Bordetella pertussis is the causative agent of human whooping cough, a highly contagious respiratory disease which despite vaccination programs remains the major cause of infant morbidity and mortality. The requirement of the RNA chaperone Hfq for virulence of B. pertussis suggested that Hfq-dependent small regulatory RNAs are involved in the modulation of gene expression. High-throughput RNA sequencing revealed hundreds of putative noncoding RNAs including the RgtA sRNA. Abundance of RgtA is strongly decreased in the absence of the Hfq protein and its expression is modulated by the activities of the two-component regulatory system BvgAS and another response regulator RisA. Whereas RgtA levels were elevated under modulatory conditions or in the absence of bvg genes, deletion of the risA gene completely abolished RgtA expression. Profiling of the ΔrgtA mutant in the ΔbvgA genetic background identified the BP3831 gene encoding a periplasmic amino acid-binding protein of an ABC transporter as a possible target gene. The results of site-directed mutagenesis and in silico analysis indicate that RgtA base-pairs with the region upstream of the start codon of the BP3831 mRNA and thereby weakens the BP3831 protein production. Furthermore, our data suggest that the function of the BP3831 protein is related to transport of glutamate, an important metabolite in the B. pertussis physiology. We propose that the BvgAS/RisA interplay regulates the expression of RgtA which upon infection, when glutamate might be scarce, attenuates translation of the glutamate transporter and thereby assists in adaptation of the pathogen to other sources of energy.

Department of Chromosome Biology of the University of Vienna A 1030 Vienna Austria

Genomics Core Facility European Molecular Biology Laboratory 69117 Heidelberg Germany

Institute for Theoretical Chemistry University of Vienna A 1090 Vienna Austria

Institute of Microbiology v v i Laboratory of post transcriptional control of gene expression 14220 Prague Czech Republic

Université de Lille CNRS Inserm CHU Lille Institut Pasteur de Lille U1019 UMR8204 CIIL Center for Infection and Immunity of Lille F 59000 Lille France

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Amman F, D'Halluin A, Antoine R, Huot L, Bibova I, Keidel K, Slupek S, Bouquet P, Coutte L, Caboche S, et al. 2018. Primary transcriptome analysis reveals importance of IS elements for the shaping of the transcriptional landscape of Bordetella pertussis. RNA Biol 10.1080/15476286.2018.1462655. PubMed DOI PMC

Antal M, Bordeau V, Douchin V, Felden B. 2005. A small bacterial RNA regulates a putative ABC transporter. J Biol Chem 280: 7901–7908. PubMed

Bibova I, Skopova K, Masin J, Cerny O, Hot D, Sebo P, Vecerek B. 2013. The RNA chaperone Hfq is required for virulence of Bordetella pertussis. Infect Immun 81: 4081–4090. PubMed PMC

Bibova I, Hot D, Keidel K, Amman F, Slupek S, Cerny O, Gross R, Vecerek B. 2015. Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality. RNA Biol 12: 175–185. PubMed PMC

Bobrovskyy M, Vanderpool CK, Richards GR. 2015. Small RNAs regulate primary and secondary metabolism in gram-negative bacteria. Microbiol Spectr 3 10.1128/microbiolspec.MBP-0009-2014 PubMed DOI

Branco Dos Santos F, Olivier BG, Boele J, Smessaert V, De Rop P, Krumpochova P, Klau GW, Giera M, Dehottay P, Teusink B, et al. 2017. Probing the genome-scale metabolic landscape of Bordetella pertussis, the causative agent of whooping cough. Appl Environ Microbiol 10.1128/AEM.01528-17 PubMed DOI PMC

Brennan RG, Link TM. 2007. Hfq structure, function and ligand binding. Curr Opin Microbiol 10: 125–133. PubMed

Burdin N, Handy LK, Plotkin SA. 2017. What is wrong with pertussis vaccine immunity? The problem of waning effectiveness of pertussis vaccines. Cold Spring Harb Perspect Biol 9: a029454. PubMed PMC

Carrier MC, Bourassa JS, Masse E. 2017. Cellular homeostasis: a small RNA at the crossroads of iron and photosynthesis. Curr Biol 27: R380–R383. PubMed

Chen Q, Ng V, Warfel JM, Merkel TJ, Stibitz S. 2017. Activation of Bvg-repressed genes in Bordetella pertussis by RisA requires cross talk from noncooperonic histidine kinase RisK. J Bacteriol 199: e00475-17. PubMed PMC

Cherry JD. 2010. The present and future control of pertussis. Clin Infect Dis 51: 663–667. PubMed

Cotter PA, Jones AM. 2003. Phosphorelay control of virulence gene expression in Bordetella. Trends Microbiol 11: 367–373. PubMed

Cotter PA, Miller JF. 1994. BvgAS-mediated signal transduction: analysis of phase-locked regulatory mutants of Bordetella bronchiseptica in a rabbit model. Infect Immun 62: 3381–3390. PubMed PMC

Coutte L, Huot L, Antoine R, Slupek S, Merkel TJ, Chen Q, Stibitz S, Hot D, Locht C. 2016. The multifaceted RisA regulon of Bordetella pertussis. Sci Rep 6: 32774. PubMed PMC

Croinin TO, Grippe VK, Merkel TJ. 2005. Activation of the vrg6 promoter of Bordetella pertussis by RisA. J Bacteriol 187: 1648–1658. PubMed PMC

Desnoyers G, Bouchard MP, Masse E. 2013. New insights into small RNA-dependent translational regulation in prokaryotes. Trends Genet 29: 92–98. PubMed

Ding Y, Davis BM, Waldor MK. 2004. Hfq is essential for Vibrio cholerae virulence and downregulates σE expression. Mol Microbiol 53: 345–354. PubMed

Geng J, Song Y, Yang L, Feng Y, Qiu Y, Li G, Guo J, Bi Y, Qu Y, Wang W, et al. 2009. Involvement of the post-transcriptional regulator Hfq in Yersinia pestis virulence. PLoS One 4: e6213. PubMed PMC

Gottesman S, McCullen CA, Guillier M, Vanderpool CK, Majdalani N, Benhammou J, Thompson KM, FitzGerald PC, Sowa NA, FitzGerald DJ. 2006. Small RNA regulators and the bacterial response to stress. Cold Spring Harb Symp Quant Biol 71: 1–11. PubMed PMC

Guillier M, Gottesman S, Storz G. 2006. Modulating the outer membrane with small RNAs. Genes Dev 20: 2338–2348. PubMed

Heininger U, Cotter PA, Fescemyer HW, Martinez de Tejada G, Yuk MH, Miller JF, Harvill ET. 2002. Comparative phenotypic analysis of the Bordetella parapertussis isolate chosen for genomic sequencing. Infect Immun 70: 3777–3784. PubMed PMC

Hoe CH, Raabe CA, Rozhdestvensky TS, Tang TH. 2013. Bacterial sRNAs: regulation in stress. Int J Med Microbiol 303: 217–229. PubMed

Holmqvist E, Wagner EGH. 2017. Impact of bacterial sRNAs in stress responses. Biochem Soc Trans 45: 1203–1212. PubMed PMC

Hot D, Slupek S, Wulbrecht B, D'Hondt A, Hubans C, Antoine R, Locht C, Lemoine Y. 2011. Detection of small RNAs in Bordetella pertussis and identification of a novel repeated genetic element. BMC Genomics 12: 207. PubMed PMC

Inatsuka CS, Xu Q, Vujkovic-Cvijin I, Wong S, Stibitz S, Miller JF, Cotter PA. 2010. Pertactin is required for Bordetella species to resist neutrophil-mediated clearance. Infect Immun 78: 2901–2909. PubMed PMC

