Cytochrome oxidase requirements in Bordetella reveal insights into evolution towards life in the mammalian respiratory tract.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis, the causal agent of human whooping cough, and B. bronchiseptica, which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica, which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis, which evolved from a B. bronchiseptica-like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1, and cyoABCD1. To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo. No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis-conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens.

Bordetella PlrSR regulatory system controls BvgAS activity and virulence in the lower respiratory tract.

Bacterial pathogens coordinate virulence using two-component regulatory systems (TCS). The Bordetella virulence gene (BvgAS) phosphorelay-type TCS controls expression of all known protein virulence factor-encoding genes and is considered the "master virulence regulator" in Bordetella pertussis, the causal agent of pertussis, and related organisms, including the broad host range pathogen Bordetella bronchiseptica We recently discovered an additional sensor kinase, PlrS [for persistence in the lower respiratory tract (LRT) sensor], which is required for B. bronchiseptica persistence in the LRT. Here, we show that PlrS is required for BvgAS to become and remain fully active in mouse lungs but not the nasal cavity, demonstrating that PlrS coordinates virulence specifically in the LRT. PlrS is required for LRT persistence even when BvgAS is rendered constitutively active, suggesting the presence of BvgAS-independent, PlrS-dependent virulence factors that are critical for bacterial survival in the LRT. We show that PlrS is also required for persistence of the human pathogen B. pertussis in the murine LRT and we provide evidence that PlrS most likely functions via the putative cognate response regulator PlrR. These data support a model in which PlrS senses conditions present in the LRT and activates PlrR, which controls expression of genes required for the maintenance of BvgAS activity and for essential BvgAS-independent functions. In addition to providing a major advance in our understanding of virulence regulation in Bordetella, which has served as a paradigm for several decades, these results indicate the existence of previously unknown virulence factors that may serve as new vaccine components and therapeutic or diagnostic targets.

A novel sensor kinase is required for Bordetella bronchiseptica to colonize the lower respiratory tract.

Bacterial virulence is influenced by the activity of two-component regulator systems (TCSs), which consist of membrane-bound sensor kinases that allow bacteria to sense the external environment and cytoplasmic, DNA-binding response regulator proteins that control appropriate gene expression. Respiratory pathogens of the Bordetella genus require the well-studied TCS BvgAS to control the expression of many genes required for colonization of the mammalian respiratory tract. Here we describe the identification of a novel gene in Bordetella bronchiseptica, plrS, the product of which shares sequence homology to several NtrY-family sensor kinases and is required for B. bronchiseptica to colonize and persist in the lower, but not upper, respiratory tract in rats and mice. The plrS gene is located immediately 5' to and presumably cotranscribed with a gene encoding a putative response regulator, supporting the idea that PlrS and the product of the downstream gene may compose a TCS. Consistent with this hypothesis, the PlrS-dependent colonization phenotype requires a conserved histidine that serves as the site of autophosphorylation in other sensor kinases, and in strains lacking plrS, the production and/or cellular localization of several immune-recognized proteins is altered in comparison to that in the wild-type strain. Because plrS is required for colonization and persistence only in the lower respiratory tract, a site where innate and adaptive immune mechanisms actively target infectious agents, we hypothesize that its role may be to allow Bordetella to resist the host immune response.

Natural-host animal models indicate functional interchangeability between the filamentous haemagglutinins of Bordetella pertussis and Bordetella bronchiseptica and reveal a role for the mature C-terminal domain, but not the RGD motif, during infection.

Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human-adapted (e.g. Bordetella pertussis) strains produce a surface-exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg-Gly-Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural-host animal models should apply to B. pertussis FHA as well. We also show that the C-terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage-like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.

In vivo-selected mutations in methyl-directed mismatch repair suppress the virulence attenuation of Salmonella dam mutant strains following intraperitoneal, but not oral, infection of naïve mice.

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-AP(r)) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-AP(r) derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naïve mice. These 2-AP(r) derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.

Characterization of the filamentous hemagglutinin-like protein FhaS in Bordetella bronchiseptica.

