Differential transcriptional profile ofCorynebacterium pseudotuberculosisin response to abiotic ...

14 Pages·2014·1.04 MB·English

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RESEARCH ARTICLE Open Access
Corynebacterium
Differential transcriptional profile of
pseudotuberculosis
in response to abiotic stresses
Anne Cybelle Pinto1, Pablo Henrique Caracciolo Gomes de Sá2, Rommel T J Ramos2, Silvanira Barbosa2,
Hivana P Melo Barbosa2, Adriana Carneiro Ribeiro2, Wanderson Marques Silva1, Flávia Souza Rocha1,
Mariana Passos Santana1, Thiago Luiz de Paula Castro1, Anderson Miyoshi1, Maria P C Schneider2, Artur Silva2
and Vasco Azevedo1*
Abstract
Background: The completion of whole-genome sequencing for Corynebacterium pseudotuberculosis strain 1002 has
contributedtomajoradvancesinresearchaimedatunderstandingthebiologyofthismicroorganism.Thisbacterium
causessignificantlosstogoatandsheepfarmersbecauseitisthecausalagentoftheinfectiousdiseasecaseous
lymphadenitis,whichmayleadtooutcomesrangingfromskininjurytoanimaldeath.Inthecurrentstudy,wesimulated
theconditionsexperiencedbythebacteriaduringhostinfection.BysequencingtranscriptsusingtheSOLiDTM3Plus
platform,weidentifiednewtargetsexpectedtopotentiatethesurvivalandreplicationofthepathogeninadverse
environments.Theseresultsmayalsoidentifypossiblecandidatesusefulforthedevelopmentofvaccines,diagnostic
kitsortherapiesaimedatthereductionoflossesinagribusiness.
Results:Underthe3simulatedconditions(acid,osmoticandthermalshockstresses),474differentiallyexpressed
genesexhibitingatleasta2-foldchangeinexpressionlevelswereidentified.Importantgenestotheinfectionprocess
wereinduced,suchasthoseinvolvedinvirulence,defenceagainstoxidativestress,adhesionandregulation,andmany
genesencodedhypotheticalproteins,indicatingthatfurtherinvestigationofthebacteriumisnecessary.Thedatawill
contributetoabetterunderstandingofthebiologyofC.pseudotuberculosisandtostudiesinvestigatingstrategiesto
controlthedisease.
Conclusions:DespitetheveterinaryimportanceofC.pseudotuberculosis,thebacteriumispoorlycharacterised;
therefore,effectivetreatmentsforcaseouslymphadenitishavebeendifficulttoestablish.ThroughtheuseofRNAseq,
theseresultsprovideabetterbiologicalunderstandingofthisbacterium,shedlightonthemostlikelysurvival
mechanismsusedbythismicroorganisminadverseenvironmentsandidentifycandidatesthatmayhelpreduceor
eveneradicatetheproblemscausedbythisdisease.
Keywords:Differentialgeneexpression,Transcripts,RNAseq,SOLID™,Stress,C.pseudotuberculosis
Background arabinogalactan and mycolic acids and (ii) the high G+C
Corynebacterium pseudotuberculosis is a Gram-positive contentofthegenome(47-74%)[1].
pathogenic bacterium belonging to the class Actino- The bacterium causes caseous lymphadenitis disease,
bacteria, which is a member of the Corynebacterium, which affects small ruminants and large animals (such
Mycobacterium, Nocardia and Rhodococcus genera as horses and cattle) worldwide and can infect humans
(the CMNR group). The CMNR group shares several [1]. Therefore, there is an urgent need to control the
common characteristics, including (i) the organisation of disease through the development of effective vaccines,
the cell wall, which is mainly composed of peptidoglycan, therapiesanddiagnostic kits.
Because C. pseudotuberculosis is a facultative intracel-
*Correspondence:[email protected] lularmicroorganismfoundpreferentiallyinmacrophages
1DepartmentofGeneralBiology,InstitutodeCiênciasBiológicas,
in the host, during the infection process, the bacterium
UniversidadeFederaldeMinasGerais,Av.AntônioCarlos,BeloHorizonte
31.270-901,Brazil
Fulllistofauthorinformationisavailableattheendofthearticle
©2014Pintoetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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is exposed to a number of environmental changes that [9]; the high concentration of cell wall lipids, which ren-
arefarfrom ideal[2]. ders the microorganism resistance to digestion by cellu-
After phagocytosis, the phagosome quickly becomes lar enzymes and allows it to persist as a facultative
acidic (pH~4.6-5.0)[3]negatively affectingthemetabol- intracellular parasite [10] and CP40, identified as an im-
ism and damaging macromolecules in the invading cell. munogenic protein that exhibits proteolytic activity as a
In addition, other intracellular stresses negatively affect serineprotease[11].
