- Email: [email protected]
Small RNA, Big Effect: Control of Flagellin Production
TIMI 1509 No. of Pages 2
Spotlight
Small RNA, Big Effect: Control of Flagellin Production Aleksandra A. MirandaCasoLuengo,1 Stefani C. Kary,1 Marc Erhardt,2 and Carsten Kröger1,* Many bacteria move in their environment using a remarkable, rotating nanomachine – the flagellum. In a recent publication, Choi et al. report a new addition to the group of flagellar regulators, a transacting small RNA (sRNA). The flagellum is a complex macromolecular appendage made up of thousands of proteins and is the most prominent extracellular structure known in bacteria. In many pathogenic microorganisms, including Salmonella enterica serovar Typhimurium, flagella have multiple functions in pathogenesis beyond their role as motility organelles, including adhesion to host cell surfaces and modulation of immune system responses [1]. Flagella allow Salmonella to effectively colonise the gastrointestinal tract in a chemotactic process, penetrate the mucosal barrier, and reach suitable invasion sites on host cells. Thus, during host colonization, flagella are required to induce inflammation and colitis, conditions that Salmonella exploits to outgrow the microbiota [2,3]. Salmonella has evolved complex, multilayered regulatory mechanisms to coordinate the correct spatiotemporal biosynthesis of flagella during the infection process. Many environmental signals are integrated at the level of expression and activity of the flagellar master regulator, FlhDC, which ultimately determines the start of flagella biosynthesis. The FlhDC complex activates expression of genes coding for components of the hook–basal-body (HBB) complex.
Upon sensing the completion of the HBB, genes coding for the long, external filament (the propeller), the motor-force generators (the engine), and the chemosensory system are expressed. The 10– 20 mm long external flagellar filament is made of several tens of thousands of subunits of a single protein, flagellin. S. Typhimurium produces two antigenically distinct flagellin proteins: FljB and FliC, and the expression of fljB and fliC is tightly regulated in a process termed flagellar phase variation. The alternative expression of the different flagellins is achieved by stochastic inversion of the promoter of the fljBA operon, which is mediated by the recombinase Hin. The fljBA operon additionally codes for FljA, a translational repressor of the primary flagellin fliC [4]. Therefore, under conditions in which the fljBA operon is expressed, only FljB-flagella are produced. In Choi et al., the authors report a new addition to the group of flagellar regulators: an sRNA that binds to fljBA mRNA resulting in RNase E-dependent degradation of the fljBA transcript (Figure 1). The sRNA is expressed from the virulence locus mgtCBR, which constitutes an extraordinary genetic region with multilayered regulatory architecture [5]. Transcription is initiated from a single promoter upstream of mgtC and
is activated by the PhoP/Q twocomponent system in response to low levels of Mg2+, mildly acidic pH, and antimicrobial peptides – conditions encountered by Salmonella during infection. The polycistronic mgtCBR mRNA encodes the MgtC virulence factor, the MgtB Mg2+ transporter, and the MgtR regulatory protein. The long leader region of mgtCBR also encodes two short proteins: the ATP responsive MgtM and the proline-rich MgtP. The two tandem short open reading frames, MgtM and MgtP, control transcription of the downstream coding region by an attenuationlike mechanism [6]. First, Choi et al. observed that a small leader RNA is produced from the same transcriptional start site that initiates the transcription of the mgtCBR operon, and is likely formed by transcriptional attenuation [5]. Most of the known trans-acting small, regulatory RNAs are expressed from intergenic regions or at the 3ʹ-end of a coding region. Despite the unusual origin, the mode of action of the mgtC leader resembles the stereotypical sRNA regulator acting in trans. In bacteria, these types of regulatory sRNA typically act by an imperfect base-pairing mechanism with its cognate target RNA that may lead to target activation or repression facilitated by an RNA-binding protein. In S. Typhimurium, at least 280 small RNAs
sRNA sRNA
mgtC
mgtB
FliC flagella
fljBA mRNA
mgtCBR mRNA mgtM mgtP
Hfq
mgtR
sRNA
FljB flagella C1362G
Figure 1. Small RNA-Mediated Post-Transcriptional Regulation of Flagellar Biogenesis. The 5ʹleader sRNA expressed from the mgtCBR virulence locus downregulates the expression of FljB-flagella by base-pairing with the fljBA mRNA. The interaction requires the RNA-binding protein Hfq and results in the RNaseE-mediated degradation of fljBA mRNA.
