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 has evolved to adapt to numerous environments, including the intracellular niche of eukaryotic cells. Small RNAs (sRNA) play important regulatory roles in changing environments, and are thus predicted to modulate L. monocytogenes adaption to the intracellular lifestyle. This study shows how the regulatory activity of an sRNA on a defined target is restricted to bacteria in the intracellular infection phase. This regulation relies on a long (234-nucleotide) 5′-UTR that bears the sRNA-binding site present in a transcript variant that is upregulated by intracellular L. monocytogenes. The concomitant increase in both the target transcript containing the long 5′-UTR and the sRNA, which is postulated to facilitate opening of the Shine-Dalgarno site, culminates in markedly higher protein levels in intracellular bacteria. The limited amounts of both the target and the regulator in extracellular bacteria ensure that production of this bacterial protein is confined mainly to the host rather than the non-host environment.