
 bacterial strains and growth conditions - c. diphtheriae strains used 
 in this study and their characteristics are listed in table i. these microorganisms were maintained in trypticase soy broth ( tsb ) 
 ( bd difco , usa ) at 37c and stored in the same medium with 20% glycerol . 

escherichia coli top10 electrocomp ( thermo fisher scientific inc 
 invitrogen , usa ) and e. coli op50 were grown in luria 
 bertani ( bd difco ) medium at 37c .
when appropriate , kanamycin 
 ( sigma - aldrich co , usa ) was added ( 50 g ml ) . 

table ibiological properties of corynebacterium 
 diphtheriaestrains used in this studystrainoriginbiovar/ sucrose 
 fermentation 
 tox gene 

 tehak2teo3 mic ( mg / ml-)k2teo3inhibition halo ( mm)bath ( % ) glass/ polystyrene 
 adherencehep-2 cells adherence 
 patternreferencesldcic - l1cdc - e8392::pcr2.1-topocdc - e8392_813mitis / suc -+interrupted0.3127  0.41 
 51  0.82- /++dathis papercdc - e8392diphtheria / pharynx ( usa)mitis / suc -++0.6223  1.1551  1.20- /++da 
 mattos - guaraldi et al .
( 2009 ) 

 a : toxicity evaluated by polymerase chain reaction for 
 tox gene ( pimenta et al . 

2008 ) ; b : p < 0.05 according to unpaired 
 t test ; bath : bacterial adherence to 
 n - hexadecane ; cdc : centres for disease control and prevention ; da : 
 diffuse adherence [ characterised by bacteria randomly distributed over the 
 surfaces of the human epithelial type 2 ( hep-2 ) cells ] ; 
 k2teo3 : potassium tellurite ; mic : minimal 
 inhibitory concentration ; - : negative ; + : positive ; + + : moderately adherent 
 to glass or polystyrene . 
 

a : toxicity evaluated by polymerase chain reaction for 
 tox gene ( pimenta et al . 

2008 ) ; b : p < 0.05 according to unpaired 
 t test ; bath : bacterial adherence to 
 n - hexadecane ; cdc : centres for disease control and prevention ; da : 
 diffuse adherence [ characterised by bacteria randomly distributed over the 
 surfaces of the human epithelial type 2 ( hep-2 ) cells ] ; 
 k2teo3 : potassium tellurite ; mic : minimal 
 inhibitory concentration ; - : negative ; + : positive ; + + : moderately adherent 
 to glass or polystyrene . 
 in silico search and three - dimensional model prediction of the putative 
 te 
  protein - anin silico search for resistance 
 determinants in c. diphtheriae strains
a hypothetical 
 protein included in the teo3 
  resistance / dicarboxylate transporter family was identified in all 
 sequenced strains .
the phyre2 server was used to predict the three - dimensional structure of the putative 
 te protein of c. diphtheriae ( kelley & sternberg 2009 , guo et 
 al .
two parameters were considered 
 to select the best model : confidence and coverage . 

disruption of the putative te 
 gene - for the chromosomal disruption of the c. 
 diphtheriae putative te gene , the topo ta 
 cloning kit ( thermo fisher scientific inc invitrogen ) was 
 used .
the cdc - e8392 strain was chosen as template and a 207 bp internal dna fragment 
 from cdce8392_0813 gene was amplified via polymerase chain reaction 
 ( pcr ) with the following primer pair : 5-tcgttttatgcgggtgc-3 and 
 5-ggtgtgcgcaatctgatg-3. amplification was performed with an initial denaturation step 
 at 94c for 2 min , followed by 35 cycles of denaturation at 94c for 1 min , annealing at 
 60c for 1 min and extension at 72c for 1 min and 30 s and a final elongation step at 
 72c for 10 min .
the dna fragment was ligated to the overhanging 3-deoxythymidine of linearised 
 pcr2.1-topo vector provided in the kit .
pcr2.1-topo- 
 cdce8392_0813  was propagated in e. coli top10 electrocomp according 
 to the manufacturer s instructions and isolated using the purelink quick dna miniprep 
 kit ( thermo fisher scientific inc invitrogen ) .
five micrograms of 
 unmethylated plasmid isolated from this e. colistrain was used to 
 transform c. diphtheriae using a genepulser ii ( bio - rad laboratories 
 inc , usa ) , as previously described ( dorella et al . 

