Progress 07/01/08 to 06/30/11
Outputs
OUTPUTS: Salmonella encodes two homologs of RyhB, a small RNA involved in iron homeostasis. In Salmonella Typhimurium, the expression of two sRNAs, RyhB-1 and RyhB-2, is negatively regulated by the Fur repressor. To identify the target mRNAs of RyhB-1 and RyhB-2, 9 predicted target genes were analyzed by quantitative RT-PCR. Our results suggest that the genes acnA, sodB, ftn, STM1273.1n, and acnB are the primary targets of at least one of these sRNAs. Study with deletion mutants showed that these sRNAs are singularly or additively involved in expression of multiple phenotypes, including acid resistance, resistance to hydrogen peroxide, and sensitivity to bactericidal antibiotics. Next, we used genomic microarrays for comprehensive identification of the target genes regulated by these sRNAs. The candidate targets were first identified by selecting genes with increased transcript signals in a double deletion strain as compared to the wild type. We found three candidate genes (flgJ, cheY, and fliF), involved in motility and chemotaxis, that are regulated only by RyhB2. Motility assay showed that the ryhB2 deletion increased motility, which was partially reduced back to wild type level by complementation, supporting the RT-PCR data. Adaptive phenotypes of enteric bacterial pathogens in response to in vivo-mimicking stress conditions are important because of their potentiality to enhance stress resistance. S. Enteritidis encounters a variety of such environments throughout its infection cycle, including high concentrations of propionate (PA) during food processing and within the gut of infected hosts. We have shown that S. Enteritidis grown to stationary phase in the presence of PA has a dramatically enhanced resistance to commonly encountered in vivo stressors, including extreme acidity and oxidative/nitrosative stresses when compared to unadapted salmonellae. However, competitive infection between PA adapted and unadapted cells within a murine model showed that adapted cells were at a distinct disadvantage in vivo, resulting in decreased caecal colonization in infected mice. In the next step, we used 2 D gel electrophoresis to examine the proteomes of PA adapted and unadapted S. Enteritidis and have identified five proteins that are upregulated in PA adapted cultures. Of these five, two significant stress-related proteins,Dps and CpxR, were shown to be upregulated at the transcriptional level as well. Unlike the wild type when adapted to PA, PA adapted S. Enteritidis ∆dps and S. Enteritidis ∆cpxR were at a clear disadvantage when challenged to a highly acidic environment. We also found evidence that in exponentially growing S. Enteritidis, Dps is vital for protection against the common killing mechanism of bactericidal antibiotics, a mechanism manifested by hydroxyl radical production via the Fenton reaction. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The above results support a model whereby RyhB-1 and RyhB-2 have a global regulatory effect on diverse cellular pathways in response to multiple environmental cues via post-transcriptional regulation of distinct sets of overlapping targets. Our results also suggest that, while long-term PA adaptation induces strong resistance to specific stresses in vitro, it also reduces the overall infectivity of the adapted cells by inhibiting the organism's colonization ability. The proteomics study affirms the contribution of Dps and CpxR in PA induced acid resistance. Finally, our study on Dps protein suggests that targeting Dps may represent a means to increase the potency of bactericidal antibiotics in S. Enteritidis.
Publications
- Calhoun, L. N., and Y. M. Kwon. 2010. The effect of long term propionate adaptation on the stress resistance of Salmonella Enteritidis. J. Appl. Microbiol. 109, 1294-1300.
- Calhoun, L. N., and Y. M. Kwon. 2010. Proteomic analysis of Salmonella enterica serovar Enteritidis following propionate adaptation. BMC Microbiolgy 10, 249.
- Calhoun, L. N. and Y. M. Kwon. 2011. The ferritin-like protein Dps protects Salmonella Enteritidis from the Fenton-mediated killing mechanism of bactericidal antibiotics. Int. J. Antimicrob. Agents 37, 261-265.
