Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA.In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.

Laboratory of post transcriptional control of gene expression Institute of Microbiology of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Mattoo S, Cherry JD.. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev. 2005;18(2):326–382. doi:10.1128/CMR.18.2.326-382.2005 PubMed DOI PMC

Melvin JA, et al. . Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol. 2014;12(4):274–288. doi:10.1038/nrmicro3235 PubMed DOI PMC

Belcher T, Dubois V, Rivera-Millot A, et al. . Pathogenicity and virulence of Bordetella pertussis and its adaptation to its strictly human host. Virulence. 2021;12(1):2608–2632. doi:10.1080/21505594.2021.1980987 PubMed DOI PMC

Yuk MH, Harvill ET, Cotter PA, et al. . Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system. Mol Microbiol. 2000;35(5):991–1004. doi:10.1046/j.1365-2958.2000.01785.x PubMed DOI

Nicholson TL, Brockmeier SL, Loving CL, et al. . The Bordetella bronchiseptica type III secretion system is required for persistence and disease severity but not transmission in swine. Infect Immun. 2014;82(3):1092–1103. doi:10.1128/IAI.01115-13 PubMed DOI PMC

Fennelly NK, Sisti F, Higgins SC, et al. . Bordetella pertussis expresses a functional type III secretion system that subverts protective innate and adaptive immune responses. Infect Immun. 2008;76(3):1257–1266. doi:10.1128/IAI.00836-07 PubMed DOI PMC

Gaillard ME, Bottero D, Castuma CE, et al. . Laboratory adaptation of Bordetella pertussis is associated with the loss of type three secretion system functionality. Infect Immun. 2011;79(9):3677–3682. doi:10.1128/IAI.00136-11 PubMed DOI PMC

Bibova I, Hot D, Keidel K, et al. . Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality. RNA Biol. 2015;12(2):175–185. doi:10.1080/15476286.2015.1017237 PubMed DOI PMC

Brickman TJ, Cummings CA, Liew S-Y, et al. . Transcriptional profiling of the iron starvation response in Bordetella pertussis provides new insights into siderophore utilization and virulence gene expression. J Bacteriol. 2011;193(18):4798–4812. doi:10.1128/JB.05136-11 PubMed DOI PMC

Hanawa T, Kamachi K, Yonezawa H, et al. . Glutamate limitation, BvgAS activation, and (p)ppGpp regulate the expression of the Bordetella pertussis type 3 secretion system. J Bacteriol. 2016;198(2):343–351. doi:10.1128/JB.00596-15 PubMed DOI PMC

Hester SE, Lui M, Nicholson T, et al. . Identification of a CO2 responsive regulon in Bordetella. PLoS One. 2012;7(10):e47635. doi:10.1371/journal.pone.0047635 PubMed DOI PMC

Gestal MC, Rivera I, Howard LK, et al. . Blood or serum exposure induce global transcriptional changes, altered antigenic profile, and increased cytotoxicity by classical Bordetellae. Front Microbiol. 2018;9:1969), doi:10.3389/fmicb.2018.01969 PubMed DOI PMC

Drzmisek J, et al. . Omics analysis of blood-responsive regulon in Bordetella pertussis identifies a novel essential T3SS substrate. Int J Mol Sci. 2021;22(2). PubMed PMC

Rivera I, Linz B, Dewan KK, et al. . Conservation of ancient genetic pathways for intracellular persistence Among animal pathogenic Bordetellae. Front Microbiol. 2019;10:2839. doi:10.3389/fmicb.2019.02839 PubMed DOI PMC

Farman MR, Petráčková D, Kumar D, et al. . Avirulent phenotype promotes Bordetella pertussis adaptation to the intramacrophage environment. Emerg Microbes Infect. 2023;12(1):e2146536. doi:10.1080/22221751.2022.2146536 PubMed DOI PMC

Petráčková D, Farman MR, Amman F, et al. . Transcriptional profiling of human macrophages during infection with Bordetella pertussis. RNA Biol. 2020;17(5):731–742. doi:10.1080/15476286.2020.1727694 PubMed DOI PMC

Dienstbier A, et al. . Comparative integrated omics analysis of the Hfq regulon in Bordetella pertussis. Int J Mol Sci. 2019;20(12). PubMed PMC