the microorganism, including oxidative thermal shock The availability of the C. pseudotuberculosis strain
and nitrosative, surface, osmotic and starvation stresses; 1002 genome (access number CP001809) has allowed
however, the bacterium manages to escape and persist in the further investigation and characterisation of the
theenvironment [2]. microorganism, which is poorly characterised despite its
To survive in this environment, the pathogen must importance to agribusiness. Therefore, to generate add-
mount an immediate and adequate, protective response itional information, thetranscriptionalprofile ofC.pseu-
that is reflected initially by transcriptional changes in dotuberculosis was analysed using cDNA sequencing
specific sets of genes [4]. In this context, sigma factors, with SOLiD™ 3 plus next-generation technology (Life
which coordinate the expression of these genes under TechnologiesTM, CA). Using this technology, we inves-
different types of stresses are important [2]; these factors tigated the molecular characteristics of the microorgan-
include sigS in E.coli, which is involved in trehalose syn- ism that allow it to persist in the host and the
thesis during osmotic stress, with trehalose serving as an mechanisms the bacteria use to escape the host immune
important osmoprotectant in this type of stress [4]. In response. Additionally, in an attempt to contribute to
Mycobacterium tuberculosis, RT-PCR (real-time poly- the elimination of caseous lymphadenitis in caprine and
merase chain reaction) was used to demonstrate the ovinepopulations,weinvestigatedalargenumberoftar-
transcriptional profile of 10 sigma factors during the ex- gets related to C. pseudotuberculosis virulence by simu-
ponential growth phase. The role of these sigma factors lating the specific conditions tolerated by the bacterium
was analysed under different conditions of stress, and a when invading the host (i.e., acidic, osmotic and high
number of the factors demonstrated increased expres- temperaturestresses).
sion in response to one kind of stimulus, whereas others
responded to more than one stimulus. The resistance to Results and discussion
different environmental stresses is associated with the The sequencing of cDNA is an attractive technology for
ability of the pathogenic bacteria to survive in the host, the investigation of gene expression in prokaryotic or-
and different sigma factors play fundamental roles in the ganisms because it provides a high level of coverage and
survival of the pathogen. For example, in M. smegmatis, high sensitivity for the detection of transcripts at consid-
sigB is involved in the response to oxidative stress, and erably lower costs compared to traditional methods [12].
sigE was shown to play a role in the regulation of genes Currently, sequencing technology is considered the gold
involved in the response to acid stress, thermal shock standard for the analysis of gene expression levels [13].
andsodium dodecylsulphate (SDS) exposure[5,6]. Therefore, because themicroorganism can survive envir-
The functions of sigma factors include involvement in onmental changes in the host during infection, we ana-
the adaptation to stress, the interaction of the bacterium lysed the transcriptional profile of C. pseudotuberculosis
with the extracellular medium and in a number of cases, strain 1002 using RNAseq technology. Acid, osmotic
with bacterial virulence [7]. and thermal shock stresses were used to identify genes
For C. pseudotuberculosis 1002, there is still no infor- involved in bacterial tolerance of these unfavourable
mation regarding the role of sigma factors in the regula- environments.
tion of genes involved in bacterial survival throughout Stress-generating agents were applied to the cells at an
the infection process. In addition, few virulence determi- optical density (OD) of 0.2 (A =0.2), and the cell
600nm
nants contributing to bacterial survival have been identi- viability analysis demonstrated a reduction in replication
fied. Therefore, investigations related to the control of of approximately 27% under thermal stress, 34% under
caseous lymphadenitis have been difficult [1]. To date, acid stress and 23% under osmotic stress (Figure 1). A
the virulence determinants most studied in C. pseudotu- lack of growth or reduced growth is normal during pe-
berculosis infection include the following: the PLD riods of environmental change as the organism attempts
(phospholipase D) protein, an exoprotein that is also to adapt to and physiologically adjust to the new envir-
considered leukotoxic, contributing to the formation of onment[14].
lesions and the destruction of caprine macrophages dur- The cDNA samples were sequenced using the SOLiD™
ing infection [8];the fagABC operon and the fagD gene, 3 Plus platform, which allowed the analysis of the gene
which play a role in the virulence of the bacterium and expression profile of the microorganism in the early
havebeenidentifiedasgenesinvolvedinironacquisition exponential phase. Using the Bioscope programme,
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Figure1Numberofviablecellsundereachcondition.Thecontrolconditionscorrespondtoapproximately6.7x107cellsmL-1ofC.
pseudotuberculosisstrain1002,withtheexceptionoftheosmoticstresscontrol,whichcorrespondsto6.0x107cellsmL-1.Red,controlconditions.
