genome analysis of selected foodborne pathogens for...

1134Current Trends in Biotechnology and PharmacyVol. 5 (2) 1134-1148 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)

Genome analysis of selected foodborne pathogens

AbstractAs the bacterial pathogens are showing

growing resistance against existing antibiotics,there is an urgent requirement of pharmaceuticalcompanies for designing new and effective drugsagainst these pathogens causing commonfoodborne diseases. We have performed in-silicogenomic analysis of two virulent foodbornepathogens Listeria monocytogenes and Bacilluscereus for identification of potential drug targetsfor novel drug discovery. Our prediction foundsmall number of genes of each pathogen to betargeted for drug designing. The gene productsare found to be involved in various importantregulatory proteins, membrane proteins andhypothetical proteins of unknown function ofbacterial species. Further using KEGG pathwayprediction various enzyme proteins were foundwhich have active role in biosynthetic andmetabolic pathways. The primary goal of thisstudy was to provide a valuable resource of drugtargets to the scientific community. The currentdata is included in “Drug Targets ProteinDatabase” developed by our group.

Key word: Antimicrobial drugs; Essentialproteins; Drug targets.

IntroductionBurden of microbial diseases and

simultaneously more antibiotic resistance shown

by bacteria on existing antibiotic drugs needs adifferent approach to find drug targets fordestroying bacteria more effectively. Thedevelopment of effective antimicrobial drugs isthe major challenge in field of pharmacogenomics.Growing number of microbial genome sequencingprojects have generated large number ofsequences. Recently, a number of computationaltools have been developed for in silico analysisof gene sequence information (1). The genomeinformation is also useful to identify potential andessential candidate of the microbial systems (2).A good target is a gene essential for bacteria tosurvive, yet cannot be found in the mammalianhost (3). An ideal drug target should provideadequate selectivity yielding a drug which isspecific or highlys elective against the pathogenwith respect to the human host. Moreover, thetarget should be involved in viability, growth andother essential metabolic processes of thepathogen at least under the condition of infection(4). Most of the previous research is revealedwith identification of essential gene on the basisof experimental data acquired from genedisruption and systematic mutagenesis studies onthe bacterium (5). There is huge chance of lethalaction on bacteria by inactivation of essentialgenes. Targeting non human homologous genesare much important to avoid possibilities ofunacceptable crossreactivity that might causes

Genome Analysis of Selected Foodborne Pathogens forIdentification of Drug Targets

Sushil Kumar Shakyawar, Arun Goyal and Vikash Kumar Dubey*

Department of Biotechnology, Indian Institute of Technology Guwahai,Guwahati-781 039, Assam, India

*For Correspondence - [email protected]


Sushil Kumar Shakyawar et al

side effect to the host (6). We have performedin-silico genomic analysis of virulent foodbornepathogens Listeria monocytogenes and Bacilluscereus for identification of potential drug targetsfor novel drug discovery. Listeriamonocytogenes causes Listeriosis a virulentfoodborne clinical infection. Similarly, Bacilluscereus a, Gram-positive, rod-shaped, betahemolytic bacterium causing causing severenausea, vomiting and diarrhoea. These data couldbe used for the identification of conserved proteinsequences which are essential for bacterialsurvival (7). These conserved proteins are veryimportant in finding drug targets in pathogens, idealantimicrobial targets should be highly conservedamong as well as essential for the pathogenicbacteria (8).

MethodsThe BLAST executable of protein

sequences for these two bacterial species weredownloaded from NCBI By using CD-HITparalog sequences at 60% identity were excludedfrom further study as earliesr described (9, 10).The essential gene sequences were taken formfrom the Database of Essential Gene (DEG). Nonhuman homologous sequences were identified byBLASTP analysis of non redundant sequencesat E-value cut-off of 10-3 against human proteinsequences (9, 10). Non human homologoussequences were aligned with sequencesdownloaded from DEG database using BLASTPat an E-value cut-off of 10-10 and 30% identity.The resultant protein sequences were consideredas essential drug target proteins. Further using

KEGG (http://www.genome.jp/kegg.html.)pathway prediction these essential drug targetproteins were analyzed whether these are activerole in various metabolic and cellular pathways.

Results and DiscussionBacteria are the greatest threat to food

safety and may cause food poisoning. Listeriamonocytogenes is a causative agent of Listeriosis,a most virulent foodborne clinical infection andleading cause of death among foodborne bacterialpathogens (11). The majority of human listeriosiscases occur in neonates, immunosupressed,pregnant women and old people. This is a majorthreat to human health (12). The recent studydescribes the way Listeria grabs onto molecularhandles on cells in the small intestine and thenswitches on those cells’ own uptake systems tohitch a ride inside (13). Similarly, Bacillus cereusis a, Gram-positive, rod-shaped, beta hemolyticbacterium causing nausea, vomiting and diarrhea.Generally speaking, Bacillus foodborne illnessesoccur due to survival of the bacterial endosporeswhen food is improperly cooked.

