BACKGROUND
corynebacterium pseudotuberculosis is a gram-positive, facultative, intracellular pathogen belonging to the corynebacterium, mycobacterium, nocardia, or cmn, group.
this group belongs to the phylum actinobacteria.
the defining characteristics of the cmn group are a specific cell wall organization, consisting of peptidoglycan, arabinogalactan, and mycolic acids, and a high chromosomal g + c content  <cit> .
c. pseudotuberculosis ovis is the etiological agent of the chronic infectious disease caseous lymphadenitis, which affects small ruminants worldwide.
as a result, c. pseudotuberculosis ovis is responsible for significant economic losses in the goat and sheep industries, mainly stemming from decreased meat, wool, and milk production, reproductive disorders, and carcass contamination  <cit> .
bacterial factors that contribute to the virulence of c. pseudotuberculosis include phospholipase d  <cit> , toxic cell wall lipids  <cit> , and the iron transporter fagabc complex  <cit> .
in silico analysis of the genome of c. pseudotuberculosis ovis  <dig>  <cit> , as well as the pan-genome analysis of  <dig> other strains of c. pseudotuberculosis <cit> , identified genes involved in the response of this pathogen to different types of stress.
recently, the functional genome of c. pseudotuberculosis ovis  <dig> was evaluated at the transcriptional level following exposure to different types of abiotic stress, including heat, osmotic, and acid stresses  <cit> .
this allowed the characterization of several genes involved in distinct biological processes that favor the survival of the pathogen under the given stress condition.
however, during the infection process, c. pseudotuberculosis encounters nitrosative stress, caused by nitric oxide , in the macrophage intracellular environment.
a reactive nitrogen species  found in mammalian systems, no is produced from l-arginine by no synthases , and is present in three isoforms: endothelial nos, neuronal nos, involved in blood pressure control and neural signaling, and inducible nos, associated with host defenses  <cit> .
the no produced during bacterial infection has antimicrobial properties, killing pathogens by causing damage to dna, rna, and proteins  <cit> .
however, several pathogens contain pathways that allow bacterial survival under nitrosative stress conditions, including no-sensitive transcriptional regulators  <cit> , dna and protein repair systems  <cit> , and antioxidant systems  <cit> .
currently, little is known about the factors involved in the resistance of c. pseudotuberculosis to nitrosative stress.
pacheco et al.
<cit>  showed that the alternative sigma  factor, σe, plays a role in the survival of c. pseudotuberculosis in the presence of rns.
a σe null strain showed increased susceptibility to nitric oxide compared with the wild-type, and, in an in vivo assay, was unable to persist in mice.
however, in inos-deficient mice, the mutant strain maintained its virulence  <cit> .
in the same study, the extracellular proteome of c. pseudotuberculosis was analyzed in response to nitrosative stress, allowing the characterization of proteins that contribute to the adaptive processes of the pathogen in this environment  <cit> .
to complement the results obtained in previous studies, and to identify factors involved in the survival of c. pseudotuberculosis under nitrosative stress conditions, we applied high-throughput proteomics using an liquid chromatograph high definition mass spectrometry   approach to evaluate the global expression of the functional genome of c. pseudotuberculosis ovis  <dig> at the protein level under nitrosative stress conditions.
methods
bacterial strain and growth conditions
c. pseudotuberculosis biovar ovis strain  <dig>  isolated from a goat, was maintained in brain heart infusion broth  at 37°c.
for stress-resistance assays, strain  <dig> was cultivated in a chemically-defined medium , containing na2hpo <dig> h2o , kh2po <dig> , nh4cl , mgso <dig> h2o , cacl <dig> ,  <dig> %  tween  <dig>  4%  mem vitamin solution , 1%  mem amino acid solution , 1%  mem non-essential amino acid solution , and  <dig> %  glucose, at 37°c  <cit> .
nitric oxide assay and preparation of whole bacterial lysates
diethylenetriamine/nitric oxide adduct  resistance of c. pseudotuberculosis was characterized as previously described  <cit> .
when strain  <dig> reached exponential growth phase  in the chemically-defined medium, the culture was divided into two aliquots , and deta/no was added to the appropriate aliquot to a final concentration of  <dig>  mm.
the growth of strain  <dig> in the presence of deta/no was then evaluated for 10 h. for proteomic analysis, protein was extracted after 1 h of exposure to deta/no.
both the control and deta/no cultures were centrifuged at  <dig>  × g for 10 min at 4°c.
the cell pellets were washed in phosphate buffered saline and then resuspended in 1 ml of lysis buffer  chaps, and 1 m dithiothreitol ).
the cells were then sonicated using five 1-min cycles on ice.
the resulting lysates were centrifuged at  <dig>  × g for 30 min at 4°c.
the extracted proteins were then submitted to centrifugation at  <dig>  × g for 10 min using a spin column with a threshold of 10 kda .
proteins were denatured with  rapigest sf surfactant at 60°c for 15 min , reduced using 10 mm dtt for 30 min at 60°c, and alkylated with 10 mm iodoacetamide in a dark chamber at 25°c for 30 min.
next, the proteins were enzymatically digested with 1: <dig>  trypsin at 37°c for 16 hours .
the digestion process was stopped by adding 10 μl of 5%  trifluoroacetic acid  .
glycogen phosphorylase was added to the digests to a final concentration of  <dig> fmol/μl as an internal standard for normalization prior to each replicate injection.
analysis was carried out using a two-dimensional reversed phase  nanouplc-ms  approach, using multiplexed hdmse label-free quantitation as described previously  <cit> .
lc-hdmse analysis and data processing
qualitative and quantitative by 2d nanouplc tandem nanoesi-hdmse  experiments were conducted using a 1-h reversed phase  acetonitrile  gradient ) at 500 nl/min on a nanoacquity uplc 2d rp × rp technology system  <cit> .
a nanoacquity uplc high strength silica  t <dig>  <dig>  μm 75 μm × 15 cm column  was used in conjunction with a rp xbridge beh <dig> c <dig> 5 μm 300 μm × 50 mm nanoflow column .
typical on-column sample loads were 250 ng of the total protein digests for each of the five fractions .
for all measurements, the mass spectrometer was operated in resolution mode, with a typical effective m/z conjoined ion-mobility resolving power of at least  <dig>  m fwhm, an ion mobility cell filled with nitrogen gas, and a cross-section resolving power at least  <dig> Ω/ΔΩ.
all analyses were performed using nano-electrospray ionization in the positive ion mode nanoesi , and a nanolockspray  ionization source.
the lock mass channel was sampled every 30 s. the mass spectrometer was calibrated with a ms/ms spectrum of -fibrinopeptide b  human solution  delivered though the reference sprayer of the nanolockspray source.
the double-charged ion  was used for initial single-point calibration, and ms/ms fragment ions of glu-fib were used to obtain the final instrument calibration.
multiplexed data-independent scanning with added specificity and selectivity of a non-linear “t-wave” ion mobility  experiments were performed using a synapt g2-s hdms mass spectrometer .
the mass spectrometer was set to switch automatically between standard ms  and elevated collision energies hdmse  applied to the transfer “t-wave” collision-induced dissociation cell with argon gas.
the trap collision cell was adjusted for 1 ev using a millisecond scan time adjusted based on the linear velocity of the chromatography peak delivered though nanoacquity uplc, to obtain a minimum of  <dig> scan points for each single peak at both low-energy and high-energy transmission, followed by an orthogonal acceleration time-of-flight from 50–2000 m/z.
the radio frequency  offset  was adjusted so that the nanouplc-hdmse data were effectively acquired from an m/z range of 400– <dig>  which ensured that any masses observed in the high energy spectra of less than 400 m/z arose from dissociations in the collision cell.
data processing
protein identification and quantitative data packaging were generated using dedicated algorithms  <cit> , and by searching against a c. pseudotuberculosis database with default parameters for ion accounting  <cit> .
the databases were reversed “on-the fly” during the database query searches, and appended to the original database to assess the false positive rate of identification.
for proper processing of spectra and database searching conditions, proteinlynxglobalserver v. <dig> .