Jungnitz H, West NP, Walker MJ, Chhatwal GS, Guzman CA. 1998. A second two-component regulatory system of Bordetella bronchiseptica required for bacterial resistance to oxidative stress, production of acid phosphatase, and in vivo persistence. Infect Immun 66: 4640–4650. PubMed PMC

Karataev GI, Sinyashina LN, Medkova AY, Semin EG, Shevtsova ZV, Matua AZ, Kondzariya IG, Amichba AA, Kubrava DT, Mikvabia ZY. 2016. Insertional inactivation of virulence operon in population of persistent Bordetella pertussis bacteria. Genetika 52: 422–430. PubMed

Kasuga T, Nakase Y, Ukishima K, Takatsu K. 1954. Studies on Haemophilis pertussis. III. Some properties of each phase of H. pertussis. Kitasato Arch Exp Med 27: 37–47. PubMed

Lacey BW. 1960. Antigenic modulation of Bordetella pertussis. J Hyg. Lond. 58: 57–93. PubMed PMC

Locht C. 1999. Molecular aspects of Bordetella pertussis pathogenesis. Int Microbiol 2: 137–144. PubMed

Lonnstedt I, Britton T. 2005. Hierarchical Bayes models for cDNA microarray gene expression. Biostatistics 6: 279–291. PubMed

Luu LDW, Octavia S, Zhong L, Raftery M, Sintchenko V, Lan R. 2018. Proteomic adaptation of Australian epidemic Bordetella pertussis. Proteomics 18: e1700237. PubMed

Mattoo S, Cherry JD. 2005. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev 18: 326–382. PubMed PMC

Melton AR, Weiss AA. 1989. Environmental regulation of expression of virulence determinants in Bordetella pertussis. J Bacteriol 171: 6206–6212. PubMed PMC

Merkel TJ, Stibitz S. 1995. Identification of a locus required for the regulation of bvg-repressed genes in Bordetella pertussis. J Bacteriol 177: 2727–2736. PubMed PMC

Merkel TJ, Barros C, Stibitz S. 1998. Characterization of the bvgR locus of Bordetella pertussis. J Bacteriol 180: 1682–1690. PubMed PMC

Moller T, Franch T, Hojrup P, Keene DR, Bachinger HP, Brennan RG, Valentin-Hansen P. 2002. Hfq: a bacterial Sm-like protein that mediates RNA–RNA interaction. Mol Cell 9: 23–30. PubMed

Mooi FR, Van Der Maas NA, De Melker HE. 2014. Pertussis resurgence: waning immunity and pathogen adaptation—two sides of the same coin. Epidemiol Infect 142: 685–694. PubMed PMC

Moon K, Bonocora RP, Kim DD, Chen Q, Wade JT, Stibitz S, Hinton DM. 2017. The BvgAS regulon of Bordetella pertussis. MBio 8: e01526-17. PubMed PMC

Nakamura MM, Liew SY, Cummings CA, Brinig MM, Dieterich C, Relman DA. 2006. Growth phase- and nutrient limitation-associated transcript abundance regulation in Bordetella pertussis. Infect Immun 74: 5537–5548. PubMed PMC

Papenfort K, Vogel J. 2010. Regulatory RNA in bacterial pathogens. Cell Host Microbe 8: 116–127. PubMed

Parkhill J, Sebaihia M, Preston A, Murphy LD, Thomson N, Harris DE, Holden MT, Churcher CM, Bentley SD, Mungall KL, et al. 2003. Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat Genet 35: 32–40. PubMed

Raguckas SE, VandenBussche HL, Jacobs C, Klepser ME. 2007. Pertussis resurgence: diagnosis, treatment, prevention, and beyond. Pharmacotherapy 27: 41–52. PubMed

Seydlova G, Beranova J, Bibova I, Dienstbier A, Drzmisek J, Masin J, Fiser R, Konopasek I, Vecerek B. 2017. The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches. J Biol Chem 292: 8048–8058. PubMed PMC