Dienstbier A, Amman F, Petráčková D, et al. . Comparative omics analysis of historic and recent isolates of Bordetella pertussis and effects of genome rearrangements on evolution. Emerg Infect Dis. 2021;27(1):57–68. doi:10.3201/eid2701.191541 PubMed DOI PMC

Galán JE, Lara-Tejero M, Marlovits TC, et al. . Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells. Annu Rev Microbiol. 2014;68:415–438. doi:10.1146/annurev-micro-092412-155725 PubMed DOI PMC

French CT, Panina EM, Yeh SH, et al. . The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts. Cell Microbiol. 2009;11(12):1735–1749. doi:10.1111/j.1462-5822.2009.01361.x PubMed DOI PMC

Kuwae A, Matsuzawa T, Ishikawa N, et al. . BopC is a novel type III effector secreted by and has a critical role in type III-dependent necrotic cell death. J Biol Chem. 2006;281(10):6589–6600. doi:10.1074/jbc.M512711200 PubMed DOI

Stockbauer KE, Foreman-Wykert AK, Miller JF.. Bordetella type III secretion induces caspase 1-independent necrosis. Cell Microbiol. 2003;5(2):123–132. doi:10.1046/j.1462-5822.2003.00260.x PubMed DOI

Bayram J, Malcova I, Sinkovec L, et al. . Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue. PLoS Pathog. 2020;16(8):e1008512. doi:10.1371/journal.ppat.1008512 PubMed DOI PMC

Kamanova J. Bordetella type III secretion injectosome and effector proteins. Front Cell Infect Microbiol. 2020;10:466. doi:10.3389/fcimb.2020.00466 PubMed DOI PMC

Nagamatsu K, Kuwae A, Konaka T, et al. . Bordetella evades the host immune system by inducing IL-10 through a type III effector, BopN. J Exp Med. 2009;206(13):3073–3088. doi:10.1084/jem.20090494 PubMed DOI PMC

Navarrete KM, et al. . Bopn is a gatekeeper of the Bordetella type III secretion system. Microbiol Spectr. 2023: e0411222. PubMed PMC

Fauconnier A, Veithen A, Gueirard P, et al. . Characterization of the type III secretion locus of Bordetella pertussis. Int J Med Microbiol. 2001;290(8):693–705. doi:10.1016/S1438-4221(01)80009-6 PubMed DOI

Mattoo S, Yuk MH, Huang LL, et al. . Regulation of type III secretion in Bordetella. Mol Microbiol. 2004;52(4):1201–1214. doi:10.1111/j.1365-2958.2004.04053.x PubMed DOI

Ahuja U, Shokeen B, Cheng N, et al. . Differential regulation of type III secretion and virulence genes in Bordetella pertussis and Bordetella bronchiseptica by a secreted anti-sigma factor. Proc Natl Acad Sci U S A. 2016;113(9):2341–2348. doi:10.1073/pnas.1600320113 PubMed DOI PMC

Moon K, et al. . The BvgAS regulon of Bordetella pertussis. mBio. 2017;8(5). PubMed PMC

Kurushima J, Kuwae A, Abe A.. The type III secreted protein BspR regulates the virulence genes in Bordetella bronchiseptica. PLoS One. 2012;7(6):e38925. doi:10.1371/journal.pone.0038925 PubMed DOI PMC

Abe A, et al. . Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion. Mol Microbiol. 1999;33(6):1162–1175. PubMed

Elliott SJ, et al. . Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol Microbiol. 1999;33(6):1176–1189. PubMed

Katsowich N, Elbaz N, Pal RR, et al. . Host cell attachment elicits posttranscriptional regulation in infecting enteropathogenic bacteria. Science. 2017;355(6326):735–739. doi:10.1126/science.aah4886 PubMed DOI

Ye F, Yang F, Yu R, et al. . Molecular basis of binding between the global post-transcriptional regulator CsrA and the T3SS chaperone CesT. Nat Commun. 2018;9(1):1196. doi:10.1038/s41467-018-03625-x PubMed DOI PMC

Bibova I, Skopova K, Masin J, et al. . The RNA chaperone Hfq is required for virulence of Bordetella pertussis. Infect Immun. 2013;81(11):4081–4090. doi:10.1128/IAI.00345-13 PubMed DOI PMC