<dig>  with identitye and expressione informatics v. <dig> .
<dig>  were used.
uniprotkb  with manually-reviewed annotations was also used, and the search conditions were based on taxonomy , maximum missed cleavages by trypsin allowed up to one, and variable carbamidomethyl, acetyl n-terminal, phosphoryl, and oxidation  modifications  <cit> .
the identitye algorithm with hi <dig> methodology was used for protein quantitation.
the search threshold for accepting each individual spectrum was set to the default value, with a false-positive value of 4%.
biological variability was addressed by analyzing each culture three times.
normalization was performed using the expressione tool with a housekeeping protein that showed no significant difference in abundance across all injections.
the proteins obtained were organized by the plgs expressione tool algorithm into a statistically significant list corresponding to increased and decreased regulation ratios among the different groups.
the quantitation values were averaged over all of the samples, and the quoted standard deviations at p ≤  <dig>  in the expressione software refer to the differences between biological replicates.
only proteins with a differential expression log <dig> ratio between the two conditions greater than or equal to  <dig>  were considered  <cit> .
bioinformatics analysis
the identified proteins were analyzed using the prediction tools surfg+ v <dig>   <cit> , to predict sub-cellular localization, and blast2go, to predict gene ontology functional annotations  <cit> .
the pips software predicted proteins present in pathogenicity islands  <cit> .
the protein-protein interaction network was constructed using interolog mapping methodology and metrics according to rezende et al.
<cit> .
a preview of the interaction network was generated using cytoscape version  <dig> .
<dig>  <cit> , with a spring-embedded layout.
cmregnet was used to predict gene regulatory networks  <cit> .
RESULTS
effects of nitric oxide on the growth of c. pseudotuberculosis
in this study, we examined the exponential growth of c. pseudotuberculosis strain  <dig> under nitrosative stress.
the growth and cell viability of strain  <dig> was monitored for 10 h with and without deta/no supplementation .
the control culture reached stationary phase by 5 h post-inoculation, while the culture containing deta/no did not reach stationary phase until approximately 10 h post-inoculation.
however, these results showed that although deta/no  affected the growth rate, c. pseudotuberculosis likely contains factors that promote survival in the presence of rns.figure  <dig>
growth and survival profile of
c. pseudotuberculosis
during no exposure.
growth of c. pseudotuberculosis after 10 h exposure to  <dig>  mm deta/no.
survival of c. pseudotuberculosis evaluated by colony forming units.
the results shown in a and b represent an average of three independent experiments.
label-free proteomic analysis of c. pseudotuberculosisgrown under nitrosative stress conditions
total proteome digests from three biological replicates of each individual condition were subjected to lc/mse.
in total, we identified more than  <dig>  peptides, with a normal distribution of 10 ppm error of the total identified peptides.
peptides as source fragments, peptides with a charge state of at least 2+, and the absence of decoys were factors considered to increase data quality.
a combined total of  <dig>  proteins were present in at least two of the three biological replicates for the two conditions tested, with an average of  <dig> peptides per protein, and a false discovery rate  of 0% when decoy detection was set at agreement of two out of three replicates.
the proteins referred to as exclusive to one condition or another was only identified in one condition within the detection limits of the experiment .
the dynamic range of the quantified proteins is about  <dig> logs, and proteins unique to one condition or another were only observed above the lod of the experiment, which was determined by the sample normalization prior to injection.
therefore, in our study, all samples were normalized using “scouting runs” taking into account the stoichiometry between the intensity and molarity proportion prior to the replicate runs per condition.
the dynamic range was similar for each sample, and the total amount of sample used in fmol was nearly the same.
we generate a graph of protein amounts of the identified proteins from all samples against protein ranks .figure  <dig>
2d nanouplc hdmse analysis showing:  dynamic range of the method based on protein abundance estimates, data points derived from lc-hdms
e
analysis.
proteins that were significantly differentially-regulated during no exposure.
the distribution of identified proteins with p <  <dig> , and differentially-regulated proteins with an i:c log <dig> ratio <  <dig>  in relation to the number of peptides identified for each protein.
proteins with p <  <dig>  and an i:c log <dig> ratio <  <dig> .
proteins with p <  <dig>  and an i:c log <dig> ratio >  <dig> .
after, analysis by plgs v <dig> .
<dig> software, the  <dig>  proteins originally identified in two out of three replicates were narrowed down to  <dig> proteins with p ≤  <dig> .
among these proteins,  <dig> were up-regulated in the presence of deta/no, while  <dig> proteins were down-regulated .
the remaining  <dig> proteins with p ≤  <dig>  and log2 <  <dig>  that were common to the two treatments are summarized in additional file  <dig>  in addition to the  <dig> identified proteins that were present under both control and stress conditions,  <dig> proteins were exclusively expressed under the control conditions, and  <dig> proteins were exclusively expressed in response to deta/no stress .
thus, our final list of proteins is composed of  <dig> proteins from c. pseudotuberculosis.table  <dig>
proteins identified as differentially-expressed following exposure to nitrosative stress
2
deta:ct
p
-value
transport
porh
cell division
cp1002_0716
dna synthesis and repair
ybab
ada
mutt
transcription
lexa
sigk
translation
fkbp
rpmi
gatc
rpli
rpsh
cell communication
cp1002_2005
gntk
regx3
detoxification
trxa
cp1002_1731
cp1002_0124
noxc
cp1002_0272
glycolysis pathways
nank
adha
iron-sulfur clusters
fdxa
antibiotic resistance
cp1002_0937
amino acid metabolism
serb
cp1002_1819
sdaa
lipid metabolism
glpq
oxidative phosphorylation
cp1002_1095
specific metabolic pathways
ino1
cp1002_0573
mmsb
ureg
nucleotide metabolism
pyre
unknown function
cp1002_0540
cp1002_0320
cp1002_1474
cp1002_0143
cp1002_0202
cp1002_1048
cp1002_0512
cp1002_0506
cp1002_0105
cp1002_1281
cp1002_1476
cp1002_0852
cp1002_0766
cp1002_1477
cp1002_0548
cp1002_1221
cp1002_0321
cp1002_0007
cp1002_1721
others
piub
cp1002_1286
pup
rimj
arsc
ratio values to: strain 1002_deta/no:strain 1002_ct, log ratio >  <dig> , p >  <dig>  = up-regulation, p <  <dig>  = down-regulation.
core-genome analysis of  <dig> strains of c. pseudotuberculosis: shared = present in two or more strains; core = present in  <dig> strains of c. pseudotuberculosis.
cyt =cytoplasmic, mem = membrane, pse = potentially surface-exposed, sec = secreted.
in silicoanalysis of lc-hdmsedata
the  <dig> proteins were then analyzed using the surfg+ tool to predict sub-cellular localization.
according with surfg+, our data set included approximately 41% of the predicted proteome of strain  <dig> .
in addition, we characterized proteins belonging to the following cell fractions: cytoplasmic  , membrane  , potentially surface-exposed  , and secreted   .figure  <dig>
correlation of
in silico
predicted data with proteome results.
percentage of coverage of the c. pseudotuberculosis  <dig> in silico proteome.
prediction of the subcellular localization of the proteins identified by lc/ms.
analysis of the differentially-regulated proteins of cells exposed to deta/no in relation to the core genome of c. pseudotuberculosis .
analysis of the exclusive proteome of cells exposed to dea/no in relation to the core-genome of c. pseudotuberculosis .
percent coverage of the core-genome of c. pseudotuberculosis in relation to the characterized proteome in vitro.
to evaluate whether the proteins identified in our proteomic analysis could represent a protein set expressed by c. pseudotuberculosis during exposure to nitrosative stress, we correlated our proteomic data with the predicted core-genomes of  <dig> c. pseudotuberculosis strains  <cit> .
of the open reading frames  coding for the differentially-regulated proteins and exclusive proteome of deta/no-exposed cells, 86%  and 82%  were identified, respectively, in the core-genome of c. pseudotuberculosis .
in addition, of the  <dig> total proteins identified from the proteome of strain  <dig> following exposure to nitrosative stress, 83%  of the orfs coding for these proteins were present in the core-genome of c. pseudotuberculosis, this result correspond approximately 46% of the predicted core-genome of c. pseudotuberculosis .
functional classification of the proteome of c. pseudotuberculosisexpressed under exposure to nitrosative stress
the strain  <dig> proteome was functionally classified using the blast2go tool  <cit> .
a large proportion of the differentially-regulated proteins and those exclusive to one condition were identified as hypothetical proteins.
according to the biological function prediction,  <dig> biological processes were classified as differentially regulated .
in addition, the analysis of the exclusive proteome of each condition revealed  <dig> common processes between the control and stress conditions .
however, seven biological processes were identified only in stress-exposed cells.
these processes were antibiotic metabolism , nucleotide metabolism , oxidative phosphorylation , translation , glycolysis pathways , iron-sulfur clusters , and starch and sucrose metabolism .
among all processes identified, dna synthesis and repair proteins  were most common.
an overview of the c. pseudotuberculosis response to nitrosative stress according with the proteins identified is shown in figure  <dig> figure  <dig>
comparison of biological processes between control and deta/no conditions.
a representation of the biological processes in relation to a set list of proteins identified as  differentially-regulated in deta/no-stressed cells and  comparison of exclusive biological process between the two test conditions.
overview of
c. pseudotuberculosis
response to nitrosative stress.
all proteins detected by proteomic analysis are marked in red .
the proteins that were grouped into of transcriptional process were evaluated by cmregnet and among regulators identified; we identified the gntr- family regulatory protein , genes regulated by gntr-type regulators are usually involved in carbohydrate metabolism.
the cmregnet analysis showed that of the four genes under the control of this regulator, the n-acetylglucosamine kinase  protein was highly expressed by c. pseudotuberculosis in response to deta/no.
we identified other regulator the lexa repressor  that was down regulated in the deta/no condition.
according with cmregnet, two proteins regulated by this repressor were detected in the deta/no proteome specific, pyridoxal biosynthesis lyase  and dna translocase .
others proteins under the control of this repressor was detected, however not presented significant differential regulation like reca protein
protein-protein interaction network
to investigate the interactions among the proteins identified as exclusive and differentially regulated in cells exposed to deta/no, we generated a protein interaction network using cytoscape.
the interactome analysis revealed  <dig> protein-protein interactions .
dnab/dna helicase , identified in the exclusive proteome for strain 1002_deta/no, and pyre/orotate phosphoribosyltransferase , which was down-regulated in strain 1002_deta/no, showed the greatest number of interactions with other proteins .
moreover, both of these proteins interact with proteins that are involved in metabolic processes, dna processes, antibiotic metabolism, cell cycling, and translation.figure  <dig>
protein-protein interactions.
protein-protein interactions of the proteins identified in deta/no-exposed cells.
exclusive proteome, circle; up-regulated, square; and down-regulated, rhombus.
the sizes of the nodes represent the degree of interaction for each gene/protein; the major nodes demonstrate greater interactions.
the colors of nodes and lines are in an increasing gradient scale from yellow to green to blue.
the networks were visualized using cytoscape.
discussion
c. pseudotuberculosis is exposed to different forms of oxidative and nitrosative stress during the infection process.
a previous study showed that c. pseudotuberculosis resists nitrosative stress generated by the no-donor deta/no, and that a low concentration of deta/no  induces a change in the extracellular proteome this pathogen  <cit> .
to better understand the physiology of c. pseudotuberculosis in response to nitrosative stress, we analyzed the proteome of whole bacterial lysates of c. pseudotuberculosis in response to exposure to deta/no .
the strain  <dig> proteome under nitrosative stress reveals proteins involved in bacterial defense against dna damage
proteomic analysis identified proteins involved in dna repair systems in both the exclusive proteome of deta/no-exposed cells and in the differentially-regulated proteome.
we detected the proteins formamidopyrimidine-dna glycosylase  , recb , and methylated-dna-protein-cysteine methyltransferase  , the genes for which were previously identified in a transcriptome analysis of strain  <dig> in response to different abiotic stresses  <cit> .
activation of these proteins in response to nitrosative stress confirms that they belong a group of general stress-response proteins in c. pseudotuberculosis.
the expression of fpg was up-regulated in response to acid stress  <cit> .
we also identified endonuclease iii  , which, in addition to fpg, is involved in the base excision repair  system of various bacteria.
this system cleaves n-glycosidic bonds from damaged bases, allowing their excision and replacement.
in salmonella enterica serovar typhimurium, the ber system repairs dna damaged by exposure to no.
in addition, an s. typhimurium strain defective in fpg demonstrated reduced virulence in a murine model  <cit> .
our interactome analysis showed that endo iii had one of the highest numbers of interactions with other proteins, including interactions with proteins involved in dna replication such zinc metalloprotease  and dna translocase , suggesting that this protein could play an important role in the defense pathway against rns.
the ada and recb protein were up-regulated in response to osmotic stress  <cit> .
ada is involved in the repair of dna-methylation damage, this protein have plays important in the pathway dna damage  <cit> .
recb is a component of the recbc system, which is part of the sos response the more regulatory network encoded by prokaryotic involved in dna repair  <cit> .
the recbc system acts in the recombination or degradative repair of arrested dna replication forks.
studies in s. typhimurium showed that recbc mutant strains are more attenuated than reca mutants in a murine model of infection  <cit> .
in addition, unlike reca mutants, recbc mutants were susceptible to rns  <cit> , indicating that recbc is highly important in the bacterial response to nitrosative stress.
the lexa repressor , which forms part of the general sos system along with reca  <cit> , was down-regulated in c. pseudotuberculosis cells exposed to deta/no.
we also detected the reca protein ; however, despite having a p-value < <dig> , the fold-change of - <dig>  showed that this protein was not activated under the experimental conditions.
studies performed in mycobacterium tuberculosis showed that reca was not induced until cells had been exposed to deta/no  for 4 h, but that hydrogen peroxide induced the immediate expression of reca <cit> , suggesting that reca is involved in the later stages of the nitrosative stress response.
nevertheless, cmregnet analysis identified other proteins that are regulated by lexa in the deta/no-specific proteome, including pyridoxal biosynthesis lyase  and dna translocase .
no-sensitive transcriptional regulators are activated in the presence of no
to activate these dna repair systems, it is essential that bacteria can detect ros and rns, and concomitantly activate the transcriptional regulators needed for the expression of genes involved in protection against these compounds.
in the deta/no-specific proteome, we detected the transcription factor whib .
the whib transcriptional family is composed of iron-sulfur  cluster proteins.
these proteins are o2- and no-sensitive, and allow the sensing of both external environmental signals and the redox state for intracellular bacteria  <cit> .
in m. tuberculosis, the reaction of the iron-sulfur cluster of whib <dig> with no generates a dinitrosyl iron complex , which activates a sensing mechanism in response to the no, consequently activating a system of defense against nitrosative stress  <cit> .
in addition, other in vivo and in vitro studies have also demonstrated that whib regulators play a role in the adaptation and survival of m. tuberculosis during exposure to redox environments .
we identified other regulators that are activated in response to environmental stimuli, such as a merr-family transcriptional regulator  and a lysr-type transcriptional regulator  .