Trends in Microbiology, Month Year, Vol. xx, No. yy
1
TIMI 1509 No. of Pages 2
have been identified [7]; however, only 10% have been functionally characterized. Overexpressing the mgtCBR leader RNA followed by transcriptomic analysis suggested that the sRNA represses fljBA, the operon encoding the FljB flagellin protein and FljA, a repressor of FliC-flagellin translation, by a base-pairing mechanism requiring the RNA-binding protein Hfq [5]. Strikingly, [5_TD$IF]the fljBA transcript was identified as the only target RNA[56_TD$IF], which is an unusual finding, because trans-acting sRNAs typically regulate a larger number of targets. A C1362G single nucleotide exchange in the fljB gene disrupting the RNA duplex formation was sufficient to upregulate production of FljB-flagella and facilitate swimming motility in lowmagnesium (i.e., mgtCBR-inducing) conditions. In murine macrophages, where Salmonella proliferates in a specialised compartment termed the Salmonellacontaining vacuole (SCV), the expression of flagella biosynthesis genes is low, while the mgtCBR operon is highly induced [8]. In agreement, Choi et al. [5] observed an absence of flagella in the wild-type strain in infected macrophages. By contrast, the fljB C1362G mutant expressed flagella while residing in the SCV and showed replication rates higher than those of wild-type cells. Mice challenged intraperitoneally with wild-type and C1362G mutant strains revealed that strains overexpressing FljB-flagella kill mice faster than the wild-type strain; however, the
2
Trends in Microbiology, Month Year, Vol. xx, No. yy
exact mechanism explaining hyperviru- example of the complex regulatory mechlence of the fljB C1362G mutant is anisms in bacteria. unclear. Because of the concomitant 1 Department of Microbiology, School of Genetics and downregulation of the fljA repressor upon Microbiology, Moyne Institute of Preventive Medicine, induction of the sRNA, the proportion of Trinity College Dublin, Dublin, Ireland 2 FliC-flagella-carrying cells should Humboldt-Universität zu Berlin, Institute for Biology – Bacterial physiology, Berlin, Germany increase. Upregulation of the fliC gene was observed when the sRNA was *Correspondence: [email protected] (C. Kröger). induced; however, it is unclear to what https://doi.org/10.1016/j.tim.2017.10.003 extent this locks the cells in a FliCReferences expressing phenotype, a phenotype that 1. Rossez, Y. et al. (2015) Bacterial flagella: twist and stick, or dodge across the Kingdoms. PLoS Pathog. 11, e1004483 provides a competitive advantage for gastrointestinal colonisation [9]. The 2. Stecher, B. et al. (2004) Flagella and chemotaxis are required for efficient induction of Salmonella enterica seroMgtC protein inhibits the action of the F[57_TD$IF] var Typhimurium colitis in streptomycin-pretreated mice. Infect. Immun. 72, 4138–4150 1FO ATP synthase inside the SCV [10], 3. Stecher, B. et al. (2008) Motility allows S. Typhimurium to which couples proton translocation to benefit from the mucosal defence. Cell. Microbiol. 10, 1166–1180 ATP synthesis and thereby prevents cytoplasm acidification. Inhibiting the accu- 4. Aldridge, P.D. et al. (2006) Regulatory protein that inhibits both synthesis and use of the target protein controls flamulation of intracellular ATP provides a gellar phase variation in Salmonella enterica. Proc. Natl. Acad. Sci. U. S. A. 103, 11340–11345 direct link between MgtC and the proton 5. Choi, E. et al. (2017) A trans-acting leader RNA from a motive force that is used to power rotary Salmonella virulence gene. Proc. Natl. Acad. Sci. U. S. A. 114, 10232–10237 movement of the flagellar nanomachine. This could mean that the presence of 6. Lee, E.-J. and Groisman, E.A. (2012) Tandem attenuators control expression of the Salmonella mgtCBR virulence flagella, rather than their rotation, might operon. Mol. Microbiol. 86, 212–224 7. Kröger, C. et al. (2013) An infection-relevant transcriptomic contribute to the hypervirulence. compendium for Salmonella enterica serovar Typhimurium. Cell Host Microbe 14, 683–695
In summary, Choi et al. broaden our understanding of how flagellar biogenesis is controlled, and provide evidence for a new post-transcriptional regulator in the form of a small RNA that represses the formation of FljB-flagella. Their work suggests that more 5ʹ-UTRs of genes than previously anticipated may harbour small, regulatory RNAs with important physiological functions. The thorough study of the mgtCBR virulence locus is a striking
8. Srikumar, S. et al. (2015) RNA-seq brings new insights to the intra-macrophage transcriptome of Salmonella Typhimurium. PLoS Pathog. 11, e1005262 9. Horstmann, J.A. et al. (2017) Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation. Cell. Microbiol. Published online April 18, 2017. http://dx.doi.org/ 10.1111/cmi.12739 10. Lee, E.-J. et al. (2013) A bacterial virulence protein promotes pathogenicity by inhibiting the bacterium’s own F1Fo ATP synthase. Cell 154, 146–156
