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Differential transcriptional profile of Corynebacterium pseudotuberculosis in response to abiotic stresses.

ABSTRACT: BACKGROUND: The completion of whole-genome sequencing for Corynebacterium pseudotuberculosis strain 1002 has contributed to major advances in research aimed at understanding the biology of this microorganism. This bacterium causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death. In the current study, we simulated the conditions experienced by the bacteria during host infection. By sequencing transcripts using the SOLiDTM 3 Plus platform, we identified new targets expected to potentiate the survival and replication of the pathogen in adverse environments. These results may also identify possible candidates useful for the development of vaccines, diagnostic kits or therapies aimed at the reduction of losses in agribusiness. RESULTS: Under the 3 simulated conditions (acid, osmotic and thermal shock stresses), 474 differentially expressed genes exhibiting at least a 2-fold change in expression levels were identified. Important genes to the infection process were induced, such as those involved in virulence, defence against oxidative stress, adhesion and regulation, and many genes encoded hypothetical proteins, indicating that further investigation of the bacterium is necessary. The data will contribute to a better understanding of the biology of C. pseudotuberculosis and to studies investigating strategies to control the disease. CONCLUSIONS: Despite the veterinary importance of C. pseudotuberculosis, the bacterium is poorly characterised; therefore, effective treatments for caseous lymphadenitis have been difficult to establish. Through the use of RNAseq, these results provide a better biological understanding of this bacterium, shed light on the most likely survival mechanisms used by this microorganism in adverse environments and identify candidates that may help reduce or even eradicate the problems caused by this disease.

SUBMITTER: Pinto AC

PROVIDER: S-EPMC3890534 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Differential transcriptional profile of Corynebacterium pseudotuberculosis in response to abiotic stresses.

Pinto Anne Cybelle AC de Sá Pablo Henrique Caracciolo Gomes PH Ramos Rommel T J RT Barbosa Silvanira S Barbosa Hivana P Melo HP Ribeiro Adriana Carneiro AC Silva Wanderson Marques WM Rocha Flávia Souza FS Santana Mariana Passos MP de Paula Castro Thiago Luiz TL Miyoshi Anderson A Schneider Maria P C MP Silva Artur A Azevedo Vasco V

BMC genomics 20140109

<h4>Background</h4>The completion of whole-genome sequencing for Corynebacterium pseudotuberculosis strain 1002 has contributed to major advances in research aimed at understanding the biology of this microorganism. This bacterium causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death. In the current study, we simulated the conditions experienced by the b ...[more]