The computational analysis of fullgenome sequences for identification of probableantimicrobial drug targets has become a new trendin pharmacogenomics. Two bacterial pathogenscausing foodborn human disease (Table1) werecovered for genome analysis and summary of theresults are listed in Table 1. The current studyhas been included in our database (14).

List of all drug targets for each pathogenis listed in Tables 2 & 3. We got adequate number

Table 1. Summary of genome analysis of selected bacterial species

Species Discase causedTotal No. of Non-paralogs Non human Number ofprotein coding homologous proteins withsequence proteins matches

in DEG(Essentialprotiens)

Bacillus cereus B. cereus food poisoning 5754 5486 4256 99Listeria maonocytogenes Listeriosis, perinatal septicemia, meningitis, 2846 2814 1973 82

encepthalitis, intrauterine infections



Tabl

e 2.

Lis

t of

pote

ntia

l dru

g ta

rget

s in

Bac

illus

cer

eus

S.G

ene

IDQ

uery

gen

e na

me

Sim

ilar

Ess

enti

al G

ene

in E

ssen

tial

Gen

e D

atab

ank

Gen

e na

me

Fun

ctio

nsN

o.

(DE

G)



Tabl

e 3.

Lis

t of

pote

ntia

l dru

g ta

rget

s in

Bac

illus

cer

eus

S.N

o.G

ene

IDQ

uery

gen

e na

me

Sim

ilar

Ess

enti

al G

ene

in E

ssen

tial

Gen

e D

atab

ank

G

ene

nam

eF

unct

ions

(D

EG)

1144


Current Trends in Biotechnology and PharmacyVol. 5 (2) 1134-1148 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)

1145



Tab

le 4

. L

ist

of p

roti

ens

(man

ily

enzy

mes

) ha

ving

act

ive

role

in

bios

ynth

etic

and

met

abol

ic p

athw

ays

of B

acil

lus

cere

us

S.N

o.

Gen

e ID

Prot

ein

nam

eR

ole

EC

num

ber

1146

of promising drug target protein in each species.Finally 82 genes in Listeria monocytogenes and99 genes in Bacillus cereus were found whichcan be used as promising drug targets. The targetsgenes identified are involved in regulator protein,membrane proteins and hypothetical proteins ofunknown function of bacterial species. No similarprotein is functional in human. Further KEGGpathway prediction shows that 20 proteins inBacillus cereus and 9 proteins in Listeriamonocytogenes were found to have active rolein biosynthetic and metabolic pathways (Table 4and Table 5). The data for total number of genesequence following non human homologoussequences and essential genes are shown in Table 1.

Recent publication of genome sequencesof many organisms and several newbioinformatics tools have facilitated identificationof antimicrobial drug targets in pharmacoge-nomics. In recent study, RecA was found animportant factor in DNA repair and the activatorof the SOS response that contributes to theresistance against acid and bile and to the abilityof L. monocytogenes to adhere and invade humanintestine epithelial cells (15). Present study reveals



Table 5. List of protiens (manily enzymes) having active role in biosynthetic and metabolic pathways of Bacillus cereusS.No. Gene ID Protein name Role EC Number

with in silico analysis of essential genes as apreliminary step to screen through genome ofthese pathogens. We found some enzyme proteinsactively involved in various processes like DNAreplication, recombination, repairing etc. thusthese enzyme/proteins can be used to deactivateRecA to inhibit further growth of L.monocytogenes. On average very less numberof genes was annoted as essential genes so toallow for experimental analysis, leading to asystematic strategy in designing novelantimicrobial active compounds. These genes areof interest for further characterization tounderstand role of these gene in survival ofspecies, as multi-drug target as they are involvedin regulator protein, membrane proteins involvedin biosynthetic and metabolic pathways andhypothetical proteins of unknown function ofbacterial species. The compounds active on thesegenes are expected to be more effective for lethalaction on bacteria without affecting host body.The long term goal of the study is to analyse severalpathogens genome and prepare an extensivedatabase of essential drug target proteins databseas scientific resource.



Acknowledgment Infrastructural facilities provided by Indian

Institute of Technology Guwahati and Financialsupport by Department of Biotechnology,Government of India are acknowledged.

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