Global transcriptomic response of Leptospira interrogans serovar Copenhageni upon exposure to serum
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ABSTRACT:
ORGANISM(S): Leptospira interrogans
SUBMITTER: Miranda Lo
PROVIDER: E-GEOD-17942 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Leptospirosis is a globally significant zoonotic disease caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since availability of iron is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. Virulence genes in some bacteria have been shown to be iron-regulated. In many bacteria, expression of iron-uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutant in one of these, la1857. We conducted microarray analysis to identify differentially expressed genes in L. interrogans serovar Manilae grown under low iron compared with normal iron conditions found in EMJH medium. We also compared the transcriptional profile of the la1857 mutant versus wild-type Manilae under normal and low iron conditions. Analysis used RNA derived from L. interrogans serovar Manilae grown under low iron (EMJH medium + 40 µM 2,2-dipyridyl) and normal iron as experimental and control samples, respectively. We have a serovar Manilae mutant in la1857, one of four predicted fur homologs. The mutant was also grown under low iron and normal iron conditions and compared with wild-type Manilae grown under low and normal iron conditions respectively. Three independent RNA samples (biological replicates) from parent and mutant strains grown with or without 2,2-dipyridyl were compared in a loop design resulting in 12 arrays. The orientation of Cy3 to Cy5 labeling was the same for replicates 1 and 3, while replicate 2 was a dye swap. Direct comparisons were made between arrays of experimental and control samples using raw data pulled from two different channels for data analysis. Data from wild-type Manilae grown under low iron versus normal iron and mutant versus wild-type under normal iron conditions is reported in the manuscript.
2010-05-05 | E-GEOD-20422 | biostudies-arrayexpress
Project description:Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with .10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil’s) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of ‘‘core’’ housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants. Transcriptome analysis of L. interrogans Copenhageni FIOCRUZ L1-130 using RNA from 2 different conditions using RNA-seq. Also, the reproducibility and robustness of data is ensured by three biological replicates from each condition.
2014-03-20 | E-GEOD-53818 | biostudies-arrayexpress
Project description:A large body of work has demonstrated that leptospires regulate and modify gene expression in response to environmental cues, as encountered during disease transmission, including changes in temperature, osmolarity, concentration of iron, the presence of serum, and interaction with macrophages. However, since leptospires are not readily amenable to genetic manipulation, the functional and biological significance of these differentially expressed genes often remains unclear. More recently, it is been shown that proteins, in response to changing environmental conditions, can be modified further by specific post-translational modifications. Indeed, both saprophytic and pathogenic leptospires have comprehensive systems to modify proteins. The paucibacillary nature of spirochetal infections, combined with the challenges associated with acquiring pathogens free from contaminating host proteins makes the study of these bacteria in a mammalian host-adapted state inherently difficult. As an alternative approach, we developed a model in which leptospires are cultivated in a dialysis membrane chamber (DMC) implanted within the peritoneal cavity of rats, where they are exposed to the environmental cues encountered during host infection. This strategy has been successfully applied to compare the transcriptome of L. interrogans cultivated within DMC with that of leptospires grown under standard in vitro conditions. In addition to determining the relative expression levels of ‘‘core’’ housekeeping genes under both growth conditions, we identified 166 genes that were differentially-expressed by L. interrogans in response to mammalian host signals. The proteome of DMC (dialysis membrane chamber) cultivated leptospires was compared to that of in vivo derived leptospires and with leptospires cultivated in vitro at 30°C or 37°C by 2-dimensional difference in gel electrophoresis (2-D DIGE). Our analysis indicates that the abundance of leptospire-proteins is modulated the expression of a range of proteins which are differentially expressed in response to mammalian host signals, and not temperature alone . In addition, we confirm that in several proteins there is a change in the presence of modify the expression of the post-translational modifications trimethyllysine and acetyllysine in response to environmental cues encountered during persistent renal colonization in a reservoir host of infection. These results provide novel insights in the proteome level changes, including to differential protein and post-translational modifications, expressed in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.
2017-08-31 | PXD006995 | Pride
Project description:DNA microarray analysis was used to compare the differential gene-expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22 kb genomic island covering a cluster of 34 genes (LA0186 to LA0219) was actively expressed in both strains but concomitantly up-expressed in strain 56601 in contrast to that of IPAV. RT-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 out of 34 CDSs encoding prophage-like proteins, of which, LA0195 is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II and III, all proximal to LA0195, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter CAT enzyme activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to LA0195 in vitro. These results suggested that LA0195 is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems resided outside of this region within the genome maybe responsible for the differential expression profiling in these two strains. Keywords: Differential expression of L.interrogans 56601 and IPAV in 37°C The microarray chip was prepared as previously described (42). In brief, the 3528 protein coding sequences (CDSs) successfully amplified by PCR were printed in triplicate on the slides as probes. Three expression profiling experiments were performed via reverse transcription using Superscript (Invitrogen) and with one experiment having the cDNA of IPAV labeled by Cy3 and of 56601 labeled by Cy5, while the other two having the cDNA of 56601 labeled by Cy3 and of IPAV labeled by Cy5. The unincorporated dye was removed using a QIAquick Nucleotide Removal Kit (Qiagen) as specified by the manufacturer's protocol. Samples were hybridized at 42°C for 16h, and then washed as described in the manual. Hybridization experiments were performed in triplicate using cDNA derived from three different cultures of virulent strain 56601 and avirulent strain IPAV grown at 37°C. The hybridized slides were scanned and analyzed by Tiffsplit (Agilent) to calculate the signal intensities and to determine the presence or absence of each CDS. The data were then normalized, and their backgrounds were defined using GeneSpring 4.0 (Silicon Genetics). The GeneSpring software was used to further analyze the transcription patterns. To identify genes with significantly altered expression levels, cutoff values for expression level ratios 1.5 were used to filter genes with changes (n-fold) greater than ±1.5 in three independent biological samples (27, 48, 50). Studentâs t test analysis of variance was used to compare the mean expression levels of the test and reference samples. Genes with significant differential expression levels (P < 0.05) were selected.
2010-05-25 | E-GEOD-10788 | biostudies-arrayexpress
Project description:Leptospira, the causative agent of leptospirosis is known to have several proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. In this study, we carried out in-depth proteomic analysis of Triton X-114 fractions of Leptospira interrogans using high-resolution LC-MS/MS. Our analysis resulted in the identification of 104 of 130 proteases predicted by MEROPS. Approximately 3.5% of the Leptospira genome complements for proteases, which include catalytic types of metallo-, serine-, cysteine-, aspartic-, threonine- and asparagine- peptidases. Comparison of proteases from different serovars revealed that M04, M09B, M14A, M75, M28A, A01 and U73 protease families are exclusively present in pathogenic form. The M23 and S33 protease families are represented with more than 14 members in Leptospira. In silico prediction and characterization of the proteases revealed that several proteases are membrane anchored and secretory, classical as well as non-classical system. This study demonstrates the diversity and complexity of proteases, while maintaining conservation across the serovars in Leptospira and their differential expression under pathogenic conditions.
2018-04-18 | PXD009050 | Pride
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Project description:RevR is a putative orphan response regulator with a high degree of similarity to YycF from Bacilus subtilis and PhoB from Clostridium kluyveri. A revR deletion mutant of C. perfringens strain 13 was generated and the transcriptome analysed using microarrays. Total RNA was isolated from exponentially growing cells from the revR mutant and the wild-type control. Gene expression levels were compared between the revR mutant and wild-type strain 13
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2014-12-22 | E-GEOD-28699 | biostudies-arrayexpress
Project description:ReeS, previously named as CPE1512, was originally annotated as the only hybrid sensor histidine kinase/response regulator in Clostridium perfringens. Further evidence suggests that ReeS is more likely to function as an orphan sensor histidine kinase. A reeS deletion mutant was constructed and the transcriptome analysed using microarrays. Total RNA was isolated from the reeS mutant and the wild-type control cells during exponential phase growth. Gene expression levels were compared between the reeS mutant and wild-type strain 13
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