PMID: 24405787

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Project description:Corynebacterium pseudotuberculosis strain 258, isolated from a caprine host in Brazil, was maintained in brain-heart-infusion broth (BHI HiMedia Laboratories Pvt. Ltda, India) at 37C, under rotation. Then, the bacteria was added to 20mL of BHI-Broth with 0.05% of 80% Tween and was maintained at 37C overnight, under rotation. The sample was then diluted 1/100 in 100mL of BHI broth, maintained at 37C, under rotation, and it was monitored until it reached the beginning of the exponential fase (A600 = 0.2). Afterwards, the sample was equally splitted in 4 aliquots of 20mL, centrifuged for 3 minutes under 8000rpm at 37C, and the pellets were re-suspendend in different medias according to the following conditions: acid stress, with HCl (fitting the pH to 5); osmotic stress, with 2M of NaCl; heat shock, where the pellet was re-suspended in pre-heated BHI (50C); and control, where the pellet was re-suspended in BHI under physiologic condition. Then, the samples for the acid stress, osmotic stress and control condition were maintained at 37C for 15 minutes, under rotation, whereas the sample under heat shock condition was maintained at 50C. Next, the samples were serially diluted from 10e-1 to 10e-6, where the samples from 10e-4 to 10e-6 were spread in BHI with agar (1.5%), which was maintained at 37C for 48hs for colony counting (duplicated samples); and the broth was centrifuged at 37C for 3 minutes under 8000rpm and the final pellet was re-suspended in 2mL of RNAlater, following the protocol of the manufacturer. Briefly, the bacteria suspended in RNAlater buffer were subjected to total RNA extraction using the ChargeSwitch total RNA cell kit (Invitrogen, USA) in accordance with the manufacturers recommendations, including the following adaptations: after the addition of the lysis buffer (Invitrogen), the material was transferred to 2mL tubes partially filled with 1mm diameter glass microbeads (Bertin Technologies). The cells were lysed mechanically using a Prescellys 24 homogenizer, set at 6,500 rpm, for 2 cycles (15 seconds per cycle) with an interval of 30 seconds between the cycles. The samples were centrifuged for 1 minute, and the supernatant transferred to fresh 2ml tubes and incubated in a dry bath at 60C for 15 minutes (representing the complete original protocol). DNAse was added to eliminate the residual genomic DNA. The elution of the total RNA from the magnetic beads was performed using 100 uL of milli-Q RNase-free water. The amount of total RNA was assessed using a Qubit 2.0 fluorometer (Invitrogen). In order to construct the library, the SOLiD Total RNA-Seq Kit was used. Briefly, after removing rRNAs, approximately 245ng of RNA was fragmented with RNAIII through incubation in a MJ Research thermocicler at 37C for 10 min and the reaction was interrupted with the addition of 90uL of nuclease-free water. The RNA was, then, concentrated through precipitation with ethanol according to the RiboMinus Concentration Module (Invitrogen - EUA), for subsequent hybridization with adapters and amplification of the cDNA library, which was obtained through reverse transcription using P1 adapters linked to the RNA extremities, according to the protocol SOLiD Total RNA-Seq Kit (Life Technologie, EUA). Next, the cDNA was denatured in polyacrylamide gel electrophoresis (6%) for obtaining fragments in the range from 150 to 250 base pairs for PCR amplification. The PCR amplifications were carried out in a MJ Research thermocycler as follow: the mixture was denatured at 95C for 30 sec, followed by 15 cycles of amplification at 95C for 30 sec, 62C for 30 sec and 72C for 30 sec, with a final extension of 72C for 7 min. Finally, the amplicons were purified using the kit PureLinkTM PCR Micro (Invitrogen - EUA), quantified in Qubit 2.0 Fluorometer-Invitrogen (EUA) and assessed through agarose electrophoresis 2%. The samples were fitted to the concentration of 60pg/mL, which is the suggested concentration for emulsion PCR. After the emulsion PCR, an enrichment reaction was performed for capturing only microbeads with fragments inserted. Next, a workflow analysis (WFA) was performed, where 15,000,000 microbeads were deposited to the slide and have undergone a binding cycle for quantitative and qualitative evaluation. According to this evaluation, 44,000,000 microbeads were deposited for sequencing, based on the manufacturer's description, generating reads of 50 nucleotides in range.

Project description:BackgroundIron is an essential micronutrient for the growth and development of virtually all living organisms, playing a pivotal role in the proliferative capability of many bacterial pathogens. The impact that the bioavailability of iron has on the transcriptional response of bacterial species in the CMNR group has been widely reported for some members of the group, but it hasn't yet been as deeply explored in Corynebacterium pseudotuberculosis. Here we describe for the first time a comprehensive RNA-seq whole transcriptome analysis of the T1 wild-type and the Cp13 mutant strains of C. pseudotuberculosis under iron restriction. The Cp13 mutant strain was generated by transposition mutagenesis of the ciuA gene, which encodes a surface siderophore-binding protein involved in the acquisition of iron. Iron-regulated acquisition systems are crucial for the pathogenesis of bacteria and are relevant targets to the design of new effective therapeutic approaches.ResultsTranscriptome analyses showed differential expression in 77 genes within the wild-type parental T1 strain and 59 genes in Cp13 mutant under iron restriction. Twenty-five of these genes had similar expression patterns in both strains, including up-regulated genes homologous to the hemin uptake hmu locus and two distinct operons encoding proteins structurally like hemin and Hb-binding surface proteins of C. diphtheriae, which were remarkably expressed at higher levels in the Cp13 mutant than in the T1 wild-type strain. These hemin transport protein genes were found to be located within genomic islands associated with known virulent factors. Down-regulated genes encoding iron and heme-containing components of the respiratory chain (including ctaCEF and qcrCAB genes) and up-regulated known iron/DtxR-regulated transcription factors, namely ripA and hrrA, were also identified differentially expressed in both strains under iron restriction.ConclusionBased on our results, it can be deduced that the transcriptional response of C. pseudotuberculosis under iron restriction involves the control of intracellular utilization of iron and the up-regulation of hemin acquisition systems. These findings provide a comprehensive analysis of the transcriptional response of C. pseudotuberculosis, adding important understanding of the gene regulatory adaptation of this pathogen and revealing target genes that can aid the development of effective therapeutic strategies against this important pathogen.

