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The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5'-UTR Variant.

Quereda JJ, Ortega AD, Pucciarelli MG, García-Del Portillo F - PLoS Genet. (2014)

Bottom Line: The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell.Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR.These findings emphasize how 5'-UTR length affects regulation by defined sRNA.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

ABSTRACT
Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5'-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5'-untranslated regions (5'-UTR) of 28 and 234 nucleotides. The transcript containing the long 5'-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5'-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5'-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR. These findings emphasize how 5'-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5'-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.

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Rli27 is a bona fide L. monocytogenes sRNA induced by intracellular bacteria.(A) Sequence alignment of the rli27 genomic region from L. monocytogenes, L. welshimeri and L. innocua. The −35 and −10 predicted sites for the rli27 promoter and the rli27 itself (grey background) are highlighted. Nucleotide sequence in orange corresponds to the predicted terminator shared by rli27 and lmo0412. Note that rli27 is absent in L. welshimeri. (B) Northern blot assay performed with total RNAs isolated from bacteria grown in BHI medium to stationary phase. L. monocytogenes strains used included EGDe (WT) and the Δrli27 mutant. 5S rRNA was used as loading control. (C) Real-time qPCR showing upregulation of Rli27 expression in intracellular bacteria. Bacteria were grown in BHI medium to exponential (log) or stationary phase, or collected from epithelial cells. Data derive from a minimum of three independent experiments. ***, P≤0.001, Student's t-test.
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pgen-1004765-g004: Rli27 is a bona fide L. monocytogenes sRNA induced by intracellular bacteria.(A) Sequence alignment of the rli27 genomic region from L. monocytogenes, L. welshimeri and L. innocua. The −35 and −10 predicted sites for the rli27 promoter and the rli27 itself (grey background) are highlighted. Nucleotide sequence in orange corresponds to the predicted terminator shared by rli27 and lmo0412. Note that rli27 is absent in L. welshimeri. (B) Northern blot assay performed with total RNAs isolated from bacteria grown in BHI medium to stationary phase. L. monocytogenes strains used included EGDe (WT) and the Δrli27 mutant. 5S rRNA was used as loading control. (C) Real-time qPCR showing upregulation of Rli27 expression in intracellular bacteria. Bacteria were grown in BHI medium to exponential (log) or stationary phase, or collected from epithelial cells. Data derive from a minimum of three independent experiments. ***, P≤0.001, Student's t-test.

Mentions: Genomic comparisons of pathogenic and non-pathogenic species are usually carried out to identify virulence genes, including sRNAs [18], [29]. We analyzed the genomic region of L. monocytogenes containing rli27 and those of the non-pathogenic species L. innocua and L. welshimeri. In L. monocytogenes, rli27 is flanked by lmo0411 and lmo0412, two genes that map in the opposite DNA strand (Fig. S2), whereas in the L. welshimeri genome, the same intergenic region has a small ORF (lwe0373) that codes for a predicted protein of unknown function (Fig. S2). We nonetheless found that Rli27 is highly conserved in L. innocua (82% identity, Fig. 4A), in contrast with a previous report [17]. The extremely variable rli27 genomic region might thus have been shaped by gain and/or loss of genes during Listeria speciation. Apart from Listeria species, BLAST searches did not identify rli27 orthologs in other bacterial species. Rli27, identified as a 131-nt sRNA [14], [18], is not predicted to encode any protein using the Small Open Reading Frame (ORF) tool in the ORF finder program (http://www.bioinformatics.org/sms2/orf_find.html).


The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5'-UTR Variant.

Quereda JJ, Ortega AD, Pucciarelli MG, García-Del Portillo F - PLoS Genet. (2014)

Rli27 is a bona fide L. monocytogenes sRNA induced by intracellular bacteria.(A) Sequence alignment of the rli27 genomic region from L. monocytogenes, L. welshimeri and L. innocua. The −35 and −10 predicted sites for the rli27 promoter and the rli27 itself (grey background) are highlighted. Nucleotide sequence in orange corresponds to the predicted terminator shared by rli27 and lmo0412. Note that rli27 is absent in L. welshimeri. (B) Northern blot assay performed with total RNAs isolated from bacteria grown in BHI medium to stationary phase. L. monocytogenes strains used included EGDe (WT) and the Δrli27 mutant. 5S rRNA was used as loading control. (C) Real-time qPCR showing upregulation of Rli27 expression in intracellular bacteria. Bacteria were grown in BHI medium to exponential (log) or stationary phase, or collected from epithelial cells. Data derive from a minimum of three independent experiments. ***, P≤0.001, Student's t-test.
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Related In: Results  -  Collection

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pgen-1004765-g004: Rli27 is a bona fide L. monocytogenes sRNA induced by intracellular bacteria.(A) Sequence alignment of the rli27 genomic region from L. monocytogenes, L. welshimeri and L. innocua. The −35 and −10 predicted sites for the rli27 promoter and the rli27 itself (grey background) are highlighted. Nucleotide sequence in orange corresponds to the predicted terminator shared by rli27 and lmo0412. Note that rli27 is absent in L. welshimeri. (B) Northern blot assay performed with total RNAs isolated from bacteria grown in BHI medium to stationary phase. L. monocytogenes strains used included EGDe (WT) and the Δrli27 mutant. 5S rRNA was used as loading control. (C) Real-time qPCR showing upregulation of Rli27 expression in intracellular bacteria. Bacteria were grown in BHI medium to exponential (log) or stationary phase, or collected from epithelial cells. Data derive from a minimum of three independent experiments. ***, P≤0.001, Student's t-test.
Mentions: Genomic comparisons of pathogenic and non-pathogenic species are usually carried out to identify virulence genes, including sRNAs [18], [29]. We analyzed the genomic region of L. monocytogenes containing rli27 and those of the non-pathogenic species L. innocua and L. welshimeri. In L. monocytogenes, rli27 is flanked by lmo0411 and lmo0412, two genes that map in the opposite DNA strand (Fig. S2), whereas in the L. welshimeri genome, the same intergenic region has a small ORF (lwe0373) that codes for a predicted protein of unknown function (Fig. S2). We nonetheless found that Rli27 is highly conserved in L. innocua (82% identity, Fig. 4A), in contrast with a previous report [17]. The extremely variable rli27 genomic region might thus have been shaped by gain and/or loss of genes during Listeria speciation. Apart from Listeria species, BLAST searches did not identify rli27 orthologs in other bacterial species. Rli27, identified as a 131-nt sRNA [14], [18], is not predicted to encode any protein using the Small Open Reading Frame (ORF) tool in the ORF finder program (http://www.bioinformatics.org/sms2/orf_find.html).

Bottom Line: The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell.Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR.These findings emphasize how 5'-UTR length affects regulation by defined sRNA.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

ABSTRACT
Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5'-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5'-untranslated regions (5'-UTR) of 28 and 234 nucleotides. The transcript containing the long 5'-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5'-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5'-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR. These findings emphasize how 5'-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5'-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.

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