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

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.

Listeria monocytogenes remodels the cell surface in the blood‐stage

“…The coverage obtained for most of these LPXTG proteins was however lower in bacteria grown in blood or plasma than in bacteria grown in laboratory media (Garcia‐del Portillo et al ., ), with the exceptions of Internalin A (Inl‐A) and Lmo0514. The increase in Lmo0514 levels in the blood‐stage was intriguing considering that L. monocytogenes also up‐regulates this surface protein in the intracellular niche of epithelial cells (Quereda et al ., ). The abundance of Lmo0514 in L. monocytogenes exposed to blood led us to investigate its role in virulence, interaction with blood cells and stress (see below).…”

“…The appearance of the cell wall‐associated protein Lmo0514 in the surface of intracellular bacteria occurs in the absence of major changes at the transcriptional level (Quereda et al ., ). This example denotes the importance of performing studies at the protein level to unravel key post‐transcriptional regulatory processes that modulate host‐pathogen interactions (Coiras et al ., ).…”

“…To date, no study has focused on changes at the proteome level when L. monocytogenes copes with the host defenses present in the blood. This is relevant considering that L. monocytogenes remodels at a large extent the cell wall proteome when it resides inside the eukaryotic cell (Garcia‐del Portillo et al ., ; Quereda et al ., ; Quereda and Pucciarelli, ). Following the transit from the extracellular environment to the eukaryotic intracellular niche, L. monocytogenes up‐regulates surface proteins such as the membrane protein ActA, which promotes actin recruitment, and the peptidoglycan‐bound proteins Internalin A (Inl‐A) and Lmo0514 (Quereda et al ., ).…”

“…This is relevant considering that L. monocytogenes remodels at a large extent the cell wall proteome when it resides inside the eukaryotic cell (Garcia‐del Portillo et al ., ; Quereda et al ., ; Quereda and Pucciarelli, ). Following the transit from the extracellular environment to the eukaryotic intracellular niche, L. monocytogenes up‐regulates surface proteins such as the membrane protein ActA, which promotes actin recruitment, and the peptidoglycan‐bound proteins Internalin A (Inl‐A) and Lmo0514 (Quereda et al ., ). L. monocytogenes uses Inl‐A to invade a large variety of eukaryotic cell types, reason for which its abundance in the cell wall of intracellular bacteria at late infection times remains intriguing (Garcia‐Del Portillo and Pucciarelli, ).…”

RNA- and protein-mediated control of Listeria monocytogenes virulence gene expression

“…The two divergent transcriptional units encode proteins of opposite functions: MogR is a transcriptional repressor of flagellum genes, whereas Fli proteins participate in the flagellum export apparatus. (D) Rli27 allows the intracellular-specific translation of an alternative transcript, inspired from Quereda et al 94 . Extracellular bacteria express mainly lmo0514-lmo0515 from the constitutive P1 promoter.…”