streptococcus agalactiae is the principal cause of bacterial sepsis , neonatal meningitis and endocarditis in parturient women .
it is also a cause of pneumonia particularly in the immunocompromised elderly ( 1 ) .
s. agalactiae commonly colonizes the gastrointestinal and urogenital tracts of humans ( 2 ) and the proteins enabling the bacteria for colonization , invasion and evasion are poorly defined ( 3 ) . the interaction between s. agalactiae and the epithelial cells
the fibronectin functions as a binding site for s. agalactiae through an insoluble phase ( 4 , 5 ) . a sequence of arginine , glycine and aspartic acid ( rgd ) in fibronectin is also involved in binding to proteins ( 6 ) .
furthermore , c5a peptidase has a high affinity to the rgd sequence of the fibronectin and plays a significant role in adhesion and invasion ( 4 ) .
however , the fibrinogen in s. agalactiae performs key role in attachment ( 7 ) and fbsa and fbsb are recognized fibrinogen - binding proteins ( 2 ) .
the fbsa binds to cell wall covalently because of lpxtg motif while the fbsb seems to be secreted due to lack of such motif ( 8) . both proteins promote the entry of the s. agalactiae into the epithelial cells ( 9 ) .
these enzymes play an important role in remodeling , turnover , division and separation of the cell wall ( 10 ) .
in critical situations , some of the peptidoglycan hydrolases(autolysin ) facilitate self - disintegration(autolysis ) of the peptidoglycan ( 11 , 12 ) .
while there is no clear evidence about the details of autolysin in s. agalactiae , s. aureus ( atl ) contains two major domains of autolysin : amidase and glucosaminidase ( 11 ) .
another autolysin of s. aureus ( aaa ) is bifunctional , and it also mediates adherence to immobilized fibrinogen and fibronectin ( 14 ) .
similar roles were found in other streptococcus species such as streptococcus pneumoniae ( 15 ) .
the s. agalactiae contains proteins which are not fully understood of their roles , like gbs1805 . in this study ,
afb , which is a novel protein encoded by gbs1805 gene in s. agalactiae was cloned , over expressed and purified . in silico analysis of afb
this experimental study was performed on functional analysis of gbs1805 gene of s. agalactiae . the strains and plasmids used in this study
the s. agalactiae isolate were grown on blood agar enrichedby 5% sheep blood . also , escherichia coli strains were cultured overnight on luria bertani ( lb ) ( merck , germany ) broth at 37 c .
bacterial strains and plasmids chromosomal dna of s. agalactiae was extracted using dna extraction kit ( promega , usa ) based on the manufacturer s instructions .
the upstream ( gcgcgccatatgatacatataactatgcagtagatgta ) and the downstream ( gcgcgcctcgagattcggataaatgtagctaactac ) primers ( 20pmol/l ) with the underlined restriction sites were used to amplify the gbs1805 gene at : 95 c for 5 min , 95 c for 1 min , 59 c for 45 seconds ( 30 cycles ) , 72 c for 45 seconds and 72 c for 10 seconds . the amplified gene and pet21a were double digested by ndei and xhoi enzymes ( takara , japan ) and ligated using t4 ligase ( takara , japan ) .
then , the recombinant vector was transformed to escherichia coli dh5. then positive clones were chosen fromthe ampicillin - supplemented lb agar plates and were confirmed by colony pcr and sequencing of plasmid .
the overexpression was optimized at 37 c , 1mm / ml iptg ( fermentas , usa ) for 4 hours oncethe culture has reached od600 of 0.6 .
the recombinant insoluble protein was purified by qiagen purification kit ( qiagen , netherland ) based on the manufacturer s instructions .
the purification process included 4 steps : cell lysis , binding , washing and elution .
12.5% sds - page gel was applied to analyze the purified recombinant protein by coomassie brilliant blue staining .
the membrane was blocked overnight ( 4 c ) with 3% skimmed milk in pbs .
subsequently , it was washed three times in a washing buffer ( tween 20 and pbs ) and incubated in his - tag antibody ( sigma , usa ) diluted in 1:1000 in pbs for 1h at room temperature . afterwards , the membrane was washed three times and dab substrate ( 3 , 3-diaminobenzidine ) ( sigma , usa ) was added to detect the recombinant protein .
western blotting was used to assess binding to biotinylated plasma proteins ( fibrinogen and fibronectin ) .
the recombinant protein was electroblotted on nitrocellulose membrane as previously mentioned . after blocking and washing the membrane ,
it was incubated in plasma protein ( fibrinogen and fibronectin ) diluted in 1:1000 for 3h at room temperature .