- Calhoun, L. N., J. N. Kim, Y. Ren, J. J. Song, and Y. M. Kwon. 2011. The DNA-binding protein Dps functions as a global regulator in starved Salmonella enterica serovar Enteritidis during starvation. Int. J. Microbiol. Res. (accepted)
- Calhoun, L. N., and Y. M. Kwon. 2011. Structure, function, and regulation of the DNA-binding protein Dps and its role in stress resistance in Salmonella enterica serovar Typhimurium: A review. J. Appl. Microbiol. 110, 375-386.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: RyhBs (ryhB1 and ryhB2) are small regulatory RNAs in Salmonella that are involved in iron homeostasis. These sRNAs regulate target mRNA expression by forming a base-pair complex with target transcripts, thereby accelerating mRNA decay and blocking the access of translation machinery. To understand the structure function relationship in RyhB1-target interactions, we introduced random single-point mutations to ryhB1 gene on Salmonella chromosome in sodB-lacZ background Five single-point mutations displaying a significant increase in β-galactosidase activity on X-gal plates under ryhB1-inducing condition were screened. We reasoned that RyhB1 mutants exhibiting defective or weakened interaction with a sodB transcript, one of the known targets, would appear as darker blue colonies on X-gal agar plates. Quantitative real-time PCR analysis showed that the five mutant strains regulated the level of nine target mRNAs (bfr, fumA, sdhD, aceE, acnA, ftn, frdA, acnB, and rpoS) in different patterns compared to wild-type RyhB1 strain. We have previously shown that two RyhB homologs of S. Typhimurium, RyhB1 and RyhB2, have distinct sets of overlapping targets through RT-PCR analysis and phenotypic assays. We used genomic microarrays for comprehensive identification of the target genes regulated by these sRNAs. Microarray analysis was conducted with cDNAs obtained from total RNAs of three deletion mutants (∆ryhB1, ∆ryhB2, and ∆ryhB1∆ryhB2) and wild type S. Typhimurium 14028 strain. The candidate targets were first identified by selecting genes with increased transcript signals in double deletion strain as compared to wild type. Then those candidates were clustered into the following 4 groups according to the pattern of increased signals: 1) higher in both single deletion backgrounds, 2) unchanged in both single deletion backgrounds, and 3) higher only in RyhB1 or 4) RyhB2 deletion background. We found three candidate genes (flgJ, cheY, and fliF), involved in motility and chemotaxis, are regulated only by RyhB2. RT-PCR analysis showed that repression of the three genes, which was lost in RyhB2 deletion strain, was restored by complementation with RyhB2-expressing plasmid. Motility assay also showed that ryhB2 deletion increased motility, which was reduced back to wild type level by complementation, supporting RT-PCR data. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This research demonstrates there are critically important nucleotides in regulatory RyhB1 that influence target mRNA recognition and stability, which may explain highly conserved sequence of RyhB1 across diverse serotypes of Salmonella enterica. In addition, our microarray results suggest the global regulatory role of RyhB1 and RyhB2 on multiple cellular pathways, including motility, at a post-transcriptional level. The results of this study suggest the complex mechanisms underlying sRNA-target interactions and the deeper understanding of the mechanisms might help us exploit sRNA as a platform for novel antibacterial strategy.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We have used multiple approaches to explore the possibility of using RNA molecules to inhibit target molecules in Salmonella that are conditionally essential for cell viability or virulence as a means to control this human pathogen. First, we performed genetic and phenotypic characterization of RyhB molecules in S. Typhimurium. RyhB molecules have a great potential as a platform to deliver chimeric sRNAs to inhibit target molecules, because RyhB interacts with multiple targets through imperfect base-pairing of short targeting region. Secondly, we have attempted to characterize the role of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) elements in S. Typhimurium in acquired immune mechanism against plasmid and bacteriophage. Third, we constructed a chimeric sRNA library in Salmonella using a plasmid expression system that express random recombinant molecules of 3 target essential genes in Salmonella. The next step will be to identify the candidate chimeric sRNAs that can effectively inhibit cell viability upon overexpression. The results on RyhB were presented at the American Society for Microbiology General Meeting held in 2009 and the 3 entire CRISPR elements in S. Typhimurium 14028s strain were sequenced and deposited in GenBank. PARTICIPANTS: Dr. Young Min Kwon was the principal