the electroporated cells were added to 1 ml of tsb and incubated for 4 
 h at 37c .
the culture was plated onto a tsb agar plate containing kanamycin and 
 maintained at 37c for 48 h. because pcr2.1-topo can not autonomously replicate in 
 c. diphtheriae , kanamycin - resistant c. diphtheriae 
 carried the vector , integratedvia recombination into the chromosomal 
 cdce8392_0813 gene and designated ldcic - l1 . to confirm the insertion of the plasmid into the chromosome of the c. 
 diphtheriae strain cdc - e8392 , pcr reactions using primers aligned at the 
 start and stop codons of the cdce8392_0813 gene (
5-tcgtatcaatgcgtgccacag-3 and 
 5-aatcgtgcgctgccatacatg-3 ) and primers aligned within the inserted plasmid [ kan#1 
 5-atgattgaacaagatggattg-3/kan#2 5-ttaataattcagaagaactc-3 and m13f / m13r ( life 
 technologies ) ] were performed as previously described ( dorella et al .
teo 
 3 
 susceptibility assays - potassium tellurite 
 ( k2teo3 ) sensitivity was evaluated through determination of the 
 minimal inhibitory concentration ( mic ) by using the disk diffusion method according to 
 the protocols of the clinical laboratory standards institute ( clsi ) for antimicrobials 
 ( clsi 2013 ) .
the mic of 
 k2teo3 was determined using bacterial cells grown for 48 h at 
 37c in trypticase soy agar ( bd difco ) suspended in mller hinton broth ( bd 
 difco ) to a final concentration of 10 colony forming units 
 ( cfu ) mland diluted 1,000-fold into medium supplemented with various 
 concentrations of k2teo3 ( sigma - aldrich co ) ranging from 0.0 - 2.5 
 mg / ml . after incubation for 48 h at 37c , the growth was visually 
 assessed .
the disk diffusion assay was performed with 10 cfu ml-1 
 bacteria spread onto cation - adjusted mller - hinton agar plates ( bd 
 difco ) .
after 48 h at 37c , the diameters of the zones were 
 determined . 

antimicrobial susceptibility testing - the sensitivity to antimicrobial 
 agents ( oxoid ltd , uk ) , penicillin ( 10 g ) , erythromycin ( 15 g ) , ampicillin ( 10 g ) , 
 gentamicin ( 10 g ) , cefotaxime ( 30 g ) , imipenem ( 10 g ) , ciprofloxacin ( 5 g ) , 
 clindamycin ( 2 g ) , rifampicin ( 30 g ) , tetracycline ( 30 g ) , linezolid ( 30 g ) and 
 vancomycin ( 5 g ) , was determined using the disk diffusion method according to clsi ( 2013 ) guidelines , as previously described 
 ( pereira et al .
the inoculum equivalent 
 to a 0.5 mcfarland standard was placed on the surface of a cation - adjusted mller - hinton 
 agar containing 5% sheep blood .
the plates were subsequently incubated at 37c for 24 h 
 and reconfirmed at 48 h. breakpoints for the susceptible strains were used in accordance 
 with the clsi for bacteria excluded from table 2a - j . because there is no defined 
 standard for interpreting these results ,
the breakpoints for s. 
 aureus , established by clsi , were considered in the cases of penicillin . 

agar diffusion growth inhibition assays for susceptibility to h 
 2 
 o 
 2- the sensitivity to h2o2 was assessed based on the methods 
 ofkim and holmes ( 2012 ) with some adaptations . 

approximately 10 cfu ml were spread onto the surface of 
 cation - adjusted mller - hinton agar plates .
a 10-l aliquot of 20 mm 
 h2o2 was spotted onto the centre of the plate and the diameter 
 of the growth inhibition zone was measured after an incubation period of 24 - 48 h. 
 nematode infection model - the assays were performed as previously 
 described with some adaptations ( browning et al . 