this regulator was also highly expressed in the transcriptional response of c. pseudotuberculosis  <dig> to acid stress  <cit> .
merr-type regulators have been described in the detoxification of toxic metal in several pathogenic and non-pathogenic bacteria  <cit> .
other studies have shown that this class of regulator plays a role in bacterial resistance to oxidative and nitrosative stress  <cit> .
lttrs are associated with the regulation of several biological processes, as well as in the adaptive response of bacteria to different types of stress  <cit> .
in vibrio cholerae, lttrs are associated with efflux pump regulation, which contribute to antimicrobial resistance, and are involved in colonization of the human host  <cit> .
in pathogens like e. coli <cit> , enterococcus faecalis <cit> , s. enterica <cit> , and pseudomonas aeruginosa <cit> , lttrs are involved in resistance to oxidative stress.
the detoxification pathways of c. pseudotuberculosisfollowing no exposure
our proteomic analysis identified proteins specifically expressed by cells exposed to deta/no that are involved in the detoxification process.
two of these proteins were thioredoxin   and glutathione peroxidase .
the thioredoxin and glutathione systems play major roles in thiol and disulfide balance, respectively  <cit> .
in pathogens such as helicobacter pylori, streptococcus pyogenes, and m. tuberculosis, this system is of great importance in combating the presence of ros/rns  <cit> .
a glyoxalase/dioxygenase  was identified in the differential proteome of cells exposed to deta/no.
this protein was previously detected in the proteome of c. pseudotuberculosis strain  <dig> in response to  <dig>  mm deta/no  <cit> .
the presence of this protein suggests that glyoxalase/dioxygenase plays a role in the resistance of this pathogen to nitrosative stress.
nevertheless, unlike p. aeruginosa, which contains a complete denitrification pathway  <cit> , the predicted genome of c. pseudotuberculosis ovis  <dig> revealed a truncated denitrification pathway.
however, we detected the nitric-oxide reductase cytochrome b   in the exclusive proteome of deta/no-stressed cells.
norb, which codes for this nitric-oxide reductase, is organized into the norcbqdef operon in paracoccus denitrificans <cit> , and into the norcbd operon in p. aeruginosa <cit> .
the c. pseudotuberculosis genome was predicted to only contain norb.
moreover, norb is located in the cp1002picp <dig> pathogenicity island, suggesting horizontal acquisition of the gene by this pathogen.
nitric-oxide reductase is an important protein in the denitrification process of some bacteria  <cit> .
in p. aeruginosa, norb plays a role in both the growth of the pathogen in the presence of no, and in its survival in macrophages  <cit> .
the flavohemoglobin hmp is involved in the no detoxification pathway in s. typhimurium, and levels of hmp are increased approximately two-fold in macrophages  <cit> .
interestingly, in n. meningitidis, norb levels are increased ten-fold in macrophages  <cit> , demonstrating the great power of this protein in the detoxification process.
metabolic profile of c. pseudotuberculosisin response to nitrosative stress
in addition to the presence of proteins involved in bacterial defense and detoxification pathways, strain  <dig> needs to undergo metabolic adaptation to favor bacterial survival.
we observed a metabolic readjustment in this pathogen in the proteomic analysis.
of the proteins involved in central carbohydrate metabolism, we detected only phosphoglycerate mutase  and n-acetylglucosamine kinase  in the proteome of deta/no-exposed cells.
other essential proteins involved in glycolysis , the pentose phosphate pathway, and the citric acid cycle were not detected.
similar results were found in a metabolomic study of v. cholerae in response to nitrosative stress  <cit> .
however, we hypothesized that c. pseudotuberculosis uses oxidative phosphorylation to obtain energy.
this is supported by the presence of cytochrome c oxidase polypeptide i , succinate dehydrogenase cytochrome b <dig> subunit , and ubiquinol-cytochrome c reductase cytochrome c subunit  in the exclusive proteome of deta/no-stressed cells, and by the up-regulation of the cytochrome oxidase assembly protein  under the same conditions.
however, this oxidative phosphorylation may be associated with the bacterial culture conditions used in this work, in which c. pseudotuberculosis was cultivated in the presence of deta/no under aerobic conditions.
studies have shown that growing m. tuberculosis in a low concentration of no with low levels of o <dig> can induce anaerobic respiration as a result of the inhibition of the respiratory proteins cytochrome c oxidase and nadh reductase by irreversible ligation of no.
the ligation of no to the respiratory proteins is an effect that may be both short-term reversible and long-term irreversible  <cit> .
thus, we suggest that activation of the oxidative phosphorylation system may be a more effective pathway for this pathogen to obtain energy  <cit> .
another metabolic adjustment was observed in relation to amino acid biosynthesis.
transporters and enzymes involved in the synthesis of methionine, tryptophan, and serine were identified.
however, the presence of these proteins can be associated with the bioavailability of these amino acids during exposure to no.
in addition, we detected two oligopeptide transport atp-binding proteins   that compose the oligopeptide permease system .
this complex is associated with the internalization of peptides from the extracellular environment to be used as a source of carbon and nitrogen in bacterial nutrition  <cit> .
we also identified proteins that are cofactors of metabolism, such as coabc , phosphopantetheine adenylyltransferase , and 2-dehydropantoate 2-reductase .
the presence of these proteins demonstrates activity in pantothenic acid metabolism and the biosynthesis of coenzyme a .
studies performed in species such as corynebacterium diphtheriae <cit> , streptococcus haemolyticus <cit> , and m. tuberculosis <cit>  showed that pantothenic acid and coa could have an important role in the growth and viability of these pathogens.
CONCLUSIONS
in this work, we applied high-throughput proteomics to characterize the proteome of c. pseudotuberculosis ovis  <dig> following exposure to no.
our proteomic analysis generated two profiles, which together validated findings from previous in silico analyses of c. pseudotuberculosis ovis  <dig>  the proteomic profile generated after the addition of the no-donor, deta/no , revealed a set of proteins that are involved in distinct biological process.
we detected proteins related to both the general stress response and to a more specific nitrosative stress response, which together form a network of factors that promote the survival of this pathogen under stress conditions.
however, more detailed studies are needed to assess the true role of these proteins in response to nitrosative stress in c. pseudotuberculosis.
in conclusion, this functional analysis of the genome of c. pseudotuberculosis shows the versatility of this pathogen in the presence of no.
moreover, the results presented in this study provide insights into the processes of resistance of c. pseudotuberculosis during exposure to nitrosative stress.
electronic supplementary material
additional file 1: table s1: complete list of proteins identified as significantly altered .
additional file 2: table s2: unique proteins identified in strain 1002_deta/no.
additional file 3: table s3: unique proteins identified in strain  <dig> control condition.
abbreviations
gguanine
ccitosine
nonitric oxide
rnsreactive nitrogen species
nosnitric oxide synthases
lc-hdmseliquid chromatograph high definition mass spectrometry
lc/msliquid chromatograph mass spectrometry
cdmchemically-defined médium
deta/nodiethylenetriamine/nitric oxide adduct
dttdithiothreitol
2d-rptwo-dimensional reversed phase
nanouplcnano ultra performance liquid chromatography
nanoesi-hdmsnano electrospray high definition mass spectrometry
hsshigh strength silica
plgsprotein lynx global server
fdrfalse discovery rate.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
wms, rdc, and ifsb performed microbiological analyses and sample preparation for proteomic analysis.
ghmfs and wms conducted the proteomic analysis.
scs and elf performed bioinformatics analysis of the data.
yll and am contributed substantially to data interpretation and revisions.
va and as participated in all steps of the project as coordinators, and critically reviewed the manuscript.
all authors read and approved the final manuscript.

