responsible for causing reactive arthritis and erythemanodosum. Enteropathogenic Yersinia " the etiological agents for yersiniosis" isgenerally ac uired by consuming raw or inade uately thermally processed pork or milk as well as vegetable products and ready-to-eat meals. 1s per the publishedannual e-reports of the European 6ood Safety 1uthority *E6S1+ on the prevalence

http://www.ncbi.nlm.nih.gov/pubmed/?term=Tan%20SY%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Dutta%20A%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Jakubovics%20NS%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Ang%20MY%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Siow%20CC%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Siow%20CC%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Mutha%20NV%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Heydari%20H%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Wee%20WY%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Wong%20GJ%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Wong%20GJ%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Choo%20SW%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pubmed/?term=Dutta%20A%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Jakubovics%20NS%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Ang%20MY%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Siow%20CC%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Siow%20CC%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Mutha%20NV%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Heydari%20H%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Wee%20WY%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Wong%20GJ%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Wong%20GJ%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Choo%20SW%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384384/http://www.ncbi.nlm.nih.gov/pubmed/?term=Tan%20SY%5Bauth%5D


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of %oonoses and foodborne pathogens in 23 2" yersiniosis was listed as the third-most-common enteropathogenic disease after campylobacteriosis andsalmonellosis.

1ll the three human pathogens share the similarity of carrying ane7trachromosomal 83-kb p9: virulence plasmid that encodes the 9sc type &&&secretion system *TTSS+" the protein microinjection apparatus andYersinia outer proteins *9ops+" a set of translocated effector proteins. The TTSS is highlyconserved among Gram-negative pathogens and is used by Yersinia pathogens toinject si7 different 9op effectors into the host cell. These 9op effectorsallowYersinia to resist phagocytosis and also interfere with signalling pathwayswithin the host cell.

1lthough the three pathogenic Yersinia species have been e7tensively studied for many years" the other Yersinia species" which includeY. frederiksenii, Y.intermedia, Y. kristensenii, Y. bercovieri, Y. massiliensis, Y. mollaretii, Y. rohdei, Y.ruckeri, and Y. aldovae have remained largely ignored and are not well studiedespecially at the genome level. These species are isolated from the environmentas well as from infected humans and do not possess typical Yersinia virulencemarkers. 6ormerly these species were grouped under Y. enterocolitica as differentbiogroups and they have been considered as Y. enterocolitica -like species in somepapers although they are clearly distinct fromY. enterolitica . 1lthough this group of species is generally considered non-pathogenic" some strains have been

associated with infections in mankind. Therefore" it is important for researchers toanalyse them further and to determine whether they have the potential to becomepathogenic" since the ac uisition of virulence genes through hori%ontal genetransfer is very common in prokaryotes.

'ith Y. pestis being already declared as re-emergent" Y. enterocolitica being listedas the third-most-common enteropathogen and several ;non-pathogenic strainsof Yersinia showing signs of infections in humans" it is clear that thegenus Yersinia is in a state of evolution and further study is re uired not only on itspathogenic strains but also on the non-pathogenic stains. &n recent years" a new

trend of collecting bacterial genomes into a single database has emerged as aneffective way to analyse their genomes.


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offers genomic and virulence factors information of some of theYersinia strains"however does not provide functionalities for comparing" clustering and visuali%ingthe virulence gene profiles of the user-selected Yersinia strains. These databases

do not provide the option for comparative pathogenomics analysis based on thevirulence factors of the strains. ,oreover" most of the e7isting biological databaseslack a user-friendly web interface which allows real-time and fast uerying andbrowsing of genomic data. &n order to provide researchers with a speciali%edplatform to access the genomic data of the pathogenic and the generallyconsidered non-pathogenic Yersinia species" we have developed the genusspecific database" 9ersinia>ase. 9ersinia>ase stores the Yersinia genomicresources along with various analysis tools particularly for comparative analysisof Yersinia strains. )ne of the features of this database is the #atho#roT which hasbeen developed in-house" with the intention of identifying the potential virulencemarkers in the Yersinia genus. This comparative analysis platform will help usersgain a deeper insight into the different species of Yersinia especially in the area of their pathogenicity. Asing the resources and the tools provided in 9ersinia>ase" wehave carried out genomic analyses on several pathogenic and non-pathogenicstrains of Yersinia . The preliminary results shown in this paper not only helped toidentify some of the genome characteristics of Yersinia but also demonstrated thesuitability and the usefulness of the database and the tools hosted by itfor Yersinia research.

