In vivo colonization profile study of Bordetella bronchiseptica in the nasal cavity

Bordetella bronchiseptica chronically infects a wide range of mammals, and resides primarily in the nasal cavity of the infected host. Multiple virulence factors of Bordetella species have been studied in the context of lower respiratory tract infections, but relatively less is known about the bacterial life cycle in the nasal cavity. Evidences were discovered for Bvg intermediate (Bvg(i)) phase expression in vivo and that the major adhesin filamentous hemagglutinin plays a major role in the colonization of B. bronchiseptica in the unciliated olfactory epithelia of the nasal cavity.     

Early colonization of the rat upper respiratory tract by temperature modulated Bordetella bronchiseptica.

The ability of nonmodulated Bvg+ phase cultures, temperature modulated Bvg- phase cultures, and a Bvg- phase-locked mutant of Bordetella bronchiseptica to colonize the rat upper respiratory tract was investigated. Initially, greater numbers of the temperature modulated Bvg- phase bacteria adhered to the nasal cavity of the rats. The temperature modulated Bvg- phase bacteria appeared to be quickly cleared to levels lower than the Bvg+ phase bacteria by 4 h after inoculation and stayed lower until 48 h after inoculation when colonization levels were equal to the Bvg+ phase bacteria. The level of colonization with the Bvg- phase-locked mutant of B. bronchiseptica was lower than both the nonmodulated Bvg+ phase and temperature modulated Bvg- phase cultures and declined over time during the experiment. These findings suggest that there may be increased adherence from an environmental phase to ensure bacteria survive initial clearance mechanisms until the switch to the Bvg+ phase occurs.     

Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract

Bordetella bronchiseptica lipopolysaccharide (LPS) expression varies depending on growth conditions, regulated by the Bvg system. A B. bronchiseptica pagP homologue was identified that is required for Bvg-mediated modification of the lipid A core region of LPS that occurs on switching from the Bvg- to the Bvg+ phase. Structural analysis demonstrated that the lipid A of a B. bronchiseptica pagP mutant differed from wild-type lipid A by the absence of a palmitate group in secondary acylation at the C3' position. The putative pagP promoter drove the expression of a green fluorescent protein (GFP) reporter gene in a Bvg-regulated fashion. These data suggest that B. bronchiseptica pagP encodes a Bvg-regulated lipid A palmitoyl transferase that mediates modification of the lipid A as part of the overall Bvg-mediated adaptation of this organism to changing environmental conditions. We also show that pagP is not required for the initial colonization of the mouse respiratory tract by B. bronchiseptica, but is required for persistence of the organism within this organ.     

Bordetella type III secretion modulates dendritic cell migration resulting in immunosuppression and bacterial persistence

Chronic bacterial infection reflects a balance between the host immune response and bacterial factors that promote colonization and immune evasion. Bordetella bronchiseptica uses a type III secretion system (TTSS) to persist in the lower respiratory tract of mice. We hypothesize that colonization is facilitated by bacteria-driven modulation of dendritic cells (DCs), which leads to an immunosuppressive adaptive host response. Migration of DCs to the draining lymph nodes of the respiratory tract was significantly increased in mice infected with wild-type B. bronchiseptica compared with mice infected with TTSS mutant bacteria. Reduced colonization by TTSS-deficient bacteria was evident by 7 days after infection, whereas colonization by wild-type bacteria remained high. This decrease in colonization correlated with peak IFN-gamma production by restimulated splenocytes from infected animals. Wild-type bacteria also elicited peak IFN-gamma production on day 7, but the quantity was significantly lower than that elicited by TTSS mutant bacteria. Additionally, wild-type bacteria elicited higher levels of the immunosuppressive cytokine IL-10 compared with the TTSS mutant bacteria. B. bronchiseptica colonization in IL-10(-/-) mice was significantly reduced compared with infections in wild-type mice. These findings suggest that B. bronchiseptica use the TTSS to rapidly drive respiratory DCs to secondary lymphoid tissues where these APCs stimulate an immunosuppressive response characterized by increased IL-10 and decreased IFN-gamma production that favors bacterial persistence.     

Suppression of DeltabipA phenotypes in Escherichia coli by abolishment of pseudouridylation at specific sites on the 23S rRNA

The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by DeltabipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.     

The Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica

Bordetella species utilize the BvgAS (Bordetella virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg+ phase, a nonvirulent Bvg- phase, and an intermediate Bvgi phase. Genes expressed in the Bvg+ phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvgi phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine that Bordetella bronchiseptica can form biofilms in vitro and that the generation of biofilm is maximal in the Bvgi phase. We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype. However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA. Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvgi phase in B. bronchiseptica.     