briefly , caenorhabditis elegans n2 was maintained on 
 plates containing nematode growth medium ( ngm ) agar for approximately six - seven days at 
 20c and used in infection assays with wild - type ( wt ) and mutant ( ldcic - l1)c . 

twenty l4 stage larval worms were infected with 20 l 
 of each bacterial strain ( obtained from an overnight culture ) on ngm plates at 20c for 
 24 h. the worms were assessed daily following infection and the dead nematodes were 
 counted and removed every 24 h for seven days . for each strain ,
biofilm formation on polystyrene surfaces - biofilm formation was 
 determined in 96 well flat - bottomed polystyrene microtitre plates based on previously 
 described methods ( gomes et al .
briefly , 
 200 l of the bacterial suspension with an optical density ( od ) of 0.2 (  = 570 nm ) in 
 tsb medium was applied to each well of the microplate .
after an incubation period of 48 h at 37c , the content of 
 each well was aspirated and washed with 0.01 m phosphate - buffered saline ( pbs ) ( ph 7.2 ) . 

the remaining attached bacteria were fixed with 99% methanol and stained with 2% v / v 
 crystal violet .
the bound dye was subsequently solubilised using 33% glacial acetic acid 
 and the od of the solution was measured at 570 nm using an enzyme immunosorbent assay 
 reader ( biorad laboratories inc ; model 550 ) . the cut - off od ( odc )
the strains were classified into the following 
 categories : nonadherent ( 0 : od  odc ) , weakly adherent ( + : odc < 
 od  2x odc ) , moderately adherent ( + + : 2x odc < od
 4x 
 odc ) or strongly adherent ( + + + : 4x odc  od ) . 

bacterial adherence to n - hexadecane ( bath ) assays - the evaluation of 
 bacterial adhesion to n - hexadecane was performed as previously 
 described ( mattos - guaraldi et al .
strains 
 with bath values > 50% were considered highly hydrophobic , 30%  bath values < 50% 
 were considered moderately hydrophobic and bath values < 30% were considered 
 hydrophilic . 

biofilm formation on glass surfaces and haemagglutination assays - the 
 haemagglutination activity of human b erythrocytes ( 0.5% ) and bacterial adhesion to 
 glass surfaces were both assayed using previously described methods ( mattos - guaraldi & formiga 1991 ) .
the 
 microorganisms were classified into the following categories for their ability to adhere 
 onto glass : + + + ( strong ) , confluent coat of cells on the sides of the tube and localised 
 adherence ( la ) onto the glass surface where the culture medium is in contact with the 
 air , + + ( intermediate ) , confluent coat of cells on the sides of the tube , + ( weak ) , la 
 onto the glass surface where the culture medium is in contact with air , and - 
 ( negative ) , no visible adherence . 

human epithelial type 2 ( hep-2 ) cell interaction assays - the cellular 
 interaction assays were performed using epithelial cells derived from a human epidermoid 
 larynx carcinoma ( hep-2 ) according to previously described protocols ( hirata jr et al .
briefly , 
 microorganisms grown in tsb were used to infect monolayers of hep-2 cells grown to 
 approximately 95% confluence .
after 3 h of interaction , the infected monolayers were 
 washed with 0.01 m pbs ( ph 7.2 ) , lysed with 0.1% triton x-100 ( sigma - aldrich co ) in pbs , 
 diluted and plated .
viable bacterial counts in the supernatant and cellular monolayer 
 lysates were subsequently determined . to determine the viable intracellular bacteria , 

the monolayers were treated with 150 g ml of penicillin ( sigma - aldrich co ) 
 for 1 h. the adherence pattern assays were performed using semi - confluent hep-2 
 monolayers grown on circular coverslips ( 13 mm diameter ) . at 3 h post - infection ,
the 
 giemsa - stained coverslips were examined using bright field microscopy and the observed 
 strains were classified into the following patterns : la , characterised by small clusters 
 of bacteria resembling micro - colonies , diffuse adherence ( da ) , characterised by bacteria 
 randomly distributed over the surfaces of the hep-2 cells , or aggregative adherence 
 characterised by clumps of bacteria with a  stacked - brick  appearance . 
 statistical analysis - each experiment was conducted in triplicate and 
 statistical analyses were performed with the appropriate tests using a graphpad prism 
 5.0 ( graphpad , usa ) .