Spotlight
Small RNA, Big Effect: Control of Flagellin Production Aleksandra A. MirandaCasoLuengo,1 Stefani C. Kary,1 Marc Erhardt,2 and Carsten Kröger1,* Many bacteria move in their environment using a remarkable, rotating nanomachine – the flagellum. In a recent publication, Choi et al. report a new addition to the group of flagellar regulators, a transacting small RNA (sRNA). The flagellum is a complex macromolecular appendage made up of thousands of proteins and is the most prominent extracellular structure known in bacteria. In many pathogenic microorganisms, including Salmonella enterica serovar Typhimurium, flagella have multiple functions in pathogenesis beyond their role as motility organelles, including adhesion to host cell surfaces and modulation of immune system responses [1]. Flagella allow Salmonella to effectively colonise the gastrointestinal tract in a chemotactic process, penetrate the mucosal barrier, and reach suitable invasion sites on host cells. Thus, during host colonization, flagella are required to induce inflammation and colitis, conditions that Salmonella exploits to outgrow the microbiota [2,3]. Salmonella has evolved complex, multilayered regulatory mechanisms to coordinate the correct spatiotemporal biosynthesis of flagella during the infection process. Many environmental signals are integrated at the level of expression and activity of the flagellar master regulator, FlhDC, which ultimately determines the start of flagella biosynthesis. The FlhDC complex activates expression of genes coding for components of the hook–basal-body (HBB) complex.
Upon sensing the completion of the HBB, genes coding for the long, external filament (the propeller), the motor-force generators (the engine), and the chemosensory system are expressed. The 10– 20 mm long external flagellar filament is made of several tens of thousands of subunits of a single protein, flagellin. S. Typhimurium produces two antigenically distinct flagellin proteins: FljB and FliC, and the expression of fljB and fliC is tightly regulated in a process termed flagellar phase variation. The alternative expression of the different flagellins is achieved by stochastic inversion of the promoter of the fljBA operon, which is mediated by the recombinase Hin. The fljBA operon additionally codes for FljA, a translational repressor of the primary flagellin fliC [4]. Therefore, under conditions in which the fljBA operon is expressed, only FljB-flagella are produced. In Choi et al., the authors report a new addition to the group of flagellar regulators: an sRNA that binds to fljBA mRNA resulting in RNase E-dependent degradation of the fljBA transcript (Figure 1). The sRNA is expressed from the virulence locus mgtCBR, which constitutes an extraordinary genetic region with multilayered regulatory architecture [5]. Transcription is initiated from a single promoter upstream of mgtC and
is activated by the PhoP/Q twocomponent system in response to low levels of Mg2+, mildly acidic pH, and antimicrobial peptides – conditions encountered by Salmonella during infection. The polycistronic mgtCBR mRNA encodes the MgtC virulence factor, the MgtB Mg2+ transporter, and the MgtR regulatory protein. The long leader region of mgtCBR also encodes two short proteins: the ATP responsive MgtM and the proline-rich MgtP. The two tandem short open reading frames, MgtM and MgtP, control transcription of the downstream coding region by an attenuationlike mechanism [6]. First, Choi et al. observed that a small leader RNA is produced from the same transcriptional start site that initiates the transcription of the mgtCBR operon, and is likely formed by transcriptional attenuation [5]. Most of the known trans-acting small, regulatory RNAs are expressed from intergenic regions or at the 3ʹ-end of a coding region. Despite the unusual origin, the mode of action of the mgtC leader resembles the stereotypical sRNA regulator acting in trans. In bacteria, these types of regulatory sRNA typically act by an imperfect base-pairing mechanism with its cognate target RNA that may lead to target activation or repression facilitated by an RNA-binding protein. In S. Typhimurium, at least 280 small RNAs
sRNA sRNA
mgtC
mgtB
FliC flagella
fljBA mRNA
mgtCBR mRNA mgtM mgtP
Hfq
mgtR
sRNA
FljB flagella C1362G
Figure 1. Small RNA-Mediated Post-Transcriptional Regulation of Flagellar Biogenesis. The 5ʹleader sRNA expressed from the mgtCBR virulence locus downregulates the expression of FljB-flagella by base-pairing with the fljBA mRNA. The interaction requires the RNA-binding protein Hfq and results in the RNaseE-mediated degradation of fljBA mRNA.