Green,thermalshockstress.Yellow,acidstress.Blue,osmoticstress.Figuretakenfrom[16].
unique readings were mapped on the genome, and gene (98.70%) transcripts under thermal stress and 2,064
expression was quantified based on the RPKM (reads (97.76%)underacidstress.
per kilobase of coding sequence per million mapped) According to the DEGseq analysis software, of the
[15]. Ribosomal transcripts were filtered using Bioscope, 2,065 transcripts produced following the osmotic stress,
and the total number of readings obtained before and 889 (43.05%) were considered differentially expressed
afterapplicationofthe filterisshowninTable1. comparedtothe control(p-value<0.001)[16](Figure 2),
Genomic coverage was inferred using the data gener- 565ofthesegeneswereinducedand324wererepressed.
ated in Bioscope, which represents the level of expres- In the thermal stress experiment, 543 (26.32%) tran-
sion in the RNAseq experiments. The osmotic stress scripts were considered differentially expressed, of which
produced the largest number of uniquely mapped tran- 374 were induced and 169 were repressed. In the acid
scripts throughout the entire genome, followed by the stress,811(39.30%)transcriptswere considereddifferen-
thermal stress, acid stress and control conditions tially expressed, of which 519 were induced and 292
(Table2). were repressed.
The automatic annotation of the genome using the Among the differentially expressed genes, the genes
Fgenes software (www.softberry.com), followed by man- exhibiting a 2-fold change in expression (at least 2x rela-
ual curation of the C. pseudotuberculosis strain 1002 tive to the control) were selected for analysis. The fold-
genome, identified 2,090 coding regions. From the change values were calculated based on the RPKM
cDNA sequencing performed using SOLiDTM at the be- value between the stress and the control, in which a
ginning of theexponential phase, 2,055transcripts active value greater than one indicated induced gene expres-
in control were identified (equivalent to 98.32% of the sion and a value less than one indicated repressed
transcribed genome) and 35 genes (1.67%) were consid- gene expression.
ered non-transcripts, exhibiting a RPKM value of 0. In a previous study reported by our group [17], se-
Under the different stress conditions, 2,065 (98.80%) quencingoftranscriptsfrom C. pseudotuberculosisstrain
transcripts were produced under osmotic stress, 2,063 Cp31 (biovar equi) was performed using an Ion Torrent
Table1Numberoftotalreadingsobtainedduring
Table2Numberofuniquereadingsmappedinthe
sequencing
genomeofC.pseudotuberculosis1002andcoverageof
pH 2M 50°C Control
thetranscriptsinthegenome
GrossData 17,393,077 18,783,810 21,622,844 25,235,478
2M 50°C pH Control
FilteredRibosomal 9,738,772 9,564,434 9,971,878 9,270,342
Uniquelymapped 2,016,131 1,633,118 1,764,047 1,650,975
Transcripts
readings
Grossdataincludereadingsofalltranscripts.Filteredribosomaltranscripts
Genomecoverage 43x 39x 37x 35x
refertothenumberofreadingsoftranscriptsnotconsideredintheanalysis.
pH-acidstress;2M-osmoticstress,50°C-thermalstressandcontrol-nostress. 2M-osmoticstress;50°C–thermalstress;pH-acidstressandcontrol-nostress.
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Figure2Numberofgenesdifferentiallyexpressedrelativetothecontrol.Red,genestranscribedundereachcondition(sumofyellowand
green).Yellow,genesinducedrelativetothecontrol.Green,genesrepressedrelativetothecontrol.2M,osmoticstress;50°C,thermalstressand
pH,acidstress.
platform (Life Technologies). In this study, a compara- molecules, whichareincreasedfollowingexposuretocer-
tive analysis between 2 rRNA depletion methodologies tainenvironments[19].Theresultsdemonstratedthatthe
wasperformed, andthe transcriptsweresubmitted toab growth of the bacterium was reduced but was not inter-
initio assembly. Both transcriptomes were then submit- rupted under these conditions; therefore, the bacterium
ted to gene ontology analysis according to biological survived in the environment. This observation was
processes and molecular functions. Data were obtained confirmed by the biosynthetic process, which was com-
only under physiological conditions using brain heart in- prisedof3genes(seeAdditionalfile1:FigureS1),indicat-
fusion (BHI) media. The authors observed that few tran- ing that the bacterium was able to invest energy into the
scripts represented genes involved in pathogenicity or replication process and survived in the unfavourable
the cell adhesion process and concluded that contact environment.
