Bordetella PlrSR regulatory system controls BvgAS PNAS PLUS activity and virulence in the lower respiratory tract M. Ashley Bonea,1, Aaron J. Wilkb,1,2, Andrew I. Peraulta, Sara A. Marlatta,3, Erich V. Schellera, Rebecca Anthouarda, Qing Chenc, Scott Stibitzc, Peggy A. Cottera,4, and Steven M. Juliob,4 aDepartment of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; bDepartment of Biology, Westmont College, Santa Barbara, CA 93108; and cDivision of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 Edited by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved January 6, 2017 (received for review June 13, 2016) Bacterial pathogens coordinate virulence using two-component to collectively as vags) and lack of expression of BvgAS-repressed regulatory systems (TCS). The Bordetella virulence gene (BvgAS) genes (called vrgs), which includes those encoding flagella in – phosphorelay-type TCS controls expression of all known protein B. bronchiseptica. The Bvg phase occurs when the bacteria are “ virulence factor-encoding genes and is considered the master vir- grown at ≤26 °C or when millimolar concentrations of MgSO4 or ulence regulator” in Bordetella pertussis, the causal agent of pertus- nicotinic acid are added to the growth medium (referred to as – sis, and related organisms, including the broad host range pathogen “modulating conditions”). The Bvg phase is characterized by Bordetella bronchiseptica. We recently discovered an additional sen- expression of vrg loci and lack of expression of vags. The Bvg- sor kinase, PlrS [for persistence in the lower respiratory tract (LRT) intermediate (Bvgi) phase occurs at intermediate temperatures B. bronchiseptica sensor], which is required for persistence in the or in the presence of low concentrations of MgSO4 or nicotinic LRT. Here, we show that PlrS is required for BvgAS to become and acid (9). It is characterized by expression of vags that contain remain fully active in mouse lungs but not the nasal cavity, demon- high-affinity BvgA binding sites at their promoters (such as fhaB, strating that PlrS coordinates virulence specifically in the LRT. PlrS is encoding FHA, fimBCD, encoding the FIM biogenesis proteins, required for LRT persistence even when BvgAS is rendered consti- and bvgAS itself), lack of expression of vags with low-affinity tutively active, suggesting the presence of BvgAS-independent, BvgA binding sites (such as cyaABDE, encoding ACT and ptxA-E, PlrS-dependent virulence factors that are critical for bacterial sur- encoding pertussis toxin), lack of expression of vrgs, and maximal MICROBIOLOGY vival in the LRT. We show that PlrS is also required for persistence expression of bipA, which encodes an outer-membrane protein of of the human pathogen B. pertussis in the murine LRT and we pro- unknown function (9–11). Although BvgAS activity is altered by vide evidence that PlrS most likely functions via the putative cog- temperature, MgSO4, and nicotinic acid in vitro, the true signals it nate response regulator PlrR. These data support a model in which senses in nature are unknown. PlrS senses conditions present in the LRT and activates PlrR, which B. pertussis and B. bronchiseptica strains containing loss-of- controls expression of genes required for the maintenance of BvgAS function mutations in bvgAS are avirulent, whereas strains con- activity and for essential BvgAS-independent functions. In addition taining mutations that render BvgAS active—even under modu- to providing a major advance in our understanding of virulence lating conditions in vitro—are indistinguishable from WT bacteria regulation in Bordetella, which has served as a paradigm for several decades, these results indicate the existence of previously unknown Significance virulence factors that may serve as new vaccine components and therapeutic or diagnostic targets. Bordetella spp. includes Bordetella pertussis, the causal agent of whooping cough. The Bordetella virulence gene (BvgAS) Bordetella | two-component system | virulence | respiratory infection | gene regulation two-component regulatory system (TCS) is considered the “master virulence regulator” in Bordetella, as it controls ex- pression of all known virulence factor-encoding genes. We aused by the human-specific, Gram-negative bacterium Bor- show here that another TCS, PlrSR, is required for BvgAS ac- Cdetella pertussis, whooping cough (aka pertussis) is reemerging tivity in the lower respiratory tract (LRT) and for virulence even in the United States and other developed countries, despite high when BvgAS is rendered constitutively active, suggesting that vaccine coverage (1, 2). Increased incidence in recent years co- it controls critical functions for bacterial survival in the LRT incides with the switch to acellular vaccines, which induce im- independently of BvgAS. Our data introduce a new layer of munity that is less durable than that induced by whole-cell Bordetella – complexity to a paradigm of virulence control that