Trends in Microbiology, Month Year, Vol. xx, No. yy
1
TIMI 1509 No. of Pages 2
have been identified [7]; however, only 10% have been functionally characterized. Overexpressing the mgtCBR leader RNA followed by transcriptomic analysis suggested that the sRNA represses fljBA, the operon encoding the FljB flagellin protein and FljA, a repressor of FliC-flagellin translation, by a base-pairing mechanism requiring the RNA-binding protein Hfq [5]. Strikingly, [5_TD$IF]the fljBA transcript was identified as the only target RNA[56_TD$IF], which is an unusual finding, because trans-acting sRNAs typically regulate a larger number of targets. A C1362G single nucleotide exchange in the fljB gene disrupting the RNA duplex formation was sufficient to upregulate production of FljB-flagella and facilitate swimming motility in lowmagnesium (i.e., mgtCBR-inducing) conditions. In murine macrophages, where Salmonella proliferates in a specialised compartment termed the Salmonellacontaining vacuole (SCV), the expression of flagella biosynthesis genes is low, while the mgtCBR operon is highly induced [8]. In agreement, Choi et al. [5] observed an absence of flagella in the wild-type strain in infected macrophages. By contrast, the fljB C1362G mutant expressed flagella while residing in the SCV and showed replication rates higher than those of wild-type cells. Mice challenged intraperitoneally with wild-type and C1362G mutant strains revealed that strains overexpressing FljB-flagella kill mice faster than the wild-type strain; however, the
2
Trends in Microbiology, Month Year, Vol. xx, No. yy
exact mechanism explaining hyperviru- example of the complex regulatory mechlence of the fljB C1362G mutant is anisms in bacteria. unclear. Because of the concomitant 1 Department of Microbiology, School of Genetics and downregulation of the fljA repressor upon Microbiology, Moyne Institute of Preventive Medicine, induction of the sRNA, the proportion of Trinity College Dublin, Dublin, Ireland 2 FliC-flagella-carrying cells should Humboldt-Universität zu Berlin, Institute for Biology – Bacterial physiology, Berlin, Germany increase. Upregulation of the fliC gene was observed when the sRNA was *Correspondence: [email protected] (C. Kröger). induced; however, it is unclear to what https://doi.org/10.1016/j.tim.2017.10.003 extent this locks the cells in a FliCReferences expressing phenotype, a phenotype that 1. Rossez, Y. et al. (2015) Bacterial flagella: twist and stick, or dodge across the Kingdoms. PLoS Pathog. 11, e1004483 provides a competitive advantage for gastrointestinal colonisation [9]. The 2. Stecher, B. et al. (2004) Flagella and chemotaxis are required for efficient induction of Salmonella enterica seroMgtC protein inhibits the action of the F[57_TD$IF] var Typhimurium colitis in streptomycin-pretreated mice. Infect. Immun. 72, 4138–4150 1FO ATP synthase inside the SCV [10], 3. Stecher, B. et al. (2008) Motility allows S. Typhimurium to which couples proton translocation to benefit from the mucosal defence. Cell. Microbiol. 10, 1166–1180 ATP synthesis and thereby prevents cytoplasm acidification. Inhibiting the accu- 4. Aldridge, P.D. et al. (2006) Regulatory protein that inhibits both synthesis and use of the target protein controls flamulation of intracellular ATP provides a gellar phase variation in Salmonella enterica. Proc. Natl. Acad. Sci. U. S. A. 103, 11340–11345 direct link between MgtC and the proton 5. Choi, E. et al. (2017) A trans-acting leader RNA from a motive force that is used to power rotary Salmonella virulence gene. Proc. Natl. Acad. Sci. U. S. A. 114, 10232–10237 movement of the flagellar nanomachine. This could mean that the presence of 6. Lee, E.-J. and Groisman, E.A. (2012) Tandem attenuators control expression of the Salmonella mgtCBR virulence flagella, rather than their rotation, might operon. Mol. Microbiol. 86, 212–224 7. Kröger, C. et al. (2013) An infection-relevant transcriptomic contribute to the hypervirulence. compendium for Salmonella enterica serovar Typhimurium. Cell Host Microbe 14, 683–695
In summary, Choi et al. broaden our understanding of how flagellar biogenesis is controlled, and provide evidence for a new post-transcriptional regulator in the form of a small RNA that represses the formation of FljB-flagella. Their work suggests that more 5ʹ-UTRs of genes than previously anticipated may harbour small, regulatory RNAs with important physiological functions. The thorough study of the mgtCBR virulence locus is a striking
8. Srikumar, S. et al. (2015) RNA-seq brings new insights to the intra-macrophage transcriptome of Salmonella Typhimurium. PLoS Pathog. 11, e1005262 9. Horstmann, J.A. et al. (2017) Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation. Cell. Microbiol. Published online April 18, 2017. http://dx.doi.org/ 10.1111/cmi.12739 10. Lee, E.-J. et al. (2013) A bacterial virulence protein promotes pathogenicity by inhibiting the bacterium’s own F1Fo ATP synthase. Cell 154, 146–156