 cdce8392_0813 protein represented a te 
 protein teha homolog - the structure and function of the putative 
 te protein in c. diphtheriae cdc - e8392 ( cdce8392_0813 
 protein ) were predicted using a protein homology / analogy recognition engine ( phyre ) . the 
 best model selected by the phyre2 server is displayed in fig .
the 302-residue protein was modelled with 100% confidence and 84% 
 coverage ( 302 residues of the sequence ) . according to analyses of this protein ,
a : best model for the 
 cdce8392_0813 protein ( 100% confidence and 84% coverage ) ; b : conserved regions 
 of cdce8392_0813 protein . 
 
 cdce8392_0813 interruption rendered the mutants more susceptible to k 
 2 
 teo 
 3 
 and h 
 2 
 o 
 2 
 toxicity , but did not modify antimicrobial susceptibility profiles - 
 the te resistance level of the mutant ldcic - l1 
 ( cdc - e8392::pcr2.1-topocdc - e8392_813 ) , constructed viahomologous 
 recombination , was determined and compared with that of the wt strain ( cdc - e8392 ) . an 
 increase ( 2x ) in the susceptibility to teo3 
 was observed for the mutant ( mic = 0.3125 mg / ml ) .
furthermore , 
 in disk diffusion assays , ldcic - l1 presented a 27.00 mm (  0.41 ) growth inhibition zone , 
 which was significantly different ( p = 0.0006 ) from the 23 mm (  1.15 ) diameter 
 inhibition zone of the wt strain .
a significant difference was also observed for the 
 sensitivity to h2o2 ( p = 0.0019 ) .
the wt strain presented a 22.00 
 mm (  0.82 ) growth inhibition zone , while the mutant presented a 24.70 mm (  0.64 ) 
 inhibition zone ( table ii ) . 

table iieffects of corynebacterium diphtheriae cdce8392_0813 gene 
 interruption on human cells interactions and on susceptibility to hydrogen 
 peroxidestrainha titrehuman epithelial type 
 2 epithelial cell 
 h2o2 inhibition halo ( mm)adherence 
 patternbacteria in 
 supernatant ( cfu ml)adherent bacteria 
 ( cfu ml)internalised bacteria 
 ( cfu ml)ldcic - l132da11.8 x 10 2.235.8 x 10 1.947.0 x 10 0.05 
 24.75  0.64 
 cdc - e839232da11.3 x 10 1.115.3 x 10 1.3010.0 x 10 0.5722.00  0.82 

a : p < 0.05 according to unpaired 
 ttest ; cfu : colony forming units ; da : diffuse adherence ; ha : 
 agglutination of human erythrocytes ( type b ) . 
 
 a : p < 0.05 according to unpaired 
 ttest ; cfu : colony forming units ; da : diffuse adherence ; ha : 
 agglutination of human erythrocytes ( type b ) .
antimicrobial susceptibility assays demonstrated identical profiles for c. 
 diphtheriae wt cdc - e8392 and ldcic - l1 mutant strains .
both strains were 
 determined to be susceptible to penicillin , erythromycin , ampicillin , gentamicin , 
 cefotaxime , imipenem , ciprofloxacin , clindamycin , rifampicin , tetracycline , linezolid 
 and vancomycin . 

cdce8392_0813 interruption affected the ability of c. diphtheriae to kill c. 
 elegans and survive within hep cells - the results of the experiments using 
 the nematode c. elegans revealed that the mutant strain ldcic - l1 
 exhibited an attenuated killing ability when compared with the wtc . 