then the membrane was washed as before and incubated in streptavidin ( roche , germany ) diluted washing buffer in 1:30000 at room temperature for 3h .
subsequently , the membrane was washed and incubated in alkaline phosphatase buffer for 5 min .
then the membrane was immersedin nbt / bcip ( roche , germany ) solution and kept in dark condition until the protein band appears .
the s. agalactiae isolate were grown on blood agar enrichedby 5% sheep blood . also , escherichia coli strains were cultured overnight on luria bertani ( lb ) ( merck , germany ) broth at 37 c .
chromosomal dna of s. agalactiae was extracted using dna extraction kit ( promega , usa ) based on the manufacturer s instructions .
the upstream ( gcgcgccatatgatacatataactatgcagtagatgta ) and the downstream ( gcgcgcctcgagattcggataaatgtagctaactac ) primers ( 20pmol/l ) with the underlined restriction sites were used to amplify the gbs1805 gene at : 95 c for 5 min , 95 c for 1 min , 59 c for 45 seconds ( 30 cycles ) , 72 c for 45 seconds and 72 c for 10 seconds .
the amplified gene and pet21a were double digested by ndei and xhoi enzymes ( takara , japan ) and ligated using t4 ligase ( takara , japan ) .
then , the recombinant vector was transformed to escherichia coli dh5. then positive clones were chosen fromthe ampicillin - supplemented lb agar plates and were confirmed by colony pcr and sequencing of plasmid .
the overexpression was optimized at 37 c , 1mm / ml iptg ( fermentas , usa ) for 4 hours oncethe culture has reached od600 of 0.6 .
the recombinant insoluble protein was purified by qiagen purification kit ( qiagen , netherland ) based on the manufacturer s instructions .
the purification process included 4 steps : cell lysis , binding , washing and elution .
12.5% sds - page gel was applied to analyze the purified recombinant protein by coomassie brilliant blue staining .
the membrane was blocked overnight ( 4 c ) with 3% skimmed milk in pbs .
subsequently , it was washed three times in a washing buffer ( tween 20 and pbs ) and incubated in his - tag antibody ( sigma , usa ) diluted in 1:1000 in pbs for 1h at room temperature . afterwards , the membrane was washed three times and dab substrate ( 3 , 3-diaminobenzidine ) ( sigma , usa ) was added to detect the recombinant protein .
western blotting was used to assess binding to biotinylated plasma proteins ( fibrinogen and fibronectin ) .
the recombinant protein was electroblotted on nitrocellulose membrane as previously mentioned . after blocking and washing the membrane ,
it was incubated in plasma protein ( fibrinogen and fibronectin ) diluted in 1:1000 for 3h at room temperature .
then the membrane was washed as before and incubated in streptavidin ( roche , germany ) diluted washing buffer in 1:30000 at room temperature for 3h .
subsequently , the membrane was washed and incubated in alkaline phosphatase buffer for 5 min .
then the membrane was immersedin nbt / bcip ( roche , germany ) solution and kept in dark condition until the protein band appears .
the gbs1805 gene was amplified by pcr ( fig . 1 ) and cloned into pet21a in e. coli dh5 as cloning host .
then , the constructed plasmid was transformed into bl21 and expressed as a hexahistidyl - tagged protein .
the recombinant protein was successfully purified using ni - nta ( qiagen , netherland ) , as predicted by bioinformatics analysis in jcvi .
a full sized 18 kda mature protein was indicated by the sds- page ( fig .
2 ) . then , the purified his6-tagged recombinant protein was confirmed by anti his - tag western blot experiment ( fig .
( m ) marker 100bp ( geneon , usa ) , ( 1 ) positive control streptococcus agalactiae nem316 , ( 24 ) clinical isolates
( m ) : unstained protein marker ( fermentas , usa ) , lane1 : purified recombinant protein ( 18 kda ) .
lane1 : prestained marker ( sinaclon , iran ) , lane2 : recombinant protein ( 18 kda ) .
lane 1 : prestained marker ( sinaclon , iran ) , lane 2 : positive control ( ant ii ) , lane 3 : recombinant protein ( 18 kda ) .
lane 1 : prestained marker ( sinaclon , iran ) , lane 2 : positive control ( ant ii ) , lane 3 : recombinant protein ( 18 kda ) .
attachment to specific receptors is the initial step in the pathogenesis of gram - positive bacteria ( 16 ) .
