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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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lmo0514 is expressed differentially from two distinct transcriptional start sites in extra- and intracellular L. monocytogenes.(A) Position of the three transcriptional start sites (TSS) predicted in silico for lmo0514 by Loh et al.[25]. Primers used to amplify the lmo0514 coding sequence are indicated (ORF, primers Lmo0514-F, Lmo0514-R, see Table S2), as well as two fragments of the 5′-UTR of different lengths, amplicon “a” (254 nt), obtained with primers UTR-B and UTR-1R (Table S2) and amplicon “b” (134 nt), obtained with primers UTR-A and UTR-1R (Table S2). Reverse transcriptase-PCR assays showing upregulation in intracellular bacteria of an lmo0514 transcript isoform with a long 5′-UTR. 16S rRNA was monitored as loading control. RNA was obtained from extracellular bacteria grown in BHI medium to exponential logarithmic phase (log), stationary phase (stat), and from intracellular bacteria. (B) 5′-RACE assay showing a TSS at position −28 relative to the ATG site in extracellular bacteria. Colored bar indicates the position of the primer lmo0514-PE-1rv (Table S2) used for this reaction. (C) 5′-RACE assay showing the production by intracellular bacteria of an lmo0514 transcript with a long 5′-UTR derived from a TSS at position −234 relative to the ATG site. Colored bar indicates the position of the primer lmo0514-PE-6rv (Table S2) used for this reaction. TAP, tobacco acid pyrophosphatase.
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pgen-1004765-g002: lmo0514 is expressed differentially from two distinct transcriptional start sites in extra- and intracellular L. monocytogenes.(A) Position of the three transcriptional start sites (TSS) predicted in silico for lmo0514 by Loh et al.[25]. Primers used to amplify the lmo0514 coding sequence are indicated (ORF, primers Lmo0514-F, Lmo0514-R, see Table S2), as well as two fragments of the 5′-UTR of different lengths, amplicon “a” (254 nt), obtained with primers UTR-B and UTR-1R (Table S2) and amplicon “b” (134 nt), obtained with primers UTR-A and UTR-1R (Table S2). Reverse transcriptase-PCR assays showing upregulation in intracellular bacteria of an lmo0514 transcript isoform with a long 5′-UTR. 16S rRNA was monitored as loading control. RNA was obtained from extracellular bacteria grown in BHI medium to exponential logarithmic phase (log), stationary phase (stat), and from intracellular bacteria. (B) 5′-RACE assay showing a TSS at position −28 relative to the ATG site in extracellular bacteria. Colored bar indicates the position of the primer lmo0514-PE-1rv (Table S2) used for this reaction. (C) 5′-RACE assay showing the production by intracellular bacteria of an lmo0514 transcript with a long 5′-UTR derived from a TSS at position −234 relative to the ATG site. Colored bar indicates the position of the primer lmo0514-PE-6rv (Table S2) used for this reaction. TAP, tobacco acid pyrophosphatase.

Mentions: To evaluate this possibility, we sought lmo0514 gene expression control mechanisms that operate specifically in intracellular bacteria. Previous in silico predictions by Loh et al. [25] indicated that lmo0514 could be expressed from three promoters at positions −26, −104 and −163. Two of these, −26 and −163, were assigned as tentatively regulated by sigma A (σA) and the third, at position −104, as controlled by sigma B (σB) [25] (Fig. 2A). The activity of these putative promoters and the presence of the different transcripts were analyzed by RT-PCR on RNA isolated from L. monocytogenes grown extracellularly and from intracellular bacteria that colonized JEG-3 epithelial cells. lmo0514 transcripts with a long 5′-UTR were detected specifically in intracellular bacteria (Fig. 2A). To confirm these findings, rapid amplification of 5′-cDNA ends (5′-RACE) assays were used to map transcriptional start sites (TSS) of lmo0514 in bacteria grown extracellularly and in bacteria isolated from eukaryotic cells. These 5′-RACE assays revealed two distinct TSS at positions −28 and −234 (Fig. 2B, C), and also confirmed expression of the long lmo0514 transcript by intracellular bacteria (Fig. 2C). Putative promoters for these TSS, which we termed P1 and P2, both bear bona fide −10 TATA boxes (Fig. 2B, C). The existence of two lmo0514 transcripts of different length was verified by northern blot (Fig. 3A), with sizes compatible with cotranscription of lmo0514 with the downstream gene lmo0515, which encodes a universal stress protein [26]. lmo0514-lm0515 cotranscription was verified by RT-PCR (Fig. S1). qRT-PCR assays confirmed that expression of the lmo0514 transcript variant with the long 234-nucleotide (nt) 5′-UTR was upregulated by ∼12-fold in intracellular bacteria (Fig. 3B). These findings suggested that the specific induction of this mRNA variant with a longer 5′-UTR in intracellular bacteria accounts for or contributes to the 6-fold increase in total lmo0514 mRNA (Fig. 1A). These data supported a model in which intracellular bacteria specifically upregulate expression from the P2 promoter, resulting in an lmo0514 transcript with a long 5′-UTR. This assumption takes into account the different ratios between the two lmo0514 transcripts when L. monocytogenes colonizes the eukaryotic cell.