Sharma CM, Darfeuille F, Plantinga TH, Vogel J. 2007. A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites. Genes Dev 21: 2804–2817. PubMed PMC

Sittka A, Pfeiffer V, Tedin K, Vogel J. 2007. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium. Mol Microbiol 63: 193–217. PubMed PMC

Smirnov A, Wang C, Drewry LL, Vogel J. 2017. Molecular mechanism of mRNA repression in trans by a ProQ-dependent small RNA. EMBO J 36: 1029–1045. PubMed PMC

Smyth GK, Yang YH, Speed T. 2003. Statistical issues in cDNA microarray data analysis. Methods Mol Biol 224: 111–136. PubMed

Sonnleitner E, Hagens S, Rosenau F, Wilhelm S, Habel A, Jager KE, Blasi U. 2003. Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Microb Pathog 35: 217–228. PubMed

Stainer DW, Scholte MJ. 1970. A simple chemically defined medium for the production of phase I Bordetella pertussis. J Gen Microbiol 63: 211–220. PubMed

Stenson TH, Allen AG, Al-Meer JA, Maskell D, Peppler MS. 2005. Bordetella pertussis risA, but not risS, is required for maximal expression of Bvg-repressed genes. Infect Immun 73: 5995–6004. PubMed PMC

Storz G, Opdyke JA, Zhang A. 2004. Controlling mRNA stability and translation with small, noncoding RNAs. Curr Opin Microbiol 7: 140–144. PubMed

Tefon BE, Maass S, Ozcengiz E, Becher D, Hecker M, Ozcengiz G. 2011. A comprehensive analysis of Bordetella pertussis surface proteome and identification of new immunogenic proteins. Vaccine 29: 3583–3595. PubMed

Updegrove TB, Zhang A, Storz G. 2016. Hfq: the flexible RNA matchmaker. Curr Opin Microbiol 30: 133–138. PubMed PMC

Vogel J, Luisi BF. 2011. Hfq and its constellation of RNA. Nat Rev Microbiol 9: 578–589. PubMed PMC

Wagner EG. 2009. Kill the messenger: bacterial antisense RNA promotes mRNA decay. Nat Struct Mol Biol 16: 804–806. PubMed

Wagner EG, Romby P. 2015. Small RNAs in bacteria and archaea: who they are, what they do, and how they do it. Adv Genet 90: 133–208. PubMed

Wassarman KM. 2002. Small RNAs in bacteria: diverse regulators of gene expression in response to environmental changes. Cell 109: 141–144. PubMed

WHO. 2006. Vaccine preventable deaths and the Global Immunization Vision and Strategy, 2006–2015. MMWR Morb Mortal Wkly Rep 55: 511–515. PubMed

Williamson YM, Moura H, Whitmon J, Woolfitt AR, Schieltz DM, Rees JC, Guo S, Kirkham H, Bouck D, Ades EW, et al. 2015. A proteomic characterization of Bordetella pertussis clinical isolates associated with a California state pertussis outbreak. Int J Proteomics 2015: 536537. PubMed PMC

Wilms I, Moller P, Stock AM, Gurski R, Lai EM, Narberhaus F. 2012. Hfq influences multiple transport systems and virulence in the plant pathogen Agrobacterium tumefaciens. J Bacteriol 194: 5209–5217. PubMed PMC

Wright PR, Georg J, Mann M, Sorescu DA, Richter AS, Lott S, Kleinkauf R, Hess WR, Backofen R. 2014. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Res 42: W119–W123. PubMed PMC

Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30: e15. PubMed PMC

Zimna K, Medina E, Jungnitz H, Guzman CA. 2001. Role played by the response regulator Ris in Bordetella bronchiseptica resistance to macrophage killing. FEMS Microbiol Lett 201: 177–180. PubMed

Zuker M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31: 3406–3415. PubMed PMC

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