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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 necessary for upregulation of Lmo0514 protein levels in intracellular bacteria by a mechanism that involves interaction with the lmo0514 long 5′-UTR isoform.(A) Real-time qPCR assays showing no changes in relative levels of the lmo0514 ORF (fragment amplified with Lmo0514-F and Lmo0514-R primers) or the isoform bearing the long 5′-UTR (fragment amplified with UTR-B and UTR-1R primers) in intracellular bacteria that lack Rli27 or are defective for the rli27-flanking lmo0411 gene. (B) Decrease in levels of Lmo0514 protein produced by intracellular bacteria, caused by absence of Rli27. No such effect was seen in lmo0411- or lmo0412-defective strains. Levels of InlA, another cell wall-bound LPXTG protein, were monitored as loading control. (C) RT-PCR assays showing expression of Rli27 and mutated versions Rli27-mut1 and Rli27-mut3 produced in trans from the pP1 plasmid. (D) Western blot assays showing the effect of the mut3 mutation in Rli27, which impedes upregulation of the Lmo0514 protein in intracellular bacteria. InlA levels were monitored as loading control. (E) Real-time qPCR showing that lack of Rli27 does not affect lmo0514 transcript stability in extracellular bacteria grown in BHI medium to stationary phase. Primers correspond to those that map in the coding region (Lmo0514-F and Lmo0514-R). Data are derived from a minimum of three independent experiments. (F) Western blot showing that Rli27 is not necessary for Lmo0514 protein production by extracellular bacteria. Due to the small amount of Lmo0514 protein produced by extracellular bacteria (see Fig. 1B), the anti-Lmo0514 antibody-treated membrane was overexposed. Levels of InlH, another cell wall-bound LPXTG protein, were monitored as loading control. Densitometry values are indicated in Western blots beneath panels B, D, and F.
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pgen-1004765-g006: Rli27 is necessary for upregulation of Lmo0514 protein levels in intracellular bacteria by a mechanism that involves interaction with the lmo0514 long 5′-UTR isoform.(A) Real-time qPCR assays showing no changes in relative levels of the lmo0514 ORF (fragment amplified with Lmo0514-F and Lmo0514-R primers) or the isoform bearing the long 5′-UTR (fragment amplified with UTR-B and UTR-1R primers) in intracellular bacteria that lack Rli27 or are defective for the rli27-flanking lmo0411 gene. (B) Decrease in levels of Lmo0514 protein produced by intracellular bacteria, caused by absence of Rli27. No such effect was seen in lmo0411- or lmo0412-defective strains. Levels of InlA, another cell wall-bound LPXTG protein, were monitored as loading control. (C) RT-PCR assays showing expression of Rli27 and mutated versions Rli27-mut1 and Rli27-mut3 produced in trans from the pP1 plasmid. (D) Western blot assays showing the effect of the mut3 mutation in Rli27, which impedes upregulation of the Lmo0514 protein in intracellular bacteria. InlA levels were monitored as loading control. (E) Real-time qPCR showing that lack of Rli27 does not affect lmo0514 transcript stability in extracellular bacteria grown in BHI medium to stationary phase. Primers correspond to those that map in the coding region (Lmo0514-F and Lmo0514-R). Data are derived from a minimum of three independent experiments. (F) Western blot showing that Rli27 is not necessary for Lmo0514 protein production by extracellular bacteria. Due to the small amount of Lmo0514 protein produced by extracellular bacteria (see Fig. 1B), the anti-Lmo0514 antibody-treated membrane was overexposed. Levels of InlH, another cell wall-bound LPXTG protein, were monitored as loading control. Densitometry values are indicated in Western blots beneath panels B, D, and F.

Mentions: To determine the biological relevance of the 5′-UTR-lmo0514-Rli27 interaction in vivo, we analyzed the specific contribution of Rli27 binding to Lmo0514 protein upregulation in bacteria that infect eukaryotic cells. We generated a Δrli27 strain and a second isogenic mutant, Δrli27C2T, which bears an artificial strong terminator between the remaining rli27 sequences. This mutant was intended to avoid polar effects on the flanking genes lmo0411 and lmo0412 (Fig. S4); we also included mutants in these flanking genes, Δlmo0411 and Δlmo0412[30]. In addition, we designed a qPCR assay specific for the lmo0514 long 5′-UTR for comparison to the lmo0514 coding region. There were no notable differences among strains in the relative levels of the long 5′-UTR region or the lmo0514 ORF (Fig. 6A). In contrast, Lmo0514 protein levels were ∼2.5- to 3-fold lower in the cell wall of the two Rli27-lacking mutant strains isolated from the eukaryotic cell (Fig. 6B). This phenotype was complemented by overproduction of wild-type or mut1 versions of Rli27 from a plasmid (Fig. 6C, D). In contrast, when we tested mut3, the Rli27 mutant bearing 14 nt changes in the major region predicted to interact with the lmo0514 5′-UTR (Fig. 5A), it did not restore Lmo0514 protein levels in intracellular bacteria (Fig. 6D). Wild-type, mut1 and mut3 Rli27 versions were all produced by the plasmid at similar levels (Fig. 6C). These data showed that Rli27 interaction with the lmo0514 long 5′-UTR was essential for induction of the protein in intracellular bacteria, and that elimination of the Rli27-lmo0514 5′-UTR interaction interfered with the Lmo0514 protein increase while levels for the long transcript isoform remained unchanged. Our findings thus supported the need for Rli27 binding for efficient Lmo0514 translation. Control qPCR experiments in extracellular bacteria showed similar lmo0514 transcript levels in this mutant series (Fig. 6E), whereas there were no marked changes in Lmo0514 protein levels (Fig. 6F).