vaccines or by infection with B. pertussis (3 5). Although closely has held for more than 30 y, and they indicate the existence of related to B. pertussis, Bordetella bronchiseptica infects nearly previously unknown bacterial factors that may serve as vaccine all mammals and typically causes more chronic, long-term re- components and therapeutic targets. spiratory infections (6). Despite these differences, B. pertussis and B. bronchiseptica produce a nearly identical set of virulence factors Author contributions: M.A.B., A.J.W., S.S., P.A.C., and S.M.J. designed research; M.A.B., A.J.W., that includes adhesins, such as filamentous hemagglutinin (FHA) A.I.P., S.A.M., E.V.S., R.A., and Q.C. performed research; M.A.B., A.J.W., P.A.C., and S.M.J. and fimbriae (FIM), and toxins, such as adenylate cyclase toxin analyzed data; and M.A.B., P.A.C., and S.M.J. wrote the paper. (ACT), and a type III secretion system (T3SS) (2). The authors declare no conflict of interest. The Bordetella virulence gene (BvgAS) phosphorelay-type two- This article is a PNAS Direct Submission. component regulatory system (TCS) is considered the “master 1M.A.B. and A.J.W. contributed equally to this work. virulence control system” in Bordetella. BvgAS differentially reg- 2Present address: Medical Scientist Training Program, Stanford University School of Med- ulates (either directly or indirectly) hundreds of genes and at least icine, Stanford, CA 94305. + 3 three distinct phenotypic phases (7, 8). The Bvg phase occurs Present address: Department of Biology and Chemistry, Azusa Pacific University, Azusa, – CA 91702. when the bacteria are grown at 37 °C in Stainer Scholte (SS) broth 4 or on Bordet–Gengou (BG) blood agar and correlates with BvgAS To whom correspondence may be addressed. Email: [email protected] or + [email protected]. activity. The Bvg phase is characterized by expression of all This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. currently known protein virulence factor-encoding genes (referred 1073/pnas.1609565114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1609565114 PNAS Early Edition | 1of9 Downloaded by guest on September 30, 2021 in their ability to cause respiratory tract infections (12–14). A Characterization of antibody responses following infection and analyses using recombination-based reporters of gene expression – indicate that modulation to the Bvg phase does not occur during – infection (12, 15, 16). Studies with strains that produce Bvg + phase factors ectopically in the Bvg phase have demonstrated the importance of BvgAS-mediated repression of gene expres- sion in vivo (14, 17). Taken together, these results have been + interpreted to indicate that the Bvg phase is both necessary and sufficient for the development of respiratory infection by Bordetella. Moreover, these data, together with the fact that the phenotypic profile of WT bacteria grown at 37 °C in SS medium or on BG agar + (Bvg phase conditions) is identical to that of mutants containing bvgS mutations that render BvgS insensitive to modulating condi- tions, have led to the conclusion that these in vitro growth condi- tions mimic, at least to some extent, those experienced by the bacteria in the respiratory tract. B In a survey of putative TCSs in B. bronchiseptica, we discov- ered a gene (BB0264) predicted to encode an NtrY-like sensor kinase that is essential for B. bronchiseptica to colonize the tra- chea of rats following low-dose, low-volume intranasal in- oculation and to persist in the lungs of mice following high-dose, large-volume intranasal inoculation (18). We named BB0264 plrS, for persistence in the lower respiratory tract (LRT) sensor. Subsequently, Hester et al. reported the identification of a CO2 responsive regulon in Bordetella, and hypothesized that CO2 gradients may be sensed by the bacteria as a cue indicating their localization in the LRT (19). Here, we show that plrS is required for increased virulence factor production in response to CO2, suggesting a functional link between PlrS and BvgAS. We in- vestigated this link, showed that plrS is required for BvgAS to be fully active in the LRT, and showed that plrS is required for C persistence in the LRT even when BvgAS is constitutively active, indicating that although BvgAS is necessary in vivo, it is not sufficient. Moreover, our data suggest the existence of genes that are expressed only in the LRT that encode previously unknown virulence factors, which may serve as new therapeutic or di- agnostic targets and vaccine components. Results PlrS Is Required for Enhanced BvgAS-Dependent Virulence-Associated Phenotypes in Response to Elevated CO2 Concentrations. BvgAS- regulated virulence-associated phenotypes include adherence to epithelial cells and macrophages, which is mediated by FHA (20, 21), hemolysis on blood-containing agar, which is mediated by ACT (22), and toxicity to various eukaryotic cell types in culture, which is mediated, at least in part, by the T3SS (23).