2 ) . 
 the data shown in table ii also indicated a 
 reduced ability of the mutant to survive in the intracytoplasmic compartment of hep-2 
 cells ( p = 0.0391 according tot test ) . strains cdc - e8392 and ldcic - l1 
 reached internalisation rates of 1.89  0.15% and 1.21  0.35% , respectively . 
 fig .
the kaplan - meier statistical test was used to determine the 
 probability of nematode survival .
survival curves were compared using the log 
 rank test and considered statistically different ( p < 0.05 ) . 
 

cdce8392_0813 interruption did not affect the hydrophobicity and adherence 
 properties of c. diphtheriae - the ability of c. 
 diphtheriae ldcic - l1 to adhere to and to form a biofilm on abiotic surfaces 
 was evaluated using glass and polystyrene adherence assays .
simultaneously , the surface 
 hydrophobicity of this strain was analysed through bath assays and haemagglutinating 
 activity .
the mutant showed nonadherence to the glass and moderate adherence to 
 polystyrene surfaces ( table i ) .
the mutant also 
 preferred a hydrophobic surface ( % bath = 51 ) ( table 
 i ) and the displayed haemagglutinating activity ( titre = 32 ) ( table ii ) .
the adherence of the mutant strain to hep cells was verified using the hep-2 lineage ; 
 the results are displayed in table ii .
similar to 
 the wt strain , the mutant strain showed a da pattern with a moderate degree of adherence 
 ( 31.9% ) after 3 h of interaction .
the adherence values were not considered to be 
 significantly different from the parental strain according to unpaired t 
 test ( p > 0.01 ) .
a number of genetic te determinants have been identified in different 
 bacterial species ( taylor et al . 1994 , liu & taylor 1999 , toptchieva et al . 2003 ) .
one of the chromosomal 
 tedeterminants is the operon tehab , originally described 
 ine .
homologues and orthologues of theteha 
 gene have been identified in other bacterial species , such asvibrio cholerae , 
 klebsiella pneumoniae , salmonella enterica andcorynebacterium 
 glutamicum ( chasteen et al . 2009 , 
 pei et al . 2013 ) .
recent studies have revealed 
 that this determinant did not confer the temechanism of v. 
 cholerae c6706 , but was found involved in antibiotic resistance and 
 intestinal colonisation ( pei et al .
2013 ) . for c. diphtheriae , a protein with similar sequence to teha in other 
 species was identified .
this protein , referred to as cdce8392_0813 in cdc - e8392 strain , 
 was predicted as a te protein teha homologue using phyre software . in the 
 present study , the contribution of te to c.
diphtheriae 
 pathogenesis was verified using the cdce8392_0813 mutant ( ldcic - l1 ) constructed through 
 chromosomal disruption . according to phyre
, the interrupted region of the cdce8392_0813 
 gene likely corresponded to the protein active site because it contained many conserved 
 residues .
gene complementation was not performed in the present study and might be 
 considered to be a limitation of this work .
the influence of the cdce8392_0813 protein in the te mechanism of strain 
 cdc - e8392 was also documented in the present study .
two different protocols used to 
 investigate the viability of ldcic - l1 in the presence of teo3 
  revealed that this gene interruption rendered the mutant more susceptible 
 to this compound . it has been previously suggested that te depends on the 
 expression of different enzymes involved in several bacterial functions , including 
 nitrate reduction , oxidative stress response and phosphate and cysteine metabolism 
 ( taylor 1999 , chasteen et al .
2014 ) . however , the data obtained in the present study suggest that 
 cdce8392_0813 participates in c. diphtheriae te and that 
 this mechanism relies on other bacterial factors .
the data also indicate that additional 
 studies are needed to identify and characterise these other bacterial factors .
a recent report indicated that teha protein participates in v. cholerae 
 antimicrobial resistance ( pei et al .
in 
 contrast , the findings presented here indicated that the teha homologue is not involved 
 in the susceptibility of c. diphtheriae to the antimicrobial agents 
 tested . it has previously been suggested that teo3 
  toxicity results , at least in part , from the generation of ros including 
 h2o2 .
however , teo3 
  resistance is likely mediatedvia resistance to oxidative 
 damage rather than the detoxification of the metal oxide itself ( toptchieva et al .
similar results were observed with a haemophilus influenzae mutant for 
 tehb ( whitby et al . 