Construction and content

Genome sequences

1 total of 242 genome se uences of Yersinia " which include both draft andcomplete genomes in 61ST1 format were downloaded from Bational iotechnology &nformation *B&+ 1ppro7imately /3C of the downloaded genomese uences belong to Y. pestis . 1ll generally considered non-pathogenicYersinia species were only available as draft genomes. 1 list of draftand complete genomes for each Yersinia species is shown in Table

Number of draft and complete genomes of each Yersinia species in YersiniaBase

Annotation of genome sequences

6or consistency" all of the downloaded genome se uences were annotated byusing @apid 1nnotation using Subsystem Technology *@1ST+ @1ST can predictopen reading frame *)@6+" function of the )@6" nucleotide se uence and aminoacid se uence in user-submitted genome. >esides @1ST" #S)@Tb version 4.3


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was used to determine the subcellular locali%ation of the proteins predicted by the@1ST engine. 'e also computed the hydrophobicity and molecular weight of the@1ST-predicted proteins using in-house developed #erl scripts. The summary of

genome annotations of each strain is shown in 1dditional file ! Table S .=atabase and web interface design

1 web interface was built by using (yperTe7t ,arkup 5anguage *(T,5+"(yperTe7t #reprocessor *#(#+" DavaScript" j uery" ase.>ioinformatics tools

'e developed several bioinformatics tools and integrated them into 9ersinia>ase.'e used #ython" #erl" >io#erl and @ languages to develop the #G< for comparingbetween two genomes through global alignment" #atho#roT for generating heatmap to visuali%e presence and absence of virulence genes in selected genomesand 9ersiniaTree to generate phylogenetic tree of the Yersinia strains based ontheir housekeeping genes and S r@B1. The use of these three popular scriptinglanguages allowed us to create comple7 pipelines" that perform back-endcalculations" aided communications between the web server and the applicationserver and also

Utility and discussion

vervie! of the database pipeline

1ll the Yersinia genomes after being downloaded from Bational iotechnology &nformation *B&+ website *http!HHwww.ncbi.nlm.nih.gov+ wereannotated using the @apid 1nnotation using Subsystem Technology *@1ST+ server

for consistency. 1ll the


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and also in-house #erl script to obtain information such as calculation of G<content *C+" predicted hydrophobicity *p(+" and molecular weight *=a+ of theencoded proteins. 1ll this information was then stored in the ,yS 5 database

server.'eb interface and functionalities of 9ersinia>ase

)n entering the home page of 9ersinia>ase" visitors can view the news Iconferences" blogs I information and the most recent published papers that arerelated to Yersinia " which we manually compile from various sources. The J>rowseKmenu allows the visitors to view the list ofYersinia species currently available in9ersinia>ase" with each J:iew StrainsK button leading the visitors to the J>rowseStrainsK page" displaying all available strains of that respective species. &n the

J>rowse StrainsK page a general description of that particular species is givenalong with a table listing the strains of that species. Each strain is linked to their corresponding ta7onomic classification page in B& and also to their page inGenome )nline =atabase *G)5=+. 6urthermore" by clicking on the J=etailsK icon"visitors can obtain more comprehensive information of that particular strain such astheir source and time of isolation" which we retrieved from B&" along with the listof )@6s" their respective function" start and stop positions in a tabular fashion inthe J>rowse )@6K page. 1part from that" each )@6 is linked to its correspondingAni#rot page along with their )@6 &= being linked to its corresponding page inB&. >y clicking on the 51ST search against the :irulence 6actor =atabase *:6=>+

>esides the >51ST search" users can also perform pairwise alignment of anytwo Yersinia genomes present in 9ersinia>ase of their choice by using the #G


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draw a heat map of the virulence genes profiles by using the #atho#roT or construct phylogenetic tree by using 9ersiniaTree.

Bro!sing Yersinia strainsThe browse page of this database summari%es a list of 2Yersinia spp." and their respective number of draft genomes and complete" as shown in Table The J:iewStrainsK button on the right of each species leads the user to a list of strains of thechosen species. &n the table" the information available to the user are strain status*draft genome or complete genome+" genome si%e in mega base pair *,bp+"percentage of G< content *C+" number of contigs" number of predicted


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that contain the keyword entered by the user and display it to the user seamlessly.)nce the user selects one item from the list" they will be presented with a tablewhich contains a list of genes that are related to the entered keyword.

#n"house developed pair!ise genome comparison tool $%GC&

1s pointed out earlier" Y. pestis has already been declared as re-emergent and Y.enterocolitica is listed as the third-most-common enteropathogen. ,any of thepreviously considered non-pathogenic strains ofYersinia have also shown signs of infections in humans. &t is evident that the genusYersinia is constantly evolvingand further study is re uired not only of the pathogenic strains but also of the non-pathogenic stains to gain a clear understanding of its biology. To have a clear ideaof the lifestyle" the evolution and an e7tended view of the gene pool of a species"

genomic information from a singleYersinia genome is unlikely to be sufficient. 6or detailed insights into the variations between different strains at the genetic level"the evolutionary changes among Yersinia species" the orthologous genes inspecies" as well as the genes that give each organism its uni ue characteristics"especially the potential regions associated with pathogenicity" a comparative studyof multipleYersinia genomes is re uired.

'ith that in mind" the #G< was developed and incorporated into 9ersinia>ase.This in-house developed online comparative genome analysis tool will allow theuser to compare two selected Yersinia genomes. Anlike conventional methods

which display alignments in a linear form" the #G< displays the alignments in acircular layout. )n entering the web interface of #G< in 9ersinia>ase" the user canchoose two Yersinia genomes of interest from the list for comparison. 1lternatively"the user can upload their Yersinia genome se uence for comparison withthe Yersinia genomes in 9ersinia>ase. Three parameters" which are minimumpercent identity *C+" merge threshold *bp+ and link threshold *bp+ can be definedmanually by the user. 5ink threshold *5T+ removes the links according to user-defined value" while merge threshold *,T+ allows merging of links based on user-defined value.