 corynebacterium pseudotuberculosis is a gram-positive, facultative, intracellular pathogen belonging to the corynebacterium, mycobacterium, nocardia, or cmn, group. this group belongs to the phylum actinobacteria. the defining characteristics of the cmn group are a specific cell wall organization, consisting of peptidoglycan, arabinogalactan, and mycolic acids, and a high chromosomal g + c content. c. pseudotuberculosis ovis is the etiological agent of the chronic infectious disease caseous lymphadenitis, which affects small ruminants worldwide. as a result, c. pseudotuberculosis ovis is responsible for significant economic losses in the goat and sheep industries, mainly stemming from decreased meat, wool, and milk production, reproductive disorders, and carcass contamination. bacterial factors that contribute to the virulence of c. pseudotuberculosis include phospholipase d, toxic cell wall lipids, and the iron transporter fagabc complex. in silico analysis of the genome of c. pseudotuberculosis ovis, as well as the pan-genome analysis of other strains of c. pseudotuberculosis, identified genes involved in the response of this pathogen to different types of stress. recently, the functional genome of c. pseudotuberculosis ovis was evaluated at the transcriptional level following exposure to different types of abiotic stress, including heat, osmotic, and acid stresses. this allowed the characterization of several genes involved in distinct biological processes that favor the survival of the pathogen under the given stress condition. however, during the infection process, c. pseudotuberculosis encounters nitrosative stress, caused by nitric oxide, in the macrophage intracellular environment. a reactive nitrogen species found in mammalian systems, no is produced from l-arginine by no synthases, and is present in three isoforms: endothelial nos, neuronal nos, involved in blood pressure control and neural signaling, and inducible nos, associated with host defenses. the no produced during bacterial infection has antimicrobial properties, killing pathogens by causing damage to dna, rna, and proteins. however, several pathogens contain pathways that allow bacterial survival under nitrosative stress conditions, including no-sensitive transcriptional regulators, dna and protein repair systems, and antioxidant systems. currently, little is known about the factors involved in the resistance of c. pseudotuberculosis to nitrosative stress. pacheco et al. showed that the alternative sigma factor, σe, plays a role in the survival of c. pseudotuberculosis in the presence of rns. a σe null strain showed increased susceptibility to nitric oxide compared with the wild-type, and, in an in vivo assay, was unable to persist in mice. however, in inos-deficient mice, the mutant strain maintained its virulence. in the same study, the extracellular proteome of c. pseudotuberculosis was analyzed in response to nitrosative stress, allowing the characterization of proteins that contribute to the adaptive processes of the pathogen in this environment. to complement the results obtained in previous studies, and to identify factors involved in the survival of c. pseudotuberculosis under nitrosative stress conditions, we applied high-throughput proteomics using an liquid chromatograph high definition mass spectrometry approach to evaluate the global expression of the functional genome of c. pseudotuberculosis ovis at the protein level under nitrosative stress conditions. methods bacterial strain and growth conditions c. pseudotuberculosis biovar ovis strain isolated from a goat, was maintained in brain heart infusion broth at 37°c. for stress-resistance assays, strain was cultivated in a chemically-defined medium, containing na2hpo h2o, kh2po, nh4cl, mgso h2o, cacl, % tween 4% mem vitamin solution, 1% mem amino acid solution, 1% mem non-essential amino acid solution, and % glucose, at 37°c. nitric oxide assay and preparation of whole bacterial lysates diethylenetriamine/nitric oxide adduct resistance of c. pseudotuberculosis was characterized as previously described. when strain reached exponential growth phase in the chemically-defined medium, the culture was divided into two aliquots, and deta/no was added to the appropriate aliquot to a final concentration of  mm. the growth of strain in the presence of deta/no was then evaluated for 10 h. for proteomic analysis, protein was extracted after 1 h of exposure to deta/no. both the control and deta/no cultures were centrifuged at  × g for 10 min at 4°c. the cell pellets were washed in phosphate buffered saline and then resuspended in 1 ml of lysis buffer chaps, and 1 m dithiothreitol ). the cells were then sonicated using five 1-min cycles on ice. the resulting lysates were centrifuged at  × g for 30 min at 4°c. the extracted proteins were then submitted to centrifugation at  × g for 10 min using a spin column with a threshold of 10 kda. proteins were denatured with rapigest sf surfactant at 60°c for 15 min, reduced using 10 mm dtt for 30 min at 60°c, and alkylated with 10 mm iodoacetamide in a dark chamber at 25°c for 30 min. next, the proteins were enzymatically digested with 1: trypsin at 37°c for 16 hours. the digestion process was stopped by adding 10 μl of 5% trifluoroacetic acid. glycogen phosphorylase was added to the digests to a final concentration of fmol/μl as an internal standard for normalization prior to each replicate injection. analysis was carried out using a two-dimensional reversed phase nanouplc-ms approach, using multiplexed hdmse label-free quantitation as described previously. lc-hdmse analysis and data processing qualitative and quantitative by 2d nanouplc tandem nanoesi-hdmse experiments were conducted using a 1-h reversed phase acetonitrile gradient ) at 500 nl/min on a nanoacquity uplc 2d rp × rp technology system. a nanoacquity uplc high strength silica t  μm 75 μm × 15 cm column was used in conjunction with a rp xbridge beh c 5 μm 300 μm × 50 mm nanoflow column. typical on-column sample loads were 250 ng of the total protein digests for each of the five fractions. for all measurements, the mass spectrometer was operated in resolution mode, with a typical effective m/z conjoined ion-mobility resolving power of at least  m fwhm, an ion mobility cell filled with nitrogen gas, and a cross-section resolving power at least Ω/ΔΩ. all analyses were performed using nano-electrospray ionization in the positive ion mode nanoesi, and a nanolockspray ionization source. the lock mass channel was sampled every 30 s. the mass spectrometer was calibrated with a ms/ms spectrum of -fibrinopeptide b human solution delivered though the reference sprayer of the nanolockspray source. the double-charged ion was used for initial single-point calibration, and ms/ms fragment ions of glu-fib were used to obtain the final instrument calibration. multiplexed data-independent scanning with added specificity and selectivity of a non-linear “t-wave” ion mobility experiments were performed using a synapt g2-s hdms mass spectrometer. the mass spectrometer was set to switch automatically between standard ms and elevated collision energies hdmse applied to the transfer “t-wave” collision-induced dissociation cell with argon gas. the trap collision cell was adjusted for 1 ev using a millisecond scan time adjusted based on the linear velocity of the chromatography peak delivered though nanoacquity uplc, to obtain a minimum of scan points for each single peak at both low-energy and high-energy transmission, followed by an orthogonal acceleration time-of-flight from 50–2000 m/z. the radio frequency offset was adjusted so that the nanouplc-hdmse data were effectively acquired from an m/z range of 400– which ensured that any masses observed in the high energy spectra of less than 400 m/z arose from dissociations in the collision cell. data processing protein identification and quantitative data packaging were generated using dedicated algorithms, and by searching against a c. pseudotuberculosis database with default parameters for ion accounting. the databases were reversed “on-the fly” during the database query searches, and appended to the original database to assess the false positive rate of identification. for proper processing of spectra and database searching conditions, proteinlynxglobalserver v.. with identitye and expressione informatics v.. were used. uniprotkb with manually-reviewed annotations was also used, and the search conditions were based on taxonomy, maximum missed cleavages by trypsin allowed up to one, and variable carbamidomethyl, acetyl n-terminal, phosphoryl, and oxidation modifications. the identitye algorithm with hi methodology was used for protein quantitation. the search threshold for accepting each individual spectrum was set to the default value, with a false-positive value of 4%. biological variability was addressed by analyzing each culture three times. normalization was performed using the expressione tool with a housekeeping protein that showed no significant difference in abundance across all injections. the proteins obtained were organized by the plgs expressione tool algorithm into a statistically significant list corresponding to increased and decreased regulation ratios among the different groups. the quantitation values were averaged over all of the samples, and the quoted standard deviations at p ≤  in the expressione software refer to the differences between biological replicates. only proteins with a differential expression log ratio between the two conditions greater than or equal to were considered. bioinformatics analysis the identified proteins were analyzed using the prediction tools surfg+ v, to predict sub-cellular localization, and blast2go, to predict gene ontology functional annotations. the pips software predicted proteins present in pathogenicity islands. the protein-protein interaction network was constructed using interolog mapping methodology and metrics according to rezende et al.. a preview of the interaction network was generated using cytoscape version., with a spring-embedded layout. cmregnet was used to predict gene regulatory networks.  