with the host may influence the induction of these The adhesion process, comprising 3 genes, was prom-
transcripts. inent under osmotic stress. Adhesion is essential for the
The present study simulated some of the environmen- initiation of the infectious process because the
tal conditions that the pathogen faces in the host. The bacterium-host interaction establishes a pathogenic rela-
celladhesion process wasamongthose mostrepresented tionship. The Cp1002_0988 gene, encoding a hypothet-
in the conditions described below, and these levels ical protein, is located within 1 of the pathogenicity
showed greater than a 2-fold change. This result indi- islands of C. pseudotuberculosis 1002, indicating its
cated that there were a greater number of transcripts importance in the development of the disease. Addition-
representing genes that participate in the cell adhesion ally, this gene exhibited a 6.8-fold change in expression
process when compared to the physiological condition. levels compared with the control (see Additional file 2:
Together, these findings demonstrate that contact with Table S1). The Cp1002_1764 and Cp1002_1765 genes,
the host most likely influences the transcription of genes also identified as encoding hypothetical proteins, exhib-
essentialforbacterial survival. ited 2.7-fold and 4-fold changes in expression, respect-
ively,comparedwith the control.
Genesinducedinthebiologicalprocessesoftheosmotic Pathogenicbacteria havedeveloped highlysophisticated
stressstimulon signal transduction systems that control the coordinated
The Blast2GO programme was used to identify the bio- expression of a number of virulence determinants in
logical processes most abundant in the cells under responsetoenvironmentalstresses,andchangesinosmo-
osmotic stress. However, for a more detailed analysis of laritycontributetotheexpressionofthegenes[20];there-
the processes (Figure 3), the CoreStImulon (CSI) fore, further studies to identify the proteins encoded by
programme [18] was used, which identified the genes these genes and toevaluate the true contribution of these
present in each of the processes determined in the proteinswillbenecessary.
Blast2GOprogramme(seeAdditionalfile1:FigureS1).
Themajority of the genes induced under osmoticstress Genesinducedinthebiologicalprocessesoftheacid
werepartoftheoxidoreductionprocess, represented by4 stressstimulon
genes (see Additional file 1: Figure S1). Normal aerobic Under acid stress conditions, the induction of genes
metabolism induces the production of active oxygen involved in the processes of cellular adhesion and
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Figure3Biologicalprocessesmostevidentamongthegenesinducedintheosmoticstimulon.FigureobtainedwiththeCSIprogram.
oxidoreduction in response to stress were of paramount made up the cellular oxidoreduction process, we ob-
importance because they are associated with virulence served the presence of genes with functions that ap-
(Figure 4) (see Additional file 3: Figure S2). The adhe- peared essential for the persistence of the bacterium in
sion processes were composed of hypothetical proteins, thisenvironment.
1 of which was characterised as a secreted protein con- The Cp1002_2043 gene exhibited a 7.8-fold change in
taining an LPxTG domain, which may be an important expression (see Additional file 4: Table S2). This gene,
vaccine candidate. Through analysing the genes that which may encode the Dps protein, was linked to the
Figure4Biologicalprocessesmostevidentamongthegenesinducedintheacidstimulon.FigureobtainedusingtheCSIprogram.
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processes of stress response and cellular oxidoreduction gene is among the genes that exhibited the highest fold-
(see Additional file 3: Figure S2). This protein protects change in expression values (Figure 5) (see Additional
the organism against oxidative stress because it stores file 5: Figure S3), and the gene may encode choline
iron in a bioavailable form, reducing the possibility of dehydrogenase. The induced gene exhibited a 5-fold
the production of reactive oxygen molecules. Under change in expression (See Additional file 6: Table S3)
acidic conditions, the production of molecules that pro- comparedtothe control.Theproteinbelongstotheoxi-
duce reactive oxygen species is increased. Reports have doreductase family, which catalyses the oxidation of
demonstrated that in cells in which the pH is decreased, choline to glycine betaine via the intermediate betaine
– –
the ratio of HOO to O increases [19], increasing the aldehyde. The protein promotes increased tolerance to
2
chance of producing hydrogen peroxide (H O ). There- hypersalinityandfreezingandcontributestotheosmotic
2 2
fore, the increased number of genes constituting the balance of the cell under stress. The osmoprotectors not
cellular oxidoreduction process is justified. Another only play a role in osmotic balance but also act as effect-
important gene in the oxidoreduction process was ive stabilisers of enzymatic function, providing protec-
Cp1002_0173, which exhibited a 4-fold change in tion against salinity, high temperatures, freezing,
expression and is presumed to encode a catalase that thawing andeven dryness[26].Under conditions of high
plays a role in reducing the concentration of H O in salinity, the osmoprotectors, together with the transport
2 2
the cell. system, function as virulence factors in certain patho-
The mechanisms used by the bacterium to resist the genic bacteria [20]. Understanding how these elements
damage caused by reactive oxygen species are essential operate under this condition of stress and their relation-
forsurvivalwithinthemacrophage[21].Therefore,these ship with pathogenesis will be important for future
proteins might promote the survival of the bacterium in studies.