Project description:Background:Due to cowpea ability to fix nitrogen in poor soils and relative tolerance to drought and salt stresses, efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Real-time quantitative PCR (qPCR) has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. In the present study, nine candidate reference genes were rigorously tested for their application in normalization of qPCR data onto roots of four distinct cowpea accessions under two abiotic stresses: root dehydration and salt (NaCl, 100 mM). In addition, the regulation of four target transcripts, under the same referred conditions was also scrutinized. Results:geNorm, NormFinder, BestKeeper, and ?Ct method results indicated a set of three statistically validated RGs for each stress condition: (I) root dehydration (actin, ubiquitin-conjugating enzyme E2 variant 1D, and a Phaseolus vulgaris unknown gene-UNK), and (II) salt (ubiquitin-conjugating enzyme E2 variant 1D, F-box protein, and UNK). The expression profile of the target transcripts suggests that flavonoids are important players in the cowpea response to the abiotic stresses analyzed, since chalcone isomerase and chalcone synthase were up-regulated in the tolerant and sensitive accessions. A lipid transfer protein also participates in the cowpea tolerance mechanisms to root dehydration and salt stress. The referred transcript was up-regulated in the two tolerant accessions and presented no differential expression in the sensitive counterparts. Chitinase B, in turn, generally related to plant defense, was an important target transcript under salt stress, being up-regulated at the tolerant, and down-regulated in the sensitive accession. Conclusions:Reference genes suitable for qPCR analyses in cowpea under root dehydration and salt stress were identified. This action will lead to a more accurate and reliable analysis of gene expression on this species. Additionally, the results obtained in this study may guide future research on gene expression in cowpea under other abiotic stress types that impose osmotic imbalance. The target genes analyzed, in turn, deserve functional evaluation due to their transcriptional regulation under stresses and biotechnological potential.

Project description:BackgroundPan-genomic studies aim, for instance, at defining the core, dispensable and unique genes within a species. A pan-genomics study for vaccine design tries to assess the best candidates for a vaccine against a specific pathogen. In this context, rather than studying genes predicted to be exported in a single genome, with pan-genomics it is possible to study genes present in different strains within the same species, such as virulence factors. The target organism of this pan-genomic work here presented is Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis (CLA) in goat and sheep, which causes significant economic losses in those herds around the world. Currently, only a few antigens against CLA are known as being the basis of commercial and still ineffective vaccines. In this regard, the here presented work analyses, in silico, five C. pseudotuberculosis genomes and gathers data to predict common exported proteins in all five genomes. These candidates were also compared to two recent C. pseudotuberculosis in vitro exoproteome results.ResultsThe complete genome of five C. pseudotuberculosis strains (1002, C231, I19, FRC41 and PAT10) were submitted to pan-genomics analysis, yielding 306, 59 and 12 gene sets, respectively, representing the core, dispensable and unique in silico predicted exported pan-genomes. These sets bear 150 genes classified as secreted (SEC) and 227 as potentially surface exposed (PSE). Our findings suggest that the main C. pseudotuberculosis in vitro exoproteome could be greater, appended by a fraction of the 35 proteins formerly predicted as making part of the variant in vitro exoproteome. These genomes were manually curated for correct methionine initiation and redeposited with a total of 1885 homogenized genes.ConclusionsThe in silico prediction of exported proteins has allowed to define a list of putative vaccine candidate genes present in all five complete C. pseudotuberculosis genomes. Moreover, it has also been possible to define the in silico predicted dispensable and unique C. pseudotuberculosis exported proteins. These results provide in silico evidence to further guide experiments in the areas of vaccines, diagnosis and drugs. The work here presented is the first whole C. pseudotuberculosis in silico predicted pan-exoproteome completed till today.

Dataset Information

Differential transcriptional profile of Corynebacterium pseudotuberculosis in response to abiotic stresses.

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Differential transcriptional profile of Corynebacterium pseudotuberculosis in response to abiotic stresses.

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