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)

lmo0514 is expressed differentially from two distinct transcriptional start sites in extra- and intracellular L. monocytogenes.(A) Position of the three transcriptional start sites (TSS) predicted in silico for lmo0514 by Loh et al.[25]. Primers used to amplify the lmo0514 coding sequence are indicated (ORF, primers Lmo0514-F, Lmo0514-R, see Table S2), as well as two fragments of the 5′-UTR of different lengths, amplicon “a” (254 nt), obtained with primers UTR-B and UTR-1R (Table S2) and amplicon “b” (134 nt), obtained with primers UTR-A and UTR-1R (Table S2). Reverse transcriptase-PCR assays showing upregulation in intracellular bacteria of an lmo0514 transcript isoform with a long 5′-UTR. 16S rRNA was monitored as loading control. RNA was obtained from extracellular bacteria grown in BHI medium to exponential logarithmic phase (log), stationary phase (stat), and from intracellular bacteria. (B) 5′-RACE assay showing a TSS at position −28 relative to the ATG site in extracellular bacteria. Colored bar indicates the position of the primer lmo0514-PE-1rv (Table S2) used for this reaction. (C) 5′-RACE assay showing the production by intracellular bacteria of an lmo0514 transcript with a long 5′-UTR derived from a TSS at position −234 relative to the ATG site. Colored bar indicates the position of the primer lmo0514-PE-6rv (Table S2) used for this reaction. TAP, tobacco acid pyrophosphatase.
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pgen-1004765-g002: lmo0514 is expressed differentially from two distinct transcriptional start sites in extra- and intracellular L. monocytogenes.(A) Position of the three transcriptional start sites (TSS) predicted in silico for lmo0514 by Loh et al.[25]. Primers used to amplify the lmo0514 coding sequence are indicated (ORF, primers Lmo0514-F, Lmo0514-R, see Table S2), as well as two fragments of the 5′-UTR of different lengths, amplicon “a” (254 nt), obtained with primers UTR-B and UTR-1R (Table S2) and amplicon “b” (134 nt), obtained with primers UTR-A and UTR-1R (Table S2). Reverse transcriptase-PCR assays showing upregulation in intracellular bacteria of an lmo0514 transcript isoform with a long 5′-UTR. 16S rRNA was monitored as loading control. RNA was obtained from extracellular bacteria grown in BHI medium to exponential logarithmic phase (log), stationary phase (stat), and from intracellular bacteria. (B) 5′-RACE assay showing a TSS at position −28 relative to the ATG site in extracellular bacteria. Colored bar indicates the position of the primer lmo0514-PE-1rv (Table S2) used for this reaction. (C) 5′-RACE assay showing the production by intracellular bacteria of an lmo0514 transcript with a long 5′-UTR derived from a TSS at position −234 relative to the ATG site. Colored bar indicates the position of the primer lmo0514-PE-6rv (Table S2) used for this reaction. TAP, tobacco acid pyrophosphatase.
Mentions: To evaluate this possibility, we sought lmo0514 gene expression control mechanisms that operate specifically in intracellular bacteria. Previous in silico predictions by Loh et al. [25] indicated that lmo0514 could be expressed from three promoters at positions −26, −104 and −163. Two of these, −26 and −163, were assigned as tentatively regulated by sigma A (σA) and the third, at position −104, as controlled by sigma B (σB) [25] (Fig. 2A). The activity of these putative promoters and the presence of the different transcripts were analyzed by RT-PCR on RNA isolated from L. monocytogenes grown extracellularly and from intracellular bacteria that colonized JEG-3 epithelial cells. lmo0514 transcripts with a long 5′-UTR were detected specifically in intracellular bacteria (Fig. 2A). To confirm these findings, rapid amplification of 5′-cDNA ends (5′-RACE) assays were used to map transcriptional start sites (TSS) of lmo0514 in bacteria grown extracellularly and in bacteria isolated from eukaryotic cells. These 5′-RACE assays revealed two distinct TSS at positions −28 and −234 (Fig. 2B, C), and also confirmed expression of the long lmo0514 transcript by intracellular bacteria (Fig. 2C). Putative promoters for these TSS, which we termed P1 and P2, both bear bona fide −10 TATA boxes (Fig. 2B, C). The existence of two lmo0514 transcripts of different length was verified by northern blot (Fig. 3A), with sizes compatible with cotranscription of lmo0514 with the downstream gene lmo0515, which encodes a universal stress protein [26]. lmo0514-lm0515 cotranscription was verified by RT-PCR (Fig. S1). qRT-PCR assays confirmed that expression of the lmo0514 transcript variant with the long 234-nucleotide (nt) 5′-UTR was upregulated by ∼12-fold in intracellular bacteria (Fig. 3B). These findings suggested that the specific induction of this mRNA variant with a longer 5′-UTR in intracellular bacteria accounts for or contributes to the 6-fold increase in total lmo0514 mRNA (Fig. 1A). These data supported a model in which intracellular bacteria specifically upregulate expression from the P2 promoter, resulting in an lmo0514 transcript with a long 5′-UTR. This assumption takes into account the different ratios between the two lmo0514 transcripts when L. monocytogenes colonizes the eukaryotic cell.

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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