theacidmediumstartingattheearly exponentialgrowth TheadhesionprocesscomprisestheCp1002_1765gene,
phase. which may encode a secreted protein. Cp1002_1765
The Cp1002_1192 msrB (methionine sulphoxide pep- exhibited a 2-fold change in expression compared
tide reductase) gene, which was 1 of the genes present with the control, and it is an important candidate for
in the oxidoreduction process, exhibited a high (16-fold) studies related to controlling the disease caseous
change in expression. Because the MsrA protein may be lymphadenitis.
required to maintain the role of adhesins, the high Among the genes involved in the process of the
increase in msrB expression suggested that the protein response to stress, the Cp1002_1895 gene, which may
contributes to the survival of the pathogen in the host, encodeaheat shockregulatoryprotein(HspR),exhibited
the resistance to oxidative stress in vitro and the adhe- the largest fold-change in expression (4x relative to the
sion capability of eukaryotic cells [22]. The methionine control). This protein acts as a negative regulator of the
(Met) residues in proteins exposed at the cell surface are expression of genes encoding chaperones and proteases
thought to be involved in capturing reactive oxygen spe- in different bacteria under physiological conditions. The
cies, and a complex comprised of MsrA and MsrB re- heat shock proteins play a key role in cellular metabol-
duces the oxidised Met residues, removing the reactive ism under all growth conditions, monitoring the folding,
oxygen species [23]. Met is the amino acid most sensi- assembly and translocation of cellular proteins [27]. In
tive to reactive oxygen species, and the oxidation of the M. tuberculosis, HspR represses the operon formed by
Met residue in a protein alters the protein structure (or the dnaK-grpE-dnaJ-hspR genes through interaction at
prevents translation), drastically affecting the function the HAIR (HspR-associated inverted repeats) region lo-
[24]. There is evidence that only the MsrB domain is cated in the 5′UTR region of the genes. In C. pseudotu-
present in C. pseudotuberculosis 1002. However, the loss berculosis, the hspR gene is located in the reverse strand,
of the msrA-msrB domain or msrB alone in Helicobacter belowthednaJ,GrpE anddnaKgenes,indicativeoftheir
pylori resulted in a reduction in virulence in mouse regulationbyHspR.
models,likelybecauseoftheoxidationofimportantpro- It has been suggested that the HspR protein acts as a
teins [25]. Therefore, the process of cellular oxidoreduc- repressor of other genes linked to virulence/pathogen-
tion may involve genes that contribute predominantly to icity [27]. A study in M. tuberculosis demonstrated that
the maintenance and persistence of the bacterium in the partial disruption of heat-shock regulation influences
mediaharmfultothe cell. virulence because the bacterium loses the ability to
establish a chronic infection [28]. Mutation of hspR in
Genesinducedinthebiologicalprocessesofthethermal the organism produced increased expression of the
shockstimulon DnaK chaperone, which is highly antigenic, resulting in
We analysed the genes involved in the oxidoreduction anenhancedimmuneresponseinthehost. Furthermore,
process under thermal stress. The Cp1002_1785 (betA) it was demonstrated that DnaK acts as co-repressor for
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Figure5Biologicalprocessesmostevidentamongthegenesinducedinthethermalstimulon.FigureobtainedusingtheCSIprogram.