investigator of the project. One Ph.D. student (Jeong Nam Kim) and one postdoctoral associate (Tieshan Jiang) conducted the experiments and data analysis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Salmonella encodes two homologs of sRNA RyhB that has been implicated in iron homeostasis. In S. Typhimurium, the expression of these sRNAs, RyhB 1 and RyhB2, are negatively regulated by Fur repressor, while stationary phase is the primary signal inducing RyhB2 expression. To identify the target mRNAs of these sRNAs, 11 predicted target genes were analyzed by quantitative RT-PCR for differential transcript levels between wild type and each of various deletion mutants (∆ryhB1, ∆ryhB2 and ∆ryhB1∆ryhB2) under the conditions that maximize the expression of both sRNAs. The result along with bioinformatic prediction suggests that the genes acnA, bfr, sodB, ftn, frdA, STM1273.1n, rpoS, and acnB are the primary targets of at least one of these sRNAs. To understand the biological roles of RyhB regulon, the same deletions in either WT or ∆fur background were used in various phenotypic assays. The result showed that these sRNAs are differentially or additively involved in expression of multiple phenotypes, including motility, acid resistance, resistance to hydrogen peroxide, and sensitivity to bactericidal antibiotics. The results support a model whereby RyhB1 and RyhB2 have global regulatory effects on diverse cellular pathways in response to multiple environmental cues via posttranscriptional regulation of distinct sets of overlapping targets. CRISPR elements are known to mediate acquired immune defense mechanisms against incoming phage and plasmids in prokaryotic species. To determine if this element has the same role in protecting Salmonella from the attack of plasmid, we measured the transformation efficiency of an engineered multi-copy plasmid containing one CRISPR spacer sequence. The result showed that the presence of spacer sequence on the plasmid reduced the transformation efficiency by 2-3 fold consistently. However, this result is not significant compared to more dramatic decrease in the efficiency of plasmid conjugation or phage infection due to CRISPR spacer regions in other bacterial species. For the next step, we will check the role of CRISPR spacer regions in protecting cells from phage infection. In the course of the experiment, we sequenced 3 entire CRISPR elements present in S. Typhimurium ATCC14028s strain and the sequences were deposited to GenBank (Accession number GU390666, GU390667, and GU390668).
Publications
- No publications reported this period
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Progress 07/01/08 to 12/31/08
Outputs
OUTPUTS: We have initiated experiments to develop novel RNA-based strategy to inhibit virulence in Salmonella. This project is in the initial stage of development and no significant outputs have been obtained yet. PARTICIPANTS: Dr. Young Min Kwon is the pricipal investigator of the project. Dr. Kwon supervises the overall design and performance of the experimetns. Mr. Jeong Nam Kim (Ph.D. student) is the main person conducting the experiments. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have been using two different approaches to develop a novel antibacterial strategy based on small noncoding RNA. First, we use sRNA RyhB in Salmonella as a backbone to develop synthetic sRNA that can inhibit translation of multiple target genes of choice. sRNA RyhB binds to its mRNA targets by imperfect base-pairing and the translation is inhibited while sRNA-target duplex is subject to degradation by RNase enzymes. Since RyhB in S. Typhimurium has multiple targets, it will provide a suitable platform to target the genes of interest by incorporating sequences for target recognition. To expedite the development process, we are currently using nonhomologous random recombination (NRR) to construct chimeric RyhB-based sRNA that can target multiple essential genes or conditionally essential genes (e.g. virulence genes). Secondly, we are exploring the possibility of using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) as a basis to develop an antibacterial strategy. CRISPR is a novel genetic element that provides acquired immune defense mechanisms for bacteria against phage infection. Although little has been known about the underlying mechanisms, recent researches showed that CRISPR system has enzymes that digest specific DNA sequences of incoming phage, plasmid or other mobile elements. Currently, we are testing if transformation of Salmonella with foreign DNA could be inhibited by the presence of CRISPR spacer sequences in the incoming DNA. These above approaches may be able to provide a novel measures to counteract infection by Salmonella and other pathogenic bacteria.
Publications
- No publications reported this period
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