&n the #G< pipeline" after the se uences are aligned through BA


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using BA :ersion 23 2 containing a total of /88M proteins+" for #atho#roT are considered as the Jknown virulence factorsK. &n the #atho#roT


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pipeline" a >51ST search is performed with the default parameters of M3Cse uence identity and M3C se uence completeness to identify the orthologs of these known virulence factors in the Yersinia genomes present in the 9ersinia>ase.

(owever the user can change these default parameters for the >51ST searchdepending on their desired levels of stringency. &n-house developed #erl scriptfilters the >51ST results generated from a >51ST search against :6=> based onuser-defined cut-off values for se uence identity and completeness to identify thevirulence genes and selects only the user desired strains. @esults from the filteringprocess will be used to tabulate the data matri7 *strains versus virulence genes+This is followed by e7ecuting @ scripts" to read the generated data matri7 andperform hierarchical clustering *complete-linkage algorithm+ of theYersinia strainsbased on their virulence gene profiles. 6inally" for the visuali%ation of the virulencegene profiles" ;pheatmap package is used to generate the heat map image whichis the end-product of #atho#roT. The use of heat map in #atho#roT for visuali%ation enables users to have a bird s eye view of the data in a graphicalrepresentation allowing users to identify and visuali%e the virulence gene profile intheir strains of interest" and enabling comparative analyses of virulence factorsamong different Yersinia strains

Yersinia 'ree: constructing Yersinia phylogenetic tree

#hylogenetic trees are important in studying the evolutionary relationships betweendifferent species" and their common ancestor. &n 9ersinia>ase" we have designed

and incorporated an automated pipeline written in #erl" 9ersiniaTree that willenable user to generate phylogenetic trees of the Yersinia strains based on their housekeeping genes and S r@B1. )ur automated pipeline 9ersiniaTree"primarily re uires two inputs from the user! * + gene marker used to construct thephylogenetic tree *2+ list of genomes in 9ersinia>ase which to be included in thetree. The automated pipeline also offers an optional feature where the users caninput their nucleotide se uence in 61ST1 format along with the se uences whichare retrieved from the database. " hsp 3"rpo >"or sodA for the construction of the phylogenetic tree. #revious studies have shownthat phylogenetic trees based on these four housekeeping genes are moreconsistent with the biochemical profiles ofYersinia species than those based onthe S r@B1 1fter user provides all the necessary inputs" the front end #(#e7ecutes the backend #erl pipeline. The first step of our automated pipeline is toselect target gene s nucleotide se uence of genomes chosen by user from 61ST1


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file where complete listings of se uences are stored" into a temporary 61ST1 file.The #erl script then e7ecutes ,166T which is used to perform multiple se uencealignment across nucleotide se uences. Be7t" the output file from ,166T is sent

to 6astTree? to construct phylogenetic tree in Bewick format" followed by visuali%ingof the tree in S:G format by using Bewick Atilities 6inally" #erl script sends out thefinal image to #(# to display the phylogenetic tree in web browser.

Yersinia genome( gene and virulence factors database

>esides the in-house developed tools" we have also integrated >51ST as well as:6=>->51ST into 9ersinia>ase. This will allow the users to perform their similaritysearch of their uery se uences against Yersinia genome se uences" genese uences and also against the virulence factors *downloaded from :6=> by

using >51STB" >51ST# or >51STF. This speciali%ed databasefor Yersinia provides faster searching against Yersinia se uences compared toB& BT or B@ databases when the user is trying to find theclosest Yersinia strains to their own uery se uence.

)eatures of strains in YersiniaBase

Generally" the si%e of theYersinia genomes was observed to be between 0.4 ,bpto 0.N ,bp. (owever" there were a few e7ceptions. 6or e7ample" the genome si%eof Y. ruckeri 1Tase. Three phylogenetic trees wereconstructed" one using the ,EG1 the second one using the phylogenetic tool of 9ersinia>ase" 9ersiniaTree and the other using the in-house developed tool#atho#roT. 'e used the neighbour-joining method" Tamura-Bei model with "333bootstrap replications to build phylogenetic tree with ,EG1 based on the gyr >gene The tree generated by 9ersiniaTree was also based on gyr >The gyr > geneinstead of S r@B1 gene was used for phylogenetic analysis as S r@B1 cannotresolve the phylogenetic relationships between closely related Yersinia species.(ousekeeping genes such as gyrB produce phylogenetic trees which closelycorrelate with the biochemical species designations. ,oreover studies have shown


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that gyr > is a better phylogenetic marker than S r@B1 in the EnterobacteriaceaeThe other tree was constructed using the in-house developed tool #atho#roT" withhierarchical clustering *complete-linkage+ algorithm based on the virulence gene

profiles of the selected Yersinia strains. The parameters for #atho#roT were set toM3 for se uence identity *C+ and M3 for se uence completeness *C+. 'e also usedthe in-house developed #G< Tool for finding the differences between subspeciesof Y. enterocolitica .