effects of nitric oxide on the growth of c. pseudotuberculosis in this study, we examined the exponential growth of c. pseudotuberculosis strain under nitrosative stress. the growth and cell viability of strain was monitored for 10 h with and without deta/no supplementation. the control culture reached stationary phase by 5 h post-inoculation, while the culture containing deta/no did not reach stationary phase until approximately 10 h post-inoculation. however, these results showed that although deta/no affected the growth rate, c. pseudotuberculosis likely contains factors that promote survival in the presence of rns.figure growth and survival profile of c. pseudotuberculosis during no exposure. growth of c. pseudotuberculosis after 10 h exposure to  mm deta/no. survival of c. pseudotuberculosis evaluated by colony forming units. the results shown in a and b represent an average of three independent experiments. label-free proteomic analysis of c. pseudotuberculosisgrown under nitrosative stress conditions total proteome digests from three biological replicates of each individual condition were subjected to lc/mse. in total, we identified more than peptides, with a normal distribution of 10 ppm error of the total identified peptides. peptides as source fragments, peptides with a charge state of at least 2+, and the absence of decoys were factors considered to increase data quality. a combined total of proteins were present in at least two of the three biological replicates for the two conditions tested, with an average of peptides per protein, and a false discovery rate of 0% when decoy detection was set at agreement of two out of three replicates. the proteins referred to as exclusive to one condition or another was only identified in one condition within the detection limits of the experiment. the dynamic range of the quantified proteins is about logs, and proteins unique to one condition or another were only observed above the lod of the experiment, which was determined by the sample normalization prior to injection. therefore, in our study, all samples were normalized using “scouting runs” taking into account the stoichiometry between the intensity and molarity proportion prior to the replicate runs per condition. the dynamic range was similar for each sample, and the total amount of sample used in fmol was nearly the same. we generate a graph of protein amounts of the identified proteins from all samples against protein ranks.figure 2d nanouplc hdmse analysis showing: dynamic range of the method based on protein abundance estimates, data points derived from lc-hdms e analysis. proteins that were significantly differentially-regulated during no exposure. the distribution of identified proteins with p < , and differentially-regulated proteins with an i:c log ratio <  in relation to the number of peptides identified for each protein. proteins with p <  and an i:c log ratio < . proteins with p <  and an i:c log ratio > . after, analysis by plgs v. software, the proteins originally identified in two out of three replicates were narrowed down to proteins with p ≤ . among these proteins, were up-regulated in the presence of deta/no, while proteins were down-regulated. the remaining proteins with p ≤  and log2 <  that were common to the two treatments are summarized in additional file in addition to the identified proteins that were present under both control and stress conditions, proteins were exclusively expressed under the control conditions, and proteins were exclusively expressed in response to deta/no stress. thus, our final list of proteins is composed of proteins from c. pseudotuberculosis.table proteins identified as differentially-expressed following exposure to nitrosative stress 2 deta:ct p -value transport porh cell division cp1002_0716 dna synthesis and repair ybab ada mutt transcription lexa sigk translation fkbp rpmi gatc rpli rpsh cell communication cp1002_2005 gntk regx3 detoxification trxa cp1002_1731 cp1002_0124 noxc cp1002_0272 glycolysis pathways nank adha iron-sulfur clusters fdxa antibiotic resistance cp1002_0937 amino acid metabolism serb cp1002_1819 sdaa lipid metabolism glpq oxidative phosphorylation cp1002_1095 specific metabolic pathways ino1 cp1002_0573 mmsb ureg nucleotide metabolism pyre unknown function cp1002_0540 cp1002_0320 cp1002_1474 cp1002_0143 cp1002_0202 cp1002_1048 cp1002_0512 cp1002_0506 cp1002_0105 cp1002_1281 cp1002_1476 cp1002_0852 cp1002_0766 cp1002_1477 cp1002_0548 cp1002_1221 cp1002_0321 cp1002_0007 cp1002_1721 others piub cp1002_1286 pup rimj arsc ratio values to: strain 1002_deta/no:strain 1002_ct, log ratio > , p >   = up-regulation, p <   = down-regulation. core-genome analysis of strains of c. pseudotuberculosis: shared = present in two or more strains; core = present in strains of c. pseudotuberculosis. cyt =cytoplasmic, mem = membrane, pse = potentially surface-exposed, sec = secreted. in silicoanalysis of lc-hdmsedata the proteins were then analyzed using the surfg+ tool to predict sub-cellular localization. according with surfg+, our data set included approximately 41% of the predicted proteome of strain. in addition, we characterized proteins belonging to the following cell fractions: cytoplasmic, membrane, potentially surface-exposed, and secreted.figure correlation of in silico predicted data with proteome results. percentage of coverage of the c. pseudotuberculosis in silico proteome. prediction of the subcellular localization of the proteins identified by lc/ms. analysis of the differentially-regulated proteins of cells exposed to deta/no in relation to the core genome of c. pseudotuberculosis. analysis of the exclusive proteome of cells exposed to dea/no in relation to the core-genome of c. pseudotuberculosis. percent coverage of the core-genome of c. pseudotuberculosis in relation to the characterized proteome in vitro. to evaluate whether the proteins identified in our proteomic analysis could represent a protein set expressed by c. pseudotuberculosis during exposure to nitrosative stress, we correlated our proteomic data with the predicted core-genomes of c. pseudotuberculosis strains. of the open reading frames coding for the differentially-regulated proteins and exclusive proteome of deta/no-exposed cells, 86% and 82% were identified, respectively, in the core-genome of c. pseudotuberculosis. in addition, of the total proteins identified from the proteome of strain following exposure to nitrosative stress, 83% of the orfs coding for these proteins were present in the core-genome of c. pseudotuberculosis, this result correspond approximately 46% of the predicted core-genome of c. pseudotuberculosis. functional classification of the proteome of c. pseudotuberculosisexpressed under exposure to nitrosative stress the strain proteome was functionally classified using the blast2go tool. a large proportion of the differentially-regulated proteins and those exclusive to one condition were identified as hypothetical proteins. according to the biological function prediction, biological processes were classified as differentially regulated. in addition, the analysis of the exclusive proteome of each condition revealed common processes between the control and stress conditions. however, seven biological processes were identified only in stress-exposed cells. these processes were antibiotic metabolism, nucleotide metabolism, oxidative phosphorylation, translation, glycolysis pathways, iron-sulfur clusters, and starch and sucrose metabolism. among all processes identified, dna synthesis and repair proteins were most common. an overview of the c. pseudotuberculosis response to nitrosative stress according with the proteins identified is shown in figure  figure comparison of biological processes between control and deta/no conditions. a representation of the biological processes in relation to a set list of proteins identified as differentially-regulated in deta/no-stressed cells and comparison of exclusive biological process between the two test conditions. overview of c. pseudotuberculosis response to nitrosative stress. all proteins detected by proteomic analysis are marked in red. the proteins that were grouped into of transcriptional process were evaluated by cmregnet and among regulators identified; we identified the gntr- family regulatory protein, genes regulated by gntr-type regulators are usually involved in carbohydrate metabolism. the cmregnet analysis showed that of the four genes under the control of this regulator, the n-acetylglucosamine kinase protein was highly expressed by c. pseudotuberculosis in response to