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Differential transcriptional profile of Corynebacterium pseudotuberculosis 258 in response to abiotic stress

ABSTRACT: Differential transcriptional profile of Corynebacterium pseudotuberculosis 258 in response to abiotic stress

PROVIDER: PRJEB38802 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR4230431.fastq.gz	Fastqsanger.gz
	ERR4230432.fastq.gz	Fastqsanger.gz
	ERR4230433.fastq.gz	Fastqsanger.gz
	ERR4230434.fastq.gz	Fastqsanger.gz

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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: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.

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Differential transcriptional profile of Corynebacterium pseudotuberculosis 258 in response to abiotic stress

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