%hylogeny of Yersinia based on gyrB

&n this paper" we constructed two phylogenetic trees based ongyrB " one using,EG1 while the other using our in-house developed tool 9ersinia Tree The treegenerated by 9ersinia Tree was found to be consistent with that of the tree

generated by the well-established tool ,EG1. >oth the trees showed similarity inthe clustering pattern. &n both the cases the strains ofY. pestis and Y. pseudotuberculosis grouped very close each other whereas Y.enterocolitica formed a separate cluster. &n case ofY. enterocolitica strains

3M.M@*r+ and 9 *both of which belong to the subspecies palearctica + groupedtogether while N3N *subspeciesenterocolitica + separated to form a differentbranch in the tree generated by 9ersiniaTree Similar type of clustering was alsoobserved in the tree by ,EG1

*volution of Y. pestis and Y. pseudotuberculosis

1part from the trees generated based on gyr > a separate tree was constructedbased on the virulence profiles of Y. pestis and Y. pseudotuberculosis strains usingthe #atho#roT Even in the tree by #atho#roT we found that Y. pestis and Y.

pseudotuberculosis formed a cluster that was clearly separated fromY.Enterocolitica and non-pathogenic Yersinia which was similar to that observed inboth the trees based on gyr >. This similarity in the clustering pattern" suggests thatthe housekeeping genes and virulence genes of Y. pestis and Y.

pseudotuberculosis evolved in a similar pathway and are distinct from theother Yersinia species. These results were in agreement with a recently published

report suggesting that Y. pestis and Y. pseudotuberculosis are close relatives"however Y. enterocolitica shows diametric separation with most environmentalspecies occupying intermediate branching positions between these two clustersThe tree also indicates that Y. pestis 1ngola is the first among the Y. pestis strainsto branch out from the closest common ancestor Y. pseudotuberculosis 9#&&&. Thisobservation is supported by a recently published report" which stated thatY.

pestis 1ngola belongs to one of the most ancient Y. pestis lineages se uenced to


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date" possessing genome characteristics which are intermediate between Y. pseudotuberculosis and the modern day Y. pestis These data suggest that Y. pestis is highly similar toY. pseudotuberculosis genetically and the virulence genes

that were lost or ac uired did not cause it to diverge far from its ancestor.(owever" Y. pestis has undergone gene reduction and has accumulatedpseudogenes since its emergence from Y. pseudotuberculosis

%hylogenetic tree constructed by from virulence gene profiles !ith%atho%ro'+

,irulence genes in Yersinia

>y calculating the number of virulence genes presents in every strain" we foundthat Y. pestis and Y. pseudotuberculosis generally have more virulence genes thanother species and the number of virulence genes in different strains is moreconsistent within these species compared to Y. enterocolitica and non-pathogenic Yersinia The number of virulence genes in non-pathogenic Yersinia varies from as low as 8 in Y. ruckeri 1T


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independent from the other two. Surprisingly"Y. enterocolitica N3N " which is thehighly virulent biotype" did not form the same cluster with the other two strains ofY.enterocolitica " but rather with 0 non-pathogenicYersinia .

Absence of yersiniabactin in Y. enterocolitica subsp+ palearctica

>esides having p9: virulence plasmid" highly pathogenic Yersinia strains alsopossess iron-regulated genes. There have been reports stating that Y.enterocolitica serotype )!N carries high pathogenicity island *(#&+" whichsynthesi%es yersiniabactin enterocolitica subsp. palearctica . The result isconsistent with previous findings and further suggests that Y.enterocolitica subsp. enterocolitica has followed a slightly different evolutionarypathway compared to subsp. palearctica and that yersiniabactin was ac uired

by Y. enterocolitica subsp. enterocolitica after it diverged from its common ancestor shared with subsp. palearctica .

1 heat map from #atho#roT showed the presence *red color+ and absence *blackcolor+ of yersiniabactin genes in selectedYersinia genomes. N3N which belongsto Y. enterocolitica subsp. enterocolitica has yersiniabactin genes while 9 and

3M.M@*r+ which.

'e further visuali%ed the difference between the two subspecies of Y.enterocolitica by using #G< Tool withY. Enterocolitica N3N as reference genome

1nother draft genome" Y. enterocolitica subsp. enterocolitica '1-4 0 was included.The start and stop position of the operon for yersiniabactin in N3N are 2N 83 0and 2N0 M / respectively *red circle region in the 6igure 6rom the three figuresgenerated by #G< we found only '1-4 0 has region mapped to the regionencoding yersiniabactin in N3N .

Genome comparisons between different subspecies of 9ersinia enterocolitica byusing #G< tool. #G< plots revealed yersiniabactin only present in 9ersiniaenterocolitica subsp. enterocolitica. *1+


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genes matched to the 1ngola genome with uery coverage more than M3C*e7cept fli ) and fli 5+" but with identity less than M3C

1 heat map showed the absence *black color+ of nine flagella genes in 1ngola" butpresent *red color+ in other selectedYersinia strains.