deta/no. we identified other regulator the lexa repressor that was down regulated in the deta/no condition. according with cmregnet, two proteins regulated by this repressor were detected in the deta/no proteome specific, pyridoxal biosynthesis lyase and dna translocase. others proteins under the control of this repressor was detected, however not presented significant differential regulation like reca protein protein-protein interaction network to investigate the interactions among the proteins identified as exclusive and differentially regulated in cells exposed to deta/no, we generated a protein interaction network using cytoscape. the interactome analysis revealed protein-protein interactions. dnab/dna helicase, identified in the exclusive proteome for strain 1002_deta/no, and pyre/orotate phosphoribosyltransferase, which was down-regulated in strain 1002_deta/no, showed the greatest number of interactions with other proteins. moreover, both of these proteins interact with proteins that are involved in metabolic processes, dna processes, antibiotic metabolism, cell cycling, and translation.figure protein-protein interactions. protein-protein interactions of the proteins identified in deta/no-exposed cells. exclusive proteome, circle; up-regulated, square; and down-regulated, rhombus. the sizes of the nodes represent the degree of interaction for each gene/protein; the major nodes demonstrate greater interactions. the colors of nodes and lines are in an increasing gradient scale from yellow to green to blue. the networks were visualized using cytoscape. discussion c. pseudotuberculosis is exposed to different forms of oxidative and nitrosative stress during the infection process. a previous study showed that c. pseudotuberculosis resists nitrosative stress generated by the no-donor deta/no, and that a low concentration of deta/no induces a change in the extracellular proteome this pathogen. to better understand the physiology of c. pseudotuberculosis in response to nitrosative stress, we analyzed the proteome of whole bacterial lysates of c. pseudotuberculosis in response to exposure to deta/no. the strain proteome under nitrosative stress reveals proteins involved in bacterial defense against dna damage proteomic analysis identified proteins involved in dna repair systems in both the exclusive proteome of deta/no-exposed cells and in the differentially-regulated proteome. we detected the proteins formamidopyrimidine-dna glycosylase, recb, and methylated-dna-protein-cysteine methyltransferase, the genes for which were previously identified in a transcriptome analysis of strain in response to different abiotic stresses. activation of these proteins in response to nitrosative stress confirms that they belong a group of general stress-response proteins in c. pseudotuberculosis. the expression of fpg was up-regulated in response to acid stress. we also identified endonuclease iii, which, in addition to fpg, is involved in the base excision repair system of various bacteria. this system cleaves n-glycosidic bonds from damaged bases, allowing their excision and replacement. in salmonella enterica serovar typhimurium, the ber system repairs dna damaged by exposure to no. in addition, an s. typhimurium strain defective in fpg demonstrated reduced virulence in a murine model. our interactome analysis showed that endo iii had one of the highest numbers of interactions with other proteins, including interactions with proteins involved in dna replication such zinc metalloprotease and dna translocase, suggesting that this protein could play an important role in the defense pathway against rns. the ada and recb protein were up-regulated in response to osmotic stress. ada is involved in the repair of dna-methylation damage, this protein have plays important in the pathway dna damage. recb is a component of the recbc system, which is part of the sos response the more regulatory network encoded by prokaryotic involved in dna repair. the recbc system acts in the recombination or degradative repair of arrested dna replication forks. studies in s. typhimurium showed that recbc mutant strains are more attenuated than reca mutants in a murine model of infection. in addition, unlike reca mutants, recbc mutants were susceptible to rns, indicating that recbc is highly important in the bacterial response to nitrosative stress. the lexa repressor, which forms part of the general sos system along with reca, was down-regulated in c. pseudotuberculosis cells exposed to deta/no. we also detected the reca protein; however, despite having a p-value <, the fold-change of - showed that this protein was not activated under the experimental conditions. studies performed in mycobacterium tuberculosis showed that reca was not induced until cells had been exposed to deta/no for 4 h, but that hydrogen peroxide induced the immediate expression of reca, suggesting that reca is involved in the later stages of the nitrosative stress response. nevertheless, cmregnet analysis identified other proteins that are regulated by lexa in the deta/no-specific proteome, including pyridoxal biosynthesis lyase and dna translocase. no-sensitive transcriptional regulators are activated in the presence of no to activate these dna repair systems, it is essential that bacteria can detect ros and rns, and concomitantly activate the transcriptional regulators needed for the expression of genes involved in protection against these compounds. in the deta/no-specific proteome, we detected the transcription factor whib. the whib transcriptional family is composed of iron-sulfur cluster proteins. these proteins are o2- and no-sensitive, and allow the sensing of both external environmental signals and the redox state for intracellular bacteria. in m. tuberculosis, the reaction of the iron-sulfur cluster of whib with no generates a dinitrosyl iron complex, which activates a sensing mechanism in response to the no, consequently activating a system of defense against nitrosative stress. in addition, other in vivo and in vitro studies have also demonstrated that whib regulators play a role in the adaptation and survival of m. tuberculosis during exposure to redox environments. we identified other regulators that are activated in response to environmental stimuli, such as a merr-family transcriptional regulator and a lysr-type transcriptional regulator. this regulator was also highly expressed in the transcriptional response of c. pseudotuberculosis to acid stress. merr-type regulators have been described in the detoxification of toxic metal in several pathogenic and non-pathogenic bacteria. other studies have shown that this class of regulator plays a role in bacterial resistance to oxidative and nitrosative stress. lttrs are associated with the regulation of several biological processes, as well as in the adaptive response of bacteria to different types of stress. in vibrio cholerae, lttrs are associated with efflux pump regulation, which contribute to antimicrobial resistance, and are involved in colonization of the human host. in pathogens like e. coli, enterococcus faecalis, s. enterica, and pseudomonas aeruginosa, lttrs are involved in resistance to oxidative stress. the detoxification pathways of c. pseudotuberculosisfollowing no exposure our proteomic analysis identified proteins specifically expressed by cells exposed to deta/no that are involved in the detoxification process. two of these proteins were thioredoxin and glutathione peroxidase. the thioredoxin and glutathione systems play major roles in thiol and disulfide balance, respectively. in pathogens such as helicobacter pylori, streptococcus pyogenes, and m. tuberculosis, this system is of great importance in combating the presence of ros/rns. a glyoxalase/dioxygenase was identified in the differential proteome of cells exposed to deta/no. this protein was previously detected in the proteome of c. pseudotuberculosis strain in response to  mm deta/no. the presence of this protein suggests that glyoxalase/dioxygenase plays a role in the resistance of this pathogen to nitrosative stress. nevertheless, unlike p. aeruginosa, which contains a complete denitrification pathway, the predicted genome of c. pseudotuberculosis ovis revealed a truncated denitrification pathway. however, we detected the nitric-oxide reductase cytochrome b in the exclusive proteome of deta/no-stressed cells. norb, which codes for this nitric-oxide reductase, is organized into the norcbqdef operon in paracoccus denitrificans, and into the norcbd operon in p. aeruginosa. the c. pseudotuberculosis genome was predicted to only contain norb. moreover, norb is located in the cp1002picp pathogenicity island, suggesting horizontal acquisition of the gene by this pathogen. nitric-oxide reductase is an important protein in the denitrification process of some bacteria. in p. aeruginosa, norb plays a role in both the growth of the pathogen in the presence of no, and in its survival in macrophages. the flavohemoglobin hmp is involved in the no detoxification pathway in s. typhimurium, and levels of hmp are increased approximately two-fold in macrophages. interestingly, in n. meningitidis, norb levels are increased ten-fold in macrophages, demonstrating the great power of this protein in the detoxification process. metabolic profile of c. pseudotuberculosisin response to nitrosative stress in