HspR; the activity of HspR is dependent on DnaK. A the proteins contribute strongly to the survival and per-
proposed mechanism for the regulation of hspR expres- sistence of the bacterium in unfavourable environments;
sion is that under conditions of heat shock, the operon therefore, these proteins are potential candidates for the
is induced, leading to the increased synthesis of DnaK development of vaccines, diagnostic kits or therapies for
and HspR. When the concentration of these proteins caseous lymphadenitis.
reaches a critical level, they bind to the promoter of the Among the shared repressed genes were those encod-
operon, resulting in repression [29]. To investigate ing proteins related mostly to energy metabolism, sugar
whether this scenario occurred in C. pseudotuberculosis, transport, amino acids that contribute greatly to the
we analysed the gene encoding DnaK and demonstrated maintenance and replication of the organism in the en-
that the expression of the gene was induced 2.5-fold vironment [30], such as argJ [31], nanK [32], opp [33]
relative to the control, which is consistent with the pro- (see Additional file 8: Table S5) and hypothetical pro-
posedmechanism(seeAdditionalfile6:TableS3). teins. These data are consistent with the colony-forming
unitexperiments,whichdemonstratedadecreaseinrep-
Distributionofgenesamongthesimulatedconditions lication in the stressful environments (Figure 1). A re-
From theVenn diagram (Figure 6) it was possible to ob- duction in growth is a survival strategy and occurs in
serve the distribution of genes per condition, demon- essentially all stressful situations because the organism’s
strating 29 genes active in all 3 simulated environments. ability to perceive the environment and to modulate the
The analysis of the genes demonstrated that a variation response-controlling mechanisms of resistance, metabol-
in the fold-change in expression, ranging from 2- to 43- ism and other processes [34] which are increased, spe-
times the control, was observed among the stresses. Of cific andsuitable for thenewconditions,isessential.
these genes, 48% encoded hypothetical proteins and
demonstrated a higher fold-change in expression (see Expressionofgenesencodingsigmafactorsunder
Additional file 7: Table S4) under all conditions. The simulatedconditions
data demonstrating the high number of unidentified In their natural environment, or in the host during the
genes reflects the lack ofinformation on C. pseudotuber- infection process, the bacterium is exposed to distur-
culosis, despite its importance in agribusiness. In the bances that require a fast and adaptive response to
host, the bacterium suffers various stresses simultan- ensure the survival of the pathogen. Therefore, it is ne-
eously; therefore, identifying the proteins and their role cessary for the bacterium to change the pattern of
in the cell is essential for a better understanding of the expression of the genes encoding proteins that directly
molecular mechanisms of the bacterium. Presumably, combat the deleterious nature of the stress [35].
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Figure6Venndiagramoftheconditionstested.Distributionofthegenesindicatingdifferentialexpression(A-inductionandB-repression)
withatleasta2-foldchangerelativetothecontrol.50°C,thermalshockstress;2M,osmoticstressandpH,acidstress.
Certain sigma factors play an important and funda- Streptomyces spp. contains several homologues of the
mental role in regulating the expression of virulence principalfactorthatarenotessentialforreplicationunder
genes [36]; therefore, it is important to analyse these normalconditions but appeartoplaya role under certain
genesindividually. growthconditions[39].
In the C. pseudotuberculosis strain 1002 genome, 8 Because sigA demonstrated changes in expression be-
genes encoding sigma factors were identified, which in- tweenthe controlandthestressconditionsinC.pseudo-
cluded the essential sigma factor SigA, non-essential and tuberculosis 1002, it may play a role as an alternative
alternative SigB and 6 alternative factors belonging to sigmafactor,regula[40]tinggenesinvolvedinthemain-
the group of extracytoplasmic factors SigC, SigD, SigE, tenanceofthebacterium.
SigH,SigKandSigM,whicharedispensableandinduced The gene encoding sigma M was considered differen-
frequently in response to specific conditions, for ex- tially expressed only under osmotic stress. This gene
ample,inresponsetostress. may not be expressed preferentially in the early expo-
Under the osmotic stress conditions, the DEGseq nential phase. Microarray analysis of C. glutamicum in
programme demonstrated that only the genes encoding the exponential phase (OD =0.2 to 0.3) demon-
610nm
sigma factors A and M were induced (were within the strated that the disruption of sigM did not affect the
established cutoff of a fold-change of at least 2) (see level of transcription of genes induced by thermal shock,
Additional file 9: Table S6). Under acidic conditions, the which implies that this sigma factor is not involved in
genes encoding sigma factors B, E and H were differen- the regulation of gene expression in response to this
tially expressed and under thermal stress conditions, the stress[40].