The uery se uence coverage and percentage of protein identity of nine missingflagella genes in 1ngola but present in other Yersinia strains T>51STB againstgenome of 1ngola

1lthough Yersinia is non-motile" all theY. pestis strains e7cept for the strain 1ngolahave a complete set of flagella associated genes" including flagella synthesis andflagella motor rotation There have been reports stating that mutations haveoccurred in the flagella and chemota7is gene clusters but se uence similarities of the Y. pestis flagella genes still can be found using >51ST" with the e7ception ofY.

pestis 1ngola. 'e suggest that" after the emergence of other Yersinia strains" manymutations have taken place in the / flagella genes of the 1ngola genome andaccumulated over time" which lead to low identity between 1ngola and otherY.

pestis . 1nother possibility is that the flagella gene cluster might have been replacedin 1ngola with that of a different species by hori%ontal gene transfer.

Conclusions

'ith the advent of ne7t generation se uencing *BGS+" we e7pect

more Yersinia strains will be published. 'e regularly keep track of the newdevelopments related to Yersinia especially release of new Yesinia genomes inB&. 'e are already in the process of adding new Yersinia genomes to the9ersinia>ase and will be updating the database as and when new genomesof Yersinia are available. 1t this moment" the database is optimi%ed for whole-genome and comparative pathogenomic analysis. (owever we hope to incorporateother data types as well along with new features and functionalities in future. 'eare working on it and we also encourage other researchers or research groups toemail us at girgOum.edu.my if they wish to share their annotations" opinions" andcurated data with us. 'e welcome the suggestions for improving this database asit will help us make this database a comprehensive one.

'-A.UCC#/N .*0 AB1'-AC'


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Yersinia Base: una plataforma de recursos y el an2lisis gen3mico para elan2lisis comparativo de Yersinia

Shi Yang Tan, Avirup Dutta, Nicholas S Jakubovics, Mia Yang Ang, Cheuk ChuenSiow, Naresh VR Mutha, a!e" e#"ari, $ei Yee $ee, %uat Jah $ong # Siew$oh Choo

Resumen:Fondo

9ersinia es un gPnero de bacterias Gram-negativas de la barra en forma depertenecientes a la familia Enterobacteriaceae. 5a mayorQa de las especies de9ersinia son ambientales y no causantes de enfermedades en mamQferos y sepueden aislar de muchas locali%aciones" como agua dulce y el suelo. 1lgunasespecies tambiPn se pueden aislar a partir de fuentes como el cuerpo del pacienteo incluso el perro heces tres de las especies de 9ersinia se sabe ue causan laenfermedad en humanos! el bacilo de la peste 9. pestis y la enterocolitica enteropatRgenos 9 pseudotuberculosis. 5as especies m s notorios y virulentas de estegPnero" 9. pestis" el agente causante de la peste" es un linaje recientemente salidode 9. pseudotuberculosis plaga" ue ha causado tres pandemias en la historiahumana $es principalmente una infecciRn %oonRtica causada por 9. pestis" unpatRgeno de roedores ue puede transmitirse a los humanos por la picadura deuna pulga infectada. 5a infecciRn puede provocar linfadenitis febril aguda tambiPnllamada peste bubRnica o puede causar septicemia" farQngea" menQngea yneumonQa mortal plaga 5a )rgani%aciRn ,undial de la Salud *),S+ ha clasificadoesta enfermedad re-emergente en algunas partes del mundo como la pestereapareciR en ,alawi" ,o%ambi ue y la &ndia en //0" en 1rgelia en 2334" y en5ibia en 233/ $. El potencial resurgimiento de la plaga es un peligro para la saludp blica a nivel mundial significativa. El problema se ha vuelto a n m s grave" conla apariciRn de cepas resistentes a m ltiples f rmacos y el uso potencial de lapeste como un arma biolRgica

9ersinia enterocolitica" el otro patRgeno humano notorio del gPnero 9ersinia es unagente %oonRtico ue causa enfermedades gastrointestinales en los sereshumanos. Esta especie tambiPn es responsable de causar artritis reactiva yeritema nudoso. EnteropatRgena 9ersinia" los agentes etiolRgicos de yersiniosis"se ad uieren generalmente por el consumo de carne de cerdo cruda oinsuficientemente tPrmicamente procesado o la leche" asQ como los productosvegetales y comidas listas para consumir. =e acuerdo con las publicacioneselectrRnicas informes anuales de la 1utoridad Europea de Seguridad 1limentaria*E6S1+ sobre la prevalencia de las %oonosis y los patRgenos transmitidos por


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alimentos en 23 2" yersiniosis fue catalogado como la enfermedadenteropatRgena tercera m s com n despuPs de la campilobacteriosis ysalmonelosis.

Todos los tres patRgenos humanos comparten la similitud de llevar a un 83-kbpl smido de virulencia de #y: e7tracromosRmico ue codifica el sistema desecreciRn de tipo &&& 9sc *SSTT+" el aparato de microinyecciRn de proteQnasand9ersinia proteQnas e7teriores *9ops+" un conjunto de proteQnas efectorastranslocados. El SSTT es altamente conservado entre los patRgenos Gram-negativos y es utili%ado por patRgenos 9ersinia para inyectar seis efectores 9opdiferentes en la cPlula huPsped. Estos efectores 9op permiten 9ersinia para resistir la fagocitosis y tambiPn interfieren con las vQas de seUali%aciRn dentro de la cPlulahuPsped.