addition to the presence of proteins involved in bacterial defense and detoxification pathways, strain needs to undergo metabolic adaptation to favor bacterial survival. we observed a metabolic readjustment in this pathogen in the proteomic analysis. of the proteins involved in central carbohydrate metabolism, we detected only phosphoglycerate mutase and n-acetylglucosamine kinase in the proteome of deta/no-exposed cells. other essential proteins involved in glycolysis, the pentose phosphate pathway, and the citric acid cycle were not detected. similar results were found in a metabolomic study of v. cholerae in response to nitrosative stress. however, we hypothesized that c. pseudotuberculosis uses oxidative phosphorylation to obtain energy. this is supported by the presence of cytochrome c oxidase polypeptide i, succinate dehydrogenase cytochrome b subunit, and ubiquinol-cytochrome c reductase cytochrome c subunit in the exclusive proteome of deta/no-stressed cells, and by the up-regulation of the cytochrome oxidase assembly protein under the same conditions. however, this oxidative phosphorylation may be associated with the bacterial culture conditions used in this work, in which c. pseudotuberculosis was cultivated in the presence of deta/no under aerobic conditions. studies have shown that growing m. tuberculosis in a low concentration of no with low levels of o can induce anaerobic respiration as a result of the inhibition of the respiratory proteins cytochrome c oxidase and nadh reductase by irreversible ligation of no. the ligation of no to the respiratory proteins is an effect that may be both short-term reversible and long-term irreversible. thus, we suggest that activation of the oxidative phosphorylation system may be a more effective pathway for this pathogen to obtain energy. another metabolic adjustment was observed in relation to amino acid biosynthesis. transporters and enzymes involved in the synthesis of methionine, tryptophan, and serine were identified. however, the presence of these proteins can be associated with the bioavailability of these amino acids during exposure to no. in addition, we detected two oligopeptide transport atp-binding proteins that compose the oligopeptide permease system. this complex is associated with the internalization of peptides from the extracellular environment to be used as a source of carbon and nitrogen in bacterial nutrition. we also identified proteins that are cofactors of metabolism, such as coabc, phosphopantetheine adenylyltransferase, and 2-dehydropantoate 2-reductase. the presence of these proteins demonstrates activity in pantothenic acid metabolism and the biosynthesis of coenzyme a. studies performed in species such as corynebacterium diphtheriae, streptococcus haemolyticus, and m. tuberculosis showed that pantothenic acid and coa could have an important role in the growth and viability of these pathogens.  in this work, we applied high-throughput proteomics to characterize the proteome of c. pseudotuberculosis ovis following exposure to no. our proteomic analysis generated two profiles, which together validated findings from previous in silico analyses of c. pseudotuberculosis ovis the proteomic profile generated after the addition of the no-donor, deta/no, revealed a set of proteins that are involved in distinct biological process. we detected proteins related to both the general stress response and to a more specific nitrosative stress response, which together form a network of factors that promote the survival of this pathogen under stress conditions. however, more detailed studies are needed to assess the true role of these proteins in response to nitrosative stress in c. pseudotuberculosis. in conclusion, this functional analysis of the genome of c. pseudotuberculosis shows the versatility of this pathogen in the presence of no. moreover, the results presented in this study provide insights into the processes of resistance of c. pseudotuberculosis during exposure to nitrosative stress. electronic supplementary material additional file 1: table s1: complete list of proteins identified as significantly altered. additional file 2: table s2: unique proteins identified in strain 1002_deta/no. additional file 3: table s3: unique proteins identified in strain control condition. abbreviations gguanine ccitosine nonitric oxide rnsreactive nitrogen species nosnitric oxide synthases lc-hdmseliquid chromatograph high definition mass spectrometry lc/msliquid chromatograph mass spectrometry cdmchemically-defined médium deta/nodiethylenetriamine/nitric oxide adduct dttdithiothreitol 2d-rptwo-dimensional reversed phase nanouplcnano ultra performance liquid chromatography nanoesi-hdmsnano electrospray high definition mass spectrometry hsshigh strength silica plgsprotein lynx global server fdrfalse discovery rate. competing interests the authors declare that they have no competing interests. authors’ contributions wms, rdc, and ifsb performed microbiological analyses and sample preparation for proteomic analysis. ghmfs and wms conducted the proteomic analysis. scs and elf performed bioinformatics analysis of the data. yll and am contributed substantially to data interpretation and revisions. va and as participated in all steps of the project as coordinators, and critically reviewed the manuscript. all authors read and approved the final manuscript. 
in silico analysis of the genome of c. pseudotuberculosis ovis predicted several genes that could influence the resistance of this pathogen to nitrosative stress. our proteomic data set suggested the activation of both a general stress response and a specific nitrosative stress response, as well as changes in proteins involved in cellular metabolism, detoxification, transcriptional regulation, and dna synthesis and repair. our proteomic analysis validated previously-determined in silico data for c. pseudotuberculosis ovis in addition, proteomic screening performed in the presence of no enabled the identification of a set of factors that can influence the resistance and survival of c. pseudotuberculosis during exposure to nitrosative stress. in total, proteins were exclusive to the proteome of deta/no-induced cells, and a further proteins were differentially regulated between the deta/no and control conditions. here, we applied high-throughput proteomics using high definition mass spectrometry to characterize the functional genome of c. pseudotuberculosis ovis in the presence of no-donor diethylenetriamine/nitric oxide adduct, with the aim of identifying proteins involved in nitrosative stress resistance. we characterized proteins, representing approximately 41% of the predicted proteome of c. pseudotuberculosis ovis following exposure to nitrosative stress. an interactomic analysis of the differential proteome of c. pseudotuberculosis in response to nitrosative stress was also performed. during the infection process, the bacterium is subjected to several stress conditions, including nitrosative stress, which is caused by nitric oxide. corynebacterium pseudotuberculosiscaseous lymphadenitisproteomicslabel-free proteomicsnitrosative stressnitric oxideissue-copyright-statement© the author 2014 corynebacterium pseudotuberculosis biovar ovis is a facultative intracellular pathogen, and the etiological agent of caseous lymphadenitis in small ruminants.
 corynebacterium pseudotuberculosis biovar ovis is a facultative intracellular pathogen, and the etiological agent of caseous lymphadenitis in small ruminants. during the infection process, the bacterium is subjected to several stress conditions, including nitrosative stress, which is caused by nitric oxide. in silico analysis of the genome of c. pseudotuberculosis ovis predicted several genes that could influence the resistance of this pathogen to nitrosative stress. here, we applied high-throughput proteomics using high definition mass spectrometry to characterize the functional genome of c. pseudotuberculosis ovis in the presence of no-donor diethylenetriamine/nitric oxide adduct, with the aim of identifying proteins involved in nitrosative stress resistance.  we characterized proteins, representing approximately 41% of the predicted proteome of c. pseudotuberculosis ovis following exposure to nitrosative stress. in total, proteins were exclusive to the proteome of deta/no-induced cells, and a further proteins were differentially regulated between the deta/no and control conditions. an interactomic analysis of the differential proteome of c. pseudotuberculosis in response to nitrosative stress was also performed. our proteomic data set suggested the activation of both a general stress response and a specific nitrosative stress response, as well as changes in proteins involved in cellular metabolism, detoxification, transcriptional regulation, and dna synthesis and repair.  our proteomic analysis validated previously-determined in silico data for c. pseudotuberculosis ovis in addition, proteomic screening performed in the presence of no enabled the identification of a set of factors that can influence the resistance and survival of c. pseudotuberculosis during exposure to nitrosative stress. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords corynebacterium pseudotuberculosiscaseous lymphadenitisproteomicslabel-free proteomicsnitrosative stressnitric oxideissue-copyright-statement© the author 2014 