genes encoding sigma factors A, D and H were consid- In M. tuberculosis, expression of sigM occurred in
ered differentially expressed; however, the expression of the stationary phase and only under conditions of
these genes were below the cutoff for the analysis thermal stress [41]. In C. glutamicum, experiments
(Figure 7). revealed that the deletion of sigM caused a reduction
It is possible that sigA encodes sigma factor RpoD, in the number of viable cells under conditions of heat
alternatively named sigma 70, which promotes the shock, cold shock and disulphide (an oxidative stress
binding of RNA polymerase to specific sites by acti- subtype) stress in the exponential growth phase
vating the transcription of most genes essential for (OD =0.7). Furthermore, experiments using real-
600nm
exponential growth in Escherichia coli (E. coli) [37]. time PCR demonstrated that the transcription of sigM
In C. pseudotuberculosis 1002, the gene contains the increased significantly after the application of the
4 domains conserved in the sigma 70 family. Sporad- stresses. These results suggest that sigma factor M is
ically, σA can act as an alternative sigma factor that involved in the stress response [42], albeit with a
is specifically required for the expression of virulence strong indication that the involvement occurs at the
genes. An investigation of the role of sigA in E. coli in late growth phase.
which the rpoD gene was induced (from a lack of amino Because there was an increase in the expression of
acids and thermal shock), suggested that the protein was sigM under osmotic stress in C. pseudotuberculosis 1002,
involved in the mechanism of recovery from stress [38]. a study of the regulon will be required to identify the
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Figure7Expressionofgenesencodingsigmafactorsunderconditionsofsimulatedstresses.Thefold-changevaluewasbasedonthe
ratiobetweenthestressRPKMandthecontrolRPKM.Greencolumnsindicatethedifferentiallyexpressedgenes,independentofthefold-change
cutoffestablishedfortheanalysis,whichwasatleast2x.Redcolumnsrepresentthegenesnotdifferentiallyexpressed(p-value>0.001),established
usingtheDEGseqprogramme.a,Osmoticstress.b,Acidstress.c,Thermalstress.
genes encoding proteins that assist in the persistence of reduced compared to wild type [50]. According to a
thebacteriuminthis hostile environment. study in Vibrio cholerae [51], rpoE mutants attenuate
Sigma B is a very common sigma factor in the stress virulence, and the ability of the bacteria to colonise the
response. The role of the protein in the response to mouseintestinesisreduced.
acidity tolerance was established in Bacillus subtilis, According to a study in C. pseudotuberculosis 1002
Brevibacterium flavum, Listeria monocytogenes and [52], compared with the wild type strain, the mutant
Staphylococcus aureus [43-45]. In B. subtilis, SigB regu- 1002 strain is more sensitive to agents that generate
lates the majority of stress responses, thereby contribut- nitrosative, acid (pH 5.5) and surface stresses, which in-
ing to the transcription of more than100 genes [46]. dicates the important role SigE plays in the persistence
Another study demonstrated a reformulation of tran- ofthebacterium inhostile environments.
scription during the infection process and the relevance The investigation of a mutant sigE in M. tuberculosis
of sigma B in controlling the expression of virulence H37Rv [53] demonstrated that the mutant was more
genes important for adaptation to the intestinal environ- sensitive to various environmental stresses, such as ther-
ment in L. monocytogenes [47]. In L. monocytogenes, σB mal shock, SDS and oxidative agents but not to acidity.
contributed to cell survival under different stress condi- In a report on M. Smegmatis [6], the mutant sigE was
tions [48], and the absence of the factor reduced the more sensitive to acidity, hydrogen peroxide, SDS and
ability of the species to invade epithelial cells [49]. It is thermal shock than the wild type. In the M. tuberculosis
known that σB contributes to virulence in several Gram- H37Rv study [53], the authors determined that sigE is
positive pathogens [2], and these observations under- required for the stress response and for the ability to
score the need to study the factor separately in C. grow and survive inside macrophages. Furthermore, it
pseudotuberculosis and to identify the regulon that con- wassuggestedthat sigEinfluencesthe levelof sigBinthe
tributes tothesurvivalandescapeofthebacterium from cell;however,theexpressionofsigBisnotcompletelyin-
thehost immunesystem. fluenced by sigE because the level of sigB was reduced in
The sigE gene, encoding the presumed factor SigE, a the mutant strain, whereas the mRNA levels of other
member of the σ70 subfamily exhibiting extracytoplas- sigma factors were notaffected.