1un ue las tres especies de 9ersinia patRgenos han sido ampliamente estudiadosdesde hace muchos aUos" la especie other9ersinia" ue incluyen frederiksenii 9." 9.intermedia" kristensenii 9." bercovieri 9." massiliensis 9." mollaretii 9." 9. rohdei" 9.ruckeri y 9. aldovae han permanecido en gran medida ignorados y no est n bienestudiados sobre todo a nivel del genoma. Estas especies est n aisladas delmedio ambiente" asQ como de los humanos infectados y no poseen marcadorestQpicos de virulencia 9ersinia. 1nteriormente estas especies se agruparon en 9.enterocolitica como diferentes biogroups y han sido considerados como 9.enterocolitica especies afines en algunos papeles aun ue son claramente distintas

de 9. enterolitica. 1un ue este grupo de especies se considera generalmente nopatRgeno" algunas cepas se han asociado con infecciones en la humanidad. #or lotanto" es importante para los investigadores para anali%ar m s a fondo ydeterminar si tienen el potencial de convertirse en patRgena" desde la ad uisiciRnde genes de virulencia mediante la transferencia hori%ontal de genes es muycom n en procariotas.


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an lisis comparativo *,>G=+ y el? Sistema &ntegrado de genomas microbianos*&,G+" ue proporcionan una amplia gama de genomas microbianos incluyendoalgunas cepas de 9ersinia para la genRmica comparativa. )tro dicha base de

datos vale la pena mencionar es ue ofrece #1T@&< genRmica y factores devirulencia informaciRn de algunas de las cepas de 9ersinia" sin embargo noproporciona funcionalidades para comparar" agrupamiento y visuali%aciRn de losperfiles de genes de virulencia de las cepas de 9ersinia seleccionados por elusuario. Estas bases de datos no proporcionan la opciRn para el an lisis#1T()GEB),&ase" hemosllevado a cabo los an lisis genRmicos en varias cepas patRgenas y no patRgenasde 9ersinia. 5os resultados preliminares muestran en este trabajo no sRlo ayudR aidentificar algunas de las caracterQsticas del genoma de 9ersinia pero tambiPndemostraron la conveniencia y la utilidad de la base de datos y las herramientasalojadas por ella para la investigaciRn 9ersinia.

Construcci3n y contenido

1ecuencias del genomaAn total de 242 secuencias del genoma de 9ersinia" ue incluyen tanto losproyectos como genomas completos en formato 61ST1 fueron descargados deiotecnolRgica *B&+ 1pro7imadamente el /3Cde las secuencias del genoma descargados pertenecen a 9. pestis. Todas lasespecies no patogPnica 9ersinia considerados generalmente sRlo estaban


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disponibles como proyectos de genomas. Ana lista de los proyectos y completasde genomas de especies 9ersinia se muestra en la Tabla

B mero de proyecto y completos genomas de cada especie 9ersinia en9ersinia>ase

Anotaci3n de secuencias del genoma

#or coherencia" todas las secuencias del genoma descargados fueron anotadosmediante anotaciRn r pida usando Subsistema TecnologQa *@1ST+ @1ST puedepredecir marco de lectura abierto *)@6+" la funciRn de la )@6" la secuencia denucleRtidos y la secuencia de amino cidos en el genoma enviado por el usuario.

1dem s de @1ST" #S)@Tb versiRn 4.3 se utili%R para determinar la locali%aciRnsubcelular de las proteQnas predichas por el motor @1ST. TambiPn se calculR lahidrofobicidad y el peso molecular de las proteQnas predichas-@1ST usandointernamente desarrollado scripts de #erl.

Base de datos y la interfa4 de dise5o !eb

Ana interfa% web fue construida usando (yperTe7t ,arkup 5anguage *(T,5+"(yperte7t #reprocessor *#(#+" DavaScript" j uery" (ojas de Estilo en io#erl y @ idiomas para desarrollar el #G<para comparar entre dos genomas a travPs de la alineaciRn global" #atho#roTpara la generaciRn de mapa de calor para visuali%ar presencia y ausencia degenes de virulencia en los genomas y 9ersiniaTree seleccionados para generar

rbol filogenPtico de las cepas de 9ersinia basados en sus genes de limpie%a yS r@B1. El uso de estos tres lenguajes de scripting populares nos permitiR crear

tuberQas complejas" ue reali%an c lculos de back-end" comunicaciones ayudadosentre el servidor web y el servidor de aplicaciones y tambiPn


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Utilidad y discusi3n

.escripci3n general de la tuber7a de la base de datos

Todos los genomas 9ersinia despuPs de ser descargado desde el iotecnolRgica *B&+ sitio web *http!HHwww.ncbi.nlm.nih.govfueron anotados utili%ando la anotaciRn r pida usando Subsistema TecnologQaservidor *@1ST+

#ara mantener la coherencia. Todos los io-#erl $y tambiPn en la casa script en #erl para obtener informaciRn como el c lculo delcontenido de G< *C+" predijo hidrofobicidad *p(+" y el peso molecular *=a+ de lasproteQnas codificadas. Entonces Toda esta informaciRn se almacena en el servidor de base de datos ,yS 5.