the most common receptors mediating adherence to epithelial cells including fibrinogen and fibronectin are parts of the extra cellular matrix ( ecm ) ( 2 , 17 ) and fibrinogen and fibronectin are known to be the major components of the ecm in a number of gram - positive bacteria ( 18 , 19 ) . in this study , afb ( autolysin , fibrinogen / fibronectin binding ) , is identified to be a novel protein of group b streptococcus capable of bindingto fibrinogen and fibronectin . s. agalactiae interacts with fibrinogen via a variety of proteins including fbsa , fbsb , fbsc , srr1 and srr2 ( 2 , 8 , 20 , 21 ) .
fibrinogen is expressed on epithelial and endothelial cell surfaces and interferes complement activation ( 22 ) , phagocytosis and polymorphonuclear activation ( 23 ) . since fbsa protects s. agalactiae against opsonophagocytosis ( 9 ) , deletion of fbsa leads to reduced proliferation of the bacterium in blood ( 7 ) .
the interaction of srr1 and fibrinogen contribute to pathogenesis and plays key role in attachment to brain microvascular endothelial cells ( 24 ) .
the binding ability to epithelial and endothelial cells , biofilm formation and invasion decreases significantly in fbsc - negative mutants ( 20 ) .
furthermore , fbsa and fbsb also participate in evasion of host defense ( 23 ) .
fibronectin , another component of ecm , produce - sa549 on cell surfaces in the respiratory tract and on vaginal and cervical cells ( 6 , 25 ) .
the well - defined fibronectin binding protein of group b streptococcus encoded by scpb gene , known asc5a peptidase , adheres to epithelial cells and involved in evasion ( 4 ) .
however , the scpb defective mutant s. agalactiae s attachment to fibronectin significantly decreaseswhen compared to the wild strain ( 6 ) .
in addition to the binding activity of afb , it seems to be a peptidoglycan hydrolase .
the possible role of the afb in the attachment process and its substantial pathogenicity is not well recognized due to the lack of afb - defective mutant s. agalactiae .
commonly , peptidoglycan hydrolases deal with cell separation , turn over , remodeling , and degradation of cell wall .
evidence shows that peptidoglycan hydrolases , autolysins , are involved in the pathogenesis and virulence of gram - positive bacteria by mediating bacterial adherence ( 26 , 27 ) .
to date numerous autolysin / adhesion proteins including aaa , atl , aas , aae , lytm , amic , etc which confirmed binding to ecm are described undergram - positive bacteria ( 11 , 1315 , 28 ) . in vivo studies
demonstrated that autolysin - defective mutants to be less virulent than wild type strains of listeria monocytogenes , s. aureus , s. pneumonia , and s. epidermidis ( 2931 ) . in silico studies of afb demonstrated high homology to amidase , and zymography confirmed the activity in vitro ( fig 5 ) .
therefore , afb can be a bifunctional protein , based on its binding affinity to two plasma proteins and amidase like activity .
the afb is a novel protein encoded by gbs1805 in s. agalactiae and is capable to bind with the plasma proteins - fibrinogen and fibronectin .
the role of the protein in adhesion and colonization of the bacterium is unknown and needs further investigation .
 background and objectives : streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis in newborns and results in pneumonia and bacteremia in adults . 
 a number of s. agalactiae components are involved in colonization of target cells . 
 destruction of peptidoglycan and division of covalently linked daughter cells is mediated by autolysins . in this study , 
 autolytic activity and plasma binding ability of afb novel recombinant protein of s. agalactiae was investigated.materials and methods : the gbs1805 gene was cloned and expressed . 
 e. coli strains dh5 and bl21 were used as cloning and expression hosts , respectively . after purification , antigenicity and binding ability to plasma proteins of the recombinant protein 
 was evaluated.results:afb , the 18kda protein was purified successfully . 
 the insoluble mature protein revealed the ability to bind to fibrinogen and fibronectin . 
 this insoluble mature protein revealed that it has the ability to bind to fibrinogen and fibronectin plasma proteins . 
 furthermore , in silico analysis demonstrated the afb has an autolytic activity.conclusions:afb is a novel protein capable of binding to fibrinogen and fibronectin . 
 this findings lay a ground work for further investigation of the role of the bacteria in adhesion and colonization to the host . 
INTRODUCTION
MATERIALS AND METHODS
Bacterial strains, plasmids and culture conditions.
PCR amplification of the 
Cloning and Overexpression.
Protein purification.
SDS-PAGE, Western blotting.
RESULTS
DISCUSSION
CONCLUSION