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 necessary for upregulation of Lmo0514 protein levels in intracellular bacteria by a mechanism that involves interaction with the lmo0514 long 5′-UTR isoform.(A) Real-time qPCR assays showing no changes in relative levels of the lmo0514 ORF (fragment amplified with Lmo0514-F and Lmo0514-R primers) or the isoform bearing the long 5′-UTR (fragment amplified with UTR-B and UTR-1R primers) in intracellular bacteria that lack Rli27 or are defective for the rli27-flanking lmo0411 gene. (B) Decrease in levels of Lmo0514 protein produced by intracellular bacteria, caused by absence of Rli27. No such effect was seen in lmo0411- or lmo0412-defective strains. Levels of InlA, another cell wall-bound LPXTG protein, were monitored as loading control. (C) RT-PCR assays showing expression of Rli27 and mutated versions Rli27-mut1 and Rli27-mut3 produced in trans from the pP1 plasmid. (D) Western blot assays showing the effect of the mut3 mutation in Rli27, which impedes upregulation of the Lmo0514 protein in intracellular bacteria. InlA levels were monitored as loading control. (E) Real-time qPCR showing that lack of Rli27 does not affect lmo0514 transcript stability in extracellular bacteria grown in BHI medium to stationary phase. Primers correspond to those that map in the coding region (Lmo0514-F and Lmo0514-R). Data are derived from a minimum of three independent experiments. (F) Western blot showing that Rli27 is not necessary for Lmo0514 protein production by extracellular bacteria. Due to the small amount of Lmo0514 protein produced by extracellular bacteria (see Fig. 1B), the anti-Lmo0514 antibody-treated membrane was overexposed. Levels of InlH, another cell wall-bound LPXTG protein, were monitored as loading control. Densitometry values are indicated in Western blots beneath panels B, D, and F.
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pgen-1004765-g006: Rli27 is necessary for upregulation of Lmo0514 protein levels in intracellular bacteria by a mechanism that involves interaction with the lmo0514 long 5′-UTR isoform.(A) Real-time qPCR assays showing no changes in relative levels of the lmo0514 ORF (fragment amplified with Lmo0514-F and Lmo0514-R primers) or the isoform bearing the long 5′-UTR (fragment amplified with UTR-B and UTR-1R primers) in intracellular bacteria that lack Rli27 or are defective for the rli27-flanking lmo0411 gene. (B) Decrease in levels of Lmo0514 protein produced by intracellular bacteria, caused by absence of Rli27. No such effect was seen in lmo0411- or lmo0412-defective strains. Levels of InlA, another cell wall-bound LPXTG protein, were monitored as loading control. (C) RT-PCR assays showing expression of Rli27 and mutated versions Rli27-mut1 and Rli27-mut3 produced in trans from the pP1 plasmid. (D) Western blot assays showing the effect of the mut3 mutation in Rli27, which impedes upregulation of the Lmo0514 protein in intracellular bacteria. InlA levels were monitored as loading control. (E) Real-time qPCR showing that lack of Rli27 does not affect lmo0514 transcript stability in extracellular bacteria grown in BHI medium to stationary phase. Primers correspond to those that map in the coding region (Lmo0514-F and Lmo0514-R). Data are derived from a minimum of three independent experiments. (F) Western blot showing that Rli27 is not necessary for Lmo0514 protein production by extracellular bacteria. Due to the small amount of Lmo0514 protein produced by extracellular bacteria (see Fig. 1B), the anti-Lmo0514 antibody-treated membrane was overexposed. Levels of InlH, another cell wall-bound LPXTG protein, were monitored as loading control. Densitometry values are indicated in Western blots beneath panels B, D, and F.
Mentions: To determine the biological relevance of the 5′-UTR-lmo0514-Rli27 interaction in vivo, we analyzed the specific contribution of Rli27 binding to Lmo0514 protein upregulation in bacteria that infect eukaryotic cells. We generated a Δrli27 strain and a second isogenic mutant, Δrli27C2T, which bears an artificial strong terminator between the remaining rli27 sequences. This mutant was intended to avoid polar effects on the flanking genes lmo0411 and lmo0412 (Fig. S4); we also included mutants in these flanking genes, Δlmo0411 and Δlmo0412[30]. In addition, we designed a qPCR assay specific for the lmo0514 long 5′-UTR for comparison to the lmo0514 coding region. There were no notable differences among strains in the relative levels of the long 5′-UTR region or the lmo0514 ORF (Fig. 6A). In contrast, Lmo0514 protein levels were ∼2.5- to 3-fold lower in the cell wall of the two Rli27-lacking mutant strains isolated from the eukaryotic cell (Fig. 6B). This phenotype was complemented by overproduction of wild-type or mut1 versions of Rli27 from a plasmid (Fig. 6C, D). In contrast, when we tested mut3, the Rli27 mutant bearing 14 nt changes in the major region predicted to interact with the lmo0514 5′-UTR (Fig. 5A), it did not restore Lmo0514 protein levels in intracellular bacteria (Fig. 6D). Wild-type, mut1 and mut3 Rli27 versions were all produced by the plasmid at similar levels (Fig. 6C). These data showed that Rli27 interaction with the lmo0514 long 5′-UTR was essential for induction of the protein in intracellular bacteria, and that elimination of the Rli27-lmo0514 5′-UTR interaction interfered with the Lmo0514 protein increase while levels for the long transcript isoform remained unchanged. Our findings thus supported the need for Rli27 binding for efficient Lmo0514 translation. Control qPCR experiments in extracellular bacteria showed similar lmo0514 transcript levels in this mutant series (Fig. 6E), whereas there were no marked changes in Lmo0514 protein levels (Fig. 6F).

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.

Show MeSH
Related in: MedlinePlus