#nterfa4 8eb y funcionalidades de YersiniaBase

1l entrar en la p gina de inicio de 9ersinia>ase" los visitantes pueden ver lasnoticias y conferencias" los blogs y la informaciRn y los m s recientes artQculospublicados relacionados con 9ersinia" ue compilamos manualmente desde variasfuentes. El men VBavegarV permite a los visitantes ver la lista de las especies de9ersinia disponibles actualmente en 9ersinia>ase" con cada botRn V:er


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acceder. El botRn =etalles de cada )@6 conduce al visitante a la p gina V=etalle)@6V mostrar la informaciRn detallada de este )@6 como su tipo" iniciar y detener posiciones" longitudes de nucleRtidos" asQ como secuencias de amino cidos. Se

ofrece m s informaciRn sobre la clasificaciRn funcional" el subsistema *si estdisponible+" filamento" locali%aciRn subcelular" la hidrofobicidad *p(+ y el pesomolecular *=a+. 5a p gina tambiPn muestra el amino cido y la secuencia denucleRtidos de la )@6 junto con el >rowser genoma. El men VGenome >rowserVvincula usuario j>rowse en 9ersinia>ase. D>rowse permite a los usuarios ver laposiciRn de cada )@6 en cada genoma gr ficamente.

TambiPn integramos herramientas internas y asQ como otras herramientas en9ersinia>ase aUadir funcionalidades a this9ersinia plataforma de investigaciRn. Elmen V(erramientasV permite al usuario reali%ar una b s ueda >51ST contra

cepas the9ersinia comisariada en 9ersinia>ase" asQ como b s ueda >51STe7clusivo contra la >ase de =atos factor de virulencia *vfdb+

1dem s de la e7plosiRn de b s ueda" los usuarios tambiPn pueden reali%ar laalineaciRn por parejas de dos genomas 9ersinia presentes en 9ersinia>ase de suelecciRn mediante el uso de la #G


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transcripciRn" locali%aciRn subcelular" hidrofobicidad *p(+" peso molecular *=a+ y elsubsistema de SEE=. 5a p gina tambiPn est e uipado con un navegador

j>rowse genoma basado en DavaScript r pido y moderno ue permitir al usuario

navegar genoma anotaciones y visuali%ar la ubicaciRn de la )@6 de la cepaselected9ersinia. En la parte superior de la p gina" el usuario puede encontrar unbotRn V=escargarV para descargar datos de anotaciRn" la secuencia deamino cidos y la secuencia de nucleRtidos de la )@6 predicho.

Avan4ada la b9squeda en tiempo real

Tiendas 9ersinia>ase grandes cantidades de datos relacionados con la 9ersinia" lamayorQa de ellos son )@6s ue se predijo desde el motor @1ST" con m s de unmillRn de genes y secuencias de codificaciRn. #or lo tanto" no es pr ctico para

navegar por la p gina de informaciRn re uerida por la p gina ya ue estodisminuir grandemente por el progreso.


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genRmica de un solo genoma 9ersinia. #ara una detallada comprensiRn de lasvariaciones entre diferentes cepas a nivel genPtico" los cambios evolutivos entrelas especies de 9ersinia" los genes ortRlogos en especies" asQ como los genes ue

dan a cada organismo sus caracterQsticas nicas" especialmente las regionespotenciales asociadas con la patogenicidad" la comparativa Se re uiere estudio devarios genomas 9ersinia.

ase" el usuario puede elegir dos 9ersiniagenomes de interPs

de la lista de comparaciRn. 1lternativamente" el usuario puede subir su secuenciadel genoma 9ersinia para la comparaciRn con los genomas de 9ersinia en9ersinia>ase. Tres par metros" ue son el porcentaje de identidad mQnimo *C+" sefusionan umbral *bp+ y el umbral de enlace *bp+ se puede definir manualmente por el usuario. Ambral de Enlace *5T+ elimina los enlaces seg n el valor definido por elusuario" mientras ue el umbral de fusiRn *T,+ permite la fusiRn de vQnculosbasados en el valor definido por el usuario.

En la tuberQa #G


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aUade en el diseUo circular generada por #G51ST generados a partir de una b s ueda >51ST contra vfdb basado


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en los valores de corte definidos por el usuario para la identidad de la secuencia yla integridad para identificar los genes de virulencia y selecciona cepas sRlo elusuario deseado. 5os resultados del proceso de filtrado se utili%ar n para tabular

la matri% de datos *cepas frente a genes de virulencia+ Esto es seguido por laejecuciRn de secuencias de comandos @" para leer la matri% de datos generada yreali%ar la agrupaciRn jer r uica *algoritmo completo-vinculaciRn+ de las cepas de9ersinia en funciRn de su perfiles de genes de virulencia. #or ltimo" para lavisuali%aciRn de los perfiles de genes de virulencia" pa uete WpheatmapW se utili%apara generar el mapa de imagen de calor ue es el producto final de #atho#roT. Eluso de mapa de calor en #atho#roT para la visuali%aciRn permite a los usuariostener una vista de p jaro de los datos en una representaciRn gr fica ue permite alos usuarios identificar y visuali%ar el perfil de genes de virulencia en sus cepas deinterPs" y ue permite an lisis comparativos de los factores de virulencia entre losdiferentes 9ersinia cepas