mic function, regulates functions related to perception In C. pseudotuberculosis 1002 under acidic conditions,
and response to changes in the periplasm and in the sigE was induced and the expression of both sigE and
extracytoplasmic environment. In Haemophilus influen- sigB was significant, whereas underconditions where the
zae, the expression of rpoE increased 102-fold after mRNA level of sigE was not considered significant, the
phagocytosis by macrophages, and the survival of Hae- sigB expression was also not significant. These sigma
mophilus influenzae containing a mutated rpoE was factors may influence each other at this exponential
Pintoetal.BMCGenomics2014,15:14 Page10of14
http://www.biomedcentral.com/1471-2164/15/14
phase; however, studies thatare morespecific will be ne- Table3IdentificationinsilicoofncRNAinthegenomeof
cessary toverifythis hypothesis. Corynebacteriumpseudotuberculosisstrain1002usingthe
The sigH gene, which encodes the extracytoplasmic RFAMprogramme
sigma factor H, was induced (within the established cut- ID Score Strand ncRNA
off value) only under the acidic conditions. In M. tuber- RF00023 136.03 + tmRNA.1
culosis, the sigH factor is involved in the response to
RF00059 57.42 + TPP.1
different stresses and it has been suggested that it regu-
RF01747 54.15 - msiK.1
lates the expression of genes involved in the intracellular
RF00080 49.16 - yybP-ykoY.1
survival of the microorganism. Additionally, proteins
that are part of the sigH facto regulon may interact with RF01746 48.99 - mraW.1
the host’s immune system, modulating its response [54].
This observation suggests that at the beginning of the C. Coverage of the transcripts was analysed in the pre-
pseudotuberculosis 1002 replication process, the expres- dicted element with the highest score. Figure 8 shows
sion of the sigH gene and its regulon are required for the coverage under the control condition and under the
thebacteriumtopersist inanacidicenvironment. stress conditions for the ncRNA tmRNA. This element
InC. glutamicum,amutation inthe sigHgene blocked exhibits dual properties, acting as both messenger RNA
the transcription of sigM, and the identification of the and carrier RNA in the rescue of stalled ribosomes [58].
sigH promoter upstream of sigM implied that the factor Under acidic and thermal shock conditions, coverage of
is under the direct transcriptional control of sigH [42]. It this ncRNA was slightly superior compared to the con-
was determined that in C. pseudotuberculosis 1002, trol condition. Therefore, it is possible that in the un-
when sigH expression increased under conditions of favourable environment, because of the reduction in
stress, sigM expression also increased, although the sigM replication, this element is required to attempt the
transcripts did not exhibit significant induction during rescue of stalled ribosomes to avoid a loss in protein
this growth phase. These results suggest that the sigH synthesis, allowing the bacterium to continue to repli-
gene responded to an environment similar to that en- cate in the environment, albeit at a slower pace. In
countered by the bacterium in the host, highlighting the Mycoplasma pneumoniae, tmRNA proved essential for
importance of identifying the regulon and the genes re- growth and in Salmonella typhimurium, this element is
sponsible for the persistence of the bacterium in the necessary for survival inside macrophages. It is believed
environment. that under conditions of stress, the cells become more
sensitive to the activity of tmRNA, suggesting the
Identificationofnon-codingRNA importance of the ability of cells to adapt and survive in
The prediction using the RFAM programme identified 5 differentenvironments[59].
non-coding RNAs (ncRNAs) in the genome of C. pseu-
dotuberculosis strain 1002 (Table 3), including ribos- Conclusion
witches (thiamine pyrophosphate [TPP] yybP-ykoY), Next-generation technology allowed the identification of
ncRNA tmRNA (SsrA)andncRNA mraW. a large number of genes presumed to be required by C.
In most cases, the ncRNAs in bacteria play a role in pseudotuberculosis 1002 for survival in unfavourable en-
regulating cellular response during environmental vironments, such as acidity, thermal shock and osmotic
change, which is beneficial to the organism, which needs stress. A number of these genes encode hypothetical
to adapt quickly and efficiently to these changes and proteins, which highlight the need for further investiga-
may be impaired by the cell because of the energy cost tion of the microorganism. Among the most relevant
involved in the expression of a large number of genes. biological processes identified under all simulated condi-
Compared to the synthesis of regulatory proteins, less tions were the processes of adhesion, stress response
energy is needed for the synthesis of a small RNA and oxidoreduction. In these processes, genes involved
(sRNA) [55]. Among the ncRNAs detected in the gen- in the virulence of the organism were affected and
ome, riboswitches (elements that control expression in shouldbeinvestigatedinmoredetailtoidentifytheroles
response to various metabolites and can function as sen- they play in the cell, especially inside the host. Further-
sors or binding sites for a receptor protein that senses a more, it is believed that the identification of these genes
change in the cellular environment) were identified may contribute to the development of vaccines that are
through the analysis of active transcripts. Furthermore, more effective, diagnostic kits and therapies for caseous
other elements that may be responsible for the synthesis lymphadenitis.
of peptidoglycan, which promotes the expression of The expression of sigma factors varied under the dif-
genes important under stress conditions [56,57], were ferentconditionsandatthebeginningoftheexponential
identified. phase, these important factors involved in the regulation