Filamentous hemagglutinin (FHA) is a large (>200 kDa), rod-shaped protein expressed by bordetellae that is both surface-associated and secreted. FHA mediates bacterial adherence to epithelial cells and macrophages in vitro and is absolutely required for tracheal colonization in vivo. The recently sequenced Bordetella bronchiseptica genome revealed the presence of a gene, fhaS, that is nearly identical to fhaB, the FHA structural gene. We show that although fhaS expression requires the BvgAS virulence control system, it is maximal only under a subset of conditions in which BvgAS is active, suggesting an additional level of regulation. We also show that, like FHA, FhaS undergoes a C-terminal proteolytic processing event and is both surface-associated and secreted and that export across the outer membrane requires the channel-forming protein FhaC. Unlike FHA, however, FhaS was unable to mediate adherence of B. bronchiseptica to epithelial cell lines in vitro and was not required for respiratory tract colonization in vivo. In a coinfection experiment, a DeltafhaS strain was out-competed by wild-type B. bronchiseptica, indicating that fhaS is expressed in vivo and that FhaS contributes to bacterial fitness in a manner revealed when the mutant must compete with wild-type bacteria. These data suggest that FHA and FhaS perform distinct functions during the Bordetella infectious cycle. A survey of various Bordetella strains revealed two distinct fhaS alleles that segregate according to pathogen host range and that B. parapertussis(hu) most likely acquired its fhaS allele from B. pertussis horizontally, suggesting fhaS may contribute to host-species specificity.

Bordetella filamentous hemagglutinin plays a critical role in immunomodulation, suggesting a mechanism for host specificity.

Bordetella pertussis, the causative agent of the acute childhood respiratory disease whooping cough, is a human-adapted variant of Bordetella bronchiseptica, which displays a broad host range and typically causes chronic, asymptomatic infections. These pathogens express a similar but not identical surface-exposed and secreted protein called filamentous hemagglutinin (FHA) that has been proposed to function as both a primary adhesin and an immunomodulator. To test the hypothesis that FHA plays an important role in determining host specificity and/or the propensity to cause acute versus chronic disease, we constructed a B. bronchiseptica strain expressing FHA from B. pertussis (FHA(Bp)) and compared it with wild-type B. bronchiseptica in several natural-host infection models. FHA(Bp) was able to substitute for FHA from B. bronchiseptica (FHA(Bb)) with regard to its ability to mediate adherence to several epithelial and macrophage-like cell lines in vitro, but it was unable to substitute for FHA(Bb) in vivo. Specifically, FHA(Bb), but not FHA(Bp), allowed B. bronchiseptica to colonize the lower respiratory tracts of rats, to modulate the inflammatory response in the lungs of immunocompetent mice, resulting in decreased lung damage and increased bacterial persistence, to induce a robust anti-Bordetella antibody response in these immunocompetent mice, and to overcome innate immunity and cause a lethal infection in immunodeficient mice. These results indicate a critical role for FHA in B. bronchiseptica-mediated immunomodulation, and they suggest a role for FHA in host specificity.

DNA adenine methylase overproduction in Yersinia pseudotuberculosis alters YopE expression and secretion and host immune responses to infection.

Yersinia pseudotuberculosis mutants that overproduce the DNA adenine methylase (Dam) are highly attenuated, confer fully protective immune responses, and secrete several Yersinia virulence proteins (Yersinia outer proteins [Yops]) under conditions that are nonpermissive for secretion in wild-type strains. We examined here the effects of Dam overproduction on Yersinia virulence determinant expression and secretion, as well as the host immune response to Yersinia antigens. Western blot analysis with convalescent antisera identified several low-calcium-responsive antigens whose synthesis was affected by Dam overproduction. One of these antigens was shown to be the type III secretion effector protein, YopE, a cytotoxin involved in antiphagocytosis. Dam overproduction disrupted both the thermal and calcium regulation of YopE synthesis and relaxed the thermal but not the calcium dependence of YopE secretion. Altered expression and/or secretion of Yersinia proteins in Dam-overproducing strains may contribute to the decreased virulence and heightened immunity observed in vaccinated hosts and may provide a means by which to deliver heterologous antigens and/or immune modulators of the inflammatory response.