Yersinia'ree: construir Yersenia 2rbol filogenase" hemos diseUadoe incorporado una tuberQa automati%ada escrito en #erl" 9ersiniaTree ue permitira los usuarios para generar rboles filogenPticos de las cepas de 9ersinia enfunciRn de sus genes de limpie%a y S r@B1. Buestra tuberQa automati%ada9ersiniaTree" re uiere principalmente dos entradas desde el usuario! * + marcador

genPtico utili%ado para construir el rbol filogenPtico *2+ 5ista de los genomas en9ersinia>ase para ser incluidos en el rbol. El gasoducto automati%ado tambiPnofrece una caracterQstica opcional" donde los usuarios pueden ingresar susecuencia de nucleRtidos en formato 61ST1" junto con las secuencias ue serecuperan de la base de datos. 1ctualmente" este oleoducto en 9ersinia>ase"permite a los usuarios elegir entre uno de cada cinco marcadores de genes" uese pueden ver en el men desplegable para construir el rbol filogenPtico. 1dem sde los 1@Br S comunes" los usuarios pueden elegir entre cual uiera de lossiguientes gyr>" hsp 3" rpo>" o soda para la construcciRn del rbol filogenPtico.Estudios anteriores han demostrado ue los rboles filogenPticos basados enestos cuatro genes de limpie%a son m s consistentes con los perfiles bio uQmicosde especies de 9ersinia ue los basados en el S r@B1 =espuPs usuarioproporciona todos los insumos necesarios" el #(# frontal ejecuta la canali%aciRn#erl backend. El primer paso de nuestra lQnea automati%ada es seleccionar lasecuencia de nucleRtidos del gen diana de los genomas elegidas por el usuariodesde un archivo 61ST1 donde se almacenan los listados completos de lassecuencias" en un archivo 61ST1 temporal. El script #erl ejecuta entonces ,166T


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ue se utili%a para reali%ar la alineaciRn de secuencias m ltiples a travPs desecuencias de nucleRtidos. 1 continuaciRn" el archivo de salida de ,166T seenvQa a 6astTree? para construir el rbol filogenPtico en formato Bewick" seguido

por la visuali%aciRn del rbol en formato S:G mediante el uso de BewickAtilidades 6inalmente" script #erl envQa la imagen final a #(# para mostrar el rbolfilogenPtico en navegador web.

9ersinia genoma" los genes y factores de virulencia base de datos

1dem s de las herramientas de desarrollo propio" tambiPn hemos integrado>51ST" asQ como vfdb->51ST en 9ersinia>ase. Esto permitir a los usuarios llevar a cabo su b s ueda de similitud de su consulta secuencias secuenciasagainst9ersinia genoma" secuencias de genes y tambiPn contra los factores de

virulencia *descargados de vfdb utili%ando >51STB" >51ST# o >51STF. Estabase de datos especiali%ada para 9ersinia ofrece m s r pido buscar contrasecuencias 9ersinia comparaciRn a bases de datos B& BT o B@ cuando elusuario est tratando de encontrar las cepas m s cercanas 9ersinia a su propiasecuencia de consulta.

0as caracter7sticas de las cepas en YersiniaBase

En general" se observR el tamaUo de los genomas de 9ersinia para estar entre 0"4a 0"N ,bp ,bp. Sin embargo" hubo algunas e7cepciones. #or ejemplo" el tamaUodel genoma de 9. ruckeri 1Tase" 9ersiniaTree y el otro usando el #atho#roTherramienta desarrollada de forma interna. Se utili%R el mPtodo de vecino aparticipar" modelo Tamura-Bei con 333 repeticiones de arran ue para construir el

rbol filogenPtico con ,EG1 basado en el gen gyr> El rbol generado por 9ersiniaTree tambiPn se basR en gen gyr>The gyr> en lugar del gen S r@B1 seutili%R para el an lisis filogenPtico como S r@B1 no puede resolver lasrelaciones filogenPticas entre las especies de 9ersinia estrechamente


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intermedios entre estos dos grupos El rbol tambiPn indica ue 9. pestis 1ngola esel primer lugar entre las cepas de 9. pestis ue se ramifican desde el ancestrocom n m s cercano 9. pseudotuberculosis 9#&&&. Esta observaciRn se apoya en un

informe recientemente publicado" ue declarR ue la 9. pestis 1ngola pertenece auna de las m s antiguas 9. pestis linajes secuenciados hasta la fecha" concaracterQsticas del genoma ue son intermedias entre 9. pseudotuberculosis y la=ayy moderna. #estis Estos datos sugieren ue la 9. pestis es muy similar a la 9.pseudotuberculosis genPticamente y los genes de virulencia ue se perdieron oad uiridos no causaron ue se aparte lejos de su ancestro. Sin embargo" 9.pestishas reducciRn gen sufrido y ha acumulado pseudogenes desde su apariciRnde 9. pseudotuberculosis

=rbol filogen


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articulo yersinia base abstract.docx

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