because the participation of teha in bacterial pathogenesis has been described for other 
 species , we evaluated the involvement of the putative tedeterminant 
 cdce8392_0813 in the virulence of diphtheria bacilli . using c. elegans 
 as an infection model
, we considered a simple but versatile animal model for analysing 
 the virulence of bacteria , including c. diphtheriae ( broadway et al . 2013 ) .
herein , we demonstrated an 
 attenuated ability of the ldcic - l1 mutant to kill nematodes .
similarly , mutations in the 
 te genes ofbacillus anthracis ( ycegh ) 
 also reduced survival in c. elegans and increased susceptibility to 
 teo3 
  compounds ( franks et al .
in addition to diphtheria toxin , adherence 
 factors such as glycoconjugates , haemagglutinin and pili have been well studied .
these 
 factors might be involved in biofilm formation on abiotic surfaces and/or adherence to 
 hep cells and extracellular components ( mattos - guaraldi 
 et al .
although these studies have demonstrated that a ter 
 determinant might assist e. coli o157:h7 in establishing an 
 infection through participation in the adherence to eukaryotic cells ( yin et al .
2009 ) , the data obtained in the present 
 study showed that cdce8392_0813 protein did not influence the adherence of c. 
 diphtheriae to epithelial cells .
furthermore , these results revealed that 
 cdce8392_0813 did not act as an adhesin or modify the expression of bacterial factors 
 involved in c. diphtheriae adhesion to abiotic surfaces .
the internalisation and intracellular survival of c. diphtheriaestrains 
 have been demonstrated for different human cells , including epithelial hep-2 cells 
 ( hirata jr et al .
2004 ) , human umbilical vein endothelial cells ( peixoto et al . 2014 ) and macrophage u937 cells 
 ( santos et al . 2010 ) .
these data showed that the 
 intracellular survival of c. diphtheriae was reduced after the 
 interruption of the cdce8392_0813 gene .
because ldcic - l1 showed increased 
 h2o2 sensitivity , it is likely that this determinant also 
 contributes to c. diphtheriae resistance to eukaryotic intracellular 
 defences , such as the production of ros , in addition to bacterial resistance to 
 teo3 
  toxicity .
the cdce8392_0813 gene interruption increased susceptibility to teo3 
  toxicity , indicating that the putative te protein 
 ( cdce8392_0813 ) ( teha ) might act as the factor responsible for the expression of 
 te in c. diphtheriaestrains .
furthermore , ter 
 determinant might contributes to the pathogenesis of this species , as a direct 
 correlation was verified between the expression of the cdce8392_0813 gene and the 
 abilities of c. diphtheriae to survive within the intracytoplasmic 
 compartments of hep cells and to kill the nematode c. elegans .
 
 corynebacterium diphtheriae , the aetiologic agent of diphtheria , 
 also represents a global medical challenge because of the existence of invasive 
 strains as causative agents of systemic infections . 
 although tellurite 
 ( teo32- ) is toxic to most microorganisms , teo32 - -resistant 
 bacteria , including c. diphtheriae , exist in 
 nature . 
 the presence of teo32 - -resistance ( ter ) 
 determinants in pathogenic bacteria might provide selective advantages in the natural 
 environment . in the present study , we investigated the role of the putative 
 ter determinant ( cdce8392_813gene ) in the virulence 
 attributes of diphtheria bacilli . 
 the disruption of cdce8392_0813 gene expression in 
 the ldcic - l1 mutant increased susceptibility to teo32- and reactive oxygen 
 species ( hydrogen peroxide ) , but not to other antimicrobial agents . 
 the ldcic - l1 
 mutant also showed a decrease in both the lethality of caenorhabditis elegans 
 and the survival inside of human epithelial cells compared to wild - type 
 strain . 
 conversely , the haemagglutinating activity and adherence to and formation of 
 biofilms on different abiotic surfaces were not regulated through the cdce8392_0813 
 gene . in conclusion 
 , the cdce8392_813 gene contributes to the ter and 
 pathogenic potential of c. diphtheriae . 
 
MATERIALS AND METHODS
RESULTS
DISCUSSION