Inatsuka CS, Xu Q, Vujkovic-Cvijin I, et al. . Pertactin is required for Bordetella species to resist neutrophil-mediated clearance. Infect Immun. 2010;78(7):2901–2909. doi:10.1128/IAI.00188-10 PubMed DOI PMC

Bolger AM, Lohse M, Usadel B.. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–2120. doi:10.1093/bioinformatics/btu170 PubMed DOI PMC

Patro R, Duggal G, Love MI, et al. . Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14(4):417–419. doi:10.1038/nmeth.4197 PubMed DOI PMC

Risso D, Ngai J, Speed TP, et al. . Normalization of RNA-Seq data using factor analysis of control genes or samples. Nat Biotechnol. 2014;32(9):896–902. doi:10.1038/nbt.2931 PubMed DOI PMC

Love MI, Huber W, Anders S.. Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2. Genome Biol. 2014;15(12):550. doi:10.1186/s13059-014-0550-8 PubMed DOI PMC

Perez-Riverol Y, Csordas A, Bai J, et al. . The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019;47(D1):D442–D450. doi:10.1093/nar/gky1106 PubMed DOI PMC

Hoffman CL, et al. . Bordetella pertussis can be motile and express flagellum-like structures. mBio. 2019;10(3). PubMed PMC

Hiramatsu Y, Nishida T, Nugraha DK, et al. . Interference of flagellar rotation up-regulates the expression of small RNA contributing to Bordetella pertussis infection. Sci Adv. 2022;8(51):eade8971. doi:10.1126/sciadv.ade8971 PubMed DOI PMC

Bart MJ, et al. . Complete genome sequences of Bordetella pertussis isolates B1917 and B1920, representing two predominant global lineages. Genome Announc. 2014;2(6). PubMed PMC

Jumper J, Jumper J, Evans R, et al. . Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–589. doi:10.1038/s41586-021-03819-2 PubMed DOI PMC

Evans R, et al. Protein complex prediction with AlphaFold-Multimer. bioRxiv, 2021: p. 2021.

Akerley BJ, Monack DM, Falkow S, et al. . The bvgAS locus negatively controls motility and synthesis of flagella in Bordetella bronchiseptica. J Bacteriol. 1992;174(3):980–990. doi:10.1128/jb.174.3.980-990.1992 PubMed DOI PMC

Nicholson TL, Conover MS, Deora R.. Transcriptome profiling reveals stage-specific production and requirement of flagella during biofilm development in Bordetella bronchiseptica. PLoS One. 2012;7(11):e49166. doi:10.1371/journal.pone.0049166 PubMed DOI PMC

Cornelis GR, Van Gijsegem F.. Assembly and function of type III secretory systems. Annu Rev Microbiol. 2000;54:735–774. doi:10.1146/annurev.micro.54.1.735 PubMed DOI

Gestal MC, et al. . Enhancement of immune response against Bordetella spp. by disrupting immunomodulation. Sci Rep. 2019;9(1):20261. doi:10.1038/s41598-019-56652-z PubMed DOI PMC

Guttenplan SB, Kearns DB.. Regulation of flagellar motility during biofilm formation. FEMS Microbiol Rev. 2013;37(6):849–871. doi:10.1111/1574-6976.12018 PubMed DOI PMC

O'Toole GA, Kolter R.. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol. 1998;30(2):295–304. doi:10.1046/j.1365-2958.1998.01062.x PubMed DOI

Watnick PI, Lauriano CM, Klose KE, et al. . The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol Microbiol. 2001;39(2):223–235. doi:10.1046/j.1365-2958.2001.02195.x PubMed DOI PMC

Reuter M, Mallett A, Pearson BM, et al. . Biofilm formation by Campylobacter jejuni is increased under aerobic conditions. Appl Environ Microbiol. 2010;76(7):2122–2128. doi:10.1128/AEM.01878-09 PubMed DOI PMC

Jerse AE, Yu J, Tall BD, et al. . A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci U S A. 1990;87(20):7839–7843. doi:10.1073/pnas.87.20.7839 PubMed DOI PMC

de Gouw D, et al. . The vaccine potential of Bordetella pertussis biofilm-derived membrane proteins. Emerg Microbes Infect. 2014;3(8):e58. PubMed PMC