Bordetella type III secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state

Bordetella bronchiseptica establishes persistent infection of the murine respiratory tract. We hypothesize that long-term colonization is mediated in part by bacteria-driven modulation of dendritic cells (DCs) leading to altered adaptive immune responses. Bone marrow-derived DCs (BMDCs) from C57BL/6 mice infected with live B. bronchiseptica exhibited high surface expression of MHCII, CD86, and CD80. However, B. bronchiseptica-infected BMDCs did not exhibit significant increases in CD40 surface expression and IL-12 secretion compared with BMDCs treated with heat-killed B. bronchiseptica. The B. bronchiseptica type III secretion system (TTSS) mediated the increase in MHCII, CD86, and CD80 surface expression, while the inhibition of CD40 and IL-12 expression was mediated by adenylate cyclase toxin (ACT). IL-6 secretion was independent of the TTSS and ACT. These phenotypic changes may result from differential regulation of MAPK signaling in DCs. Wild-type B. bronchiseptica activated the ERK 1/2 signaling pathway in a TTSS-dependent manner. Additionally, ACT was found to inhibit p38 signaling. These data suggest that B. bronchiseptica drive DC into a semimature phenotype by altering MAPK signaling. These semimature DCs may induce tolerogenic immune responses that allow the persistent colonization of B. bronchiseptica in the host respiratory tract.     

BipA is required for growth of Escherichia coli K12 at low temperature

The bipA gene encodes a ribosome-associated GTPase postulated to be involved in regulatory functions in enteropathogenic Escherichia coli. Previous studies demonstrated that BipA is tyrosine phosphorylated in EPEC strains, but not in E. coli strain K12. Results presented here indicate that BipA function is required at low temperatures in E. coli K12, suggesting a regulatory role independent of phosphorylation and of pathogenicity.     

Role of BvgA phosphorylation and DNA binding affinity in control of Bvg-mediated phenotypic phase transition in Bordetella pertussis

To investigate the mechanism by which the Bordetella BvgAS phosphorelay controls expression of at least three distinct phenotypic phases, we isolated and characterized two B. pertussis mutants that were able to express Bvg- and Bvg(i) phase phenotypes but not Bvg+ phase phenotypes. In both cases, the mutant phenotype was due to a single nucleotide change in bvgA resulting in a single amino acid substitution in BvgA. In vitro phosphorylation assays showed that BvgA containing the T194M substitution was significantly impaired in its ability to use either BvgS or acetyl phosphate as a substrate for phosphorylation. Binding studies indicated that this mutant protein was able to bind an oligonucleotide containing a high-affinity BvgA binding site in a manner similar to wild-type BvgA, but was defective for binding the fhaB promoter in the absence of RNA polymerase (RNAP). By contrast, BvgA containing the R152H substitution had wild-type phosphorylation properties but was severely defective in its ability to bind either the high-affinity BvgA binding site-containing oligonucleotide or the fhaB promoter by itself. Both mutant BvgA proteins were able to bind the fhaB promoter in the presence of RNAP however, demonstrating the profound effect that RNAP has on stabilizing the ternary complexes between promoter DNA, BvgA and RNAP. Our results are consistent with the hypothesis that BvgAS controls expression of multiple phenotypic phases by adjusting the intracellular concentration of BvgA-P and they demonstrate the additive nature of BvgA binding site affinity and protein-protein interactions at different Bvg-regulated promoters.     

Characterization of intermediate phenotypes induced by chemically undefined laboratory media in virulent Bordetella bronchiseptica strains

The expression of many virulence factors of Bordetella bronchiseptica is regulated by the bvgAS locus and reduced in response to environmental signals called modulators. Virulent strains can alternate between virulent (Bvg(+)), intermediate (Bvg(i)), and modulated (Bvg(+)mod) phenotypes. Potential vaccine antigens can be expressed by Bvg1 strains grown only in the absence of modulators. In the present study we evaluated filamentous hemagglutinin (FHA) and outer membrane protein (OMP) expression in Bvg(+) B. bronchiseptica strains grown in chemically undefined media: nutrient agar (NA), tryptic soy agar (TSA), tryptose phosphate broth (TPB), and brain-heart infusion (BHI). Our results suggest that TSA and TPB usually induce semimodulation, since Bvg(+) strains cultured in these media retained the expression of FHA and virulence-associated OMPs in the 30 kDa region, but failed to express other virulence markers such as OMPs in the regions of 90 and 200 kDa, though they expressed flagellin (avirulence marker). On the other hand, NA and BHI usually induce modulation. Thus the assayed chemically undefined media should not be used in vaccine production. Semimodulation induced by TSA and TPB can be accurately detected by SDS-PAGE Sarkosyl-insoluble OMP-enriched profiles. The reduction or absence of OMPs in the regions of 90 and 200 kDa is the most sensitive marker, and in some cases the presence of flagellin in intermediate profiles is another trait of the Bvg(i) phenotypes. Therefore these markers could be useful for selecting media for vaccine production. We also characterized the phenotype of Bvg(+) strains grown in Stainer-Scholte broth, an expensive medium, with and without glutathione, and we have detected no differences; this is the first attempt to reduce the cost of a Bordetella growth medium for veterinary vaccine production.     

Expression of the primary carbohydrate component of the Bordetella bronchiseptica biofilm matrix is dependent on growth phase but independent of Bvg regulation

We previously showed that the Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica (Y. Irie, S. Mattoo, and M. H. Yuk, J. Bacteriol. 186:5692-5698, 2004). Analyses of the extracellular components of B. bronchiseptica biofilm matrix revealed that the major sugar component in the matrix was xylose, and linkage analysis indicated a majority of it to be in a 4-linked polymeric form. The production of xylose was independent of Bvg regulation but instead was dependent on bacterial growth phase. In addition, N-acetyl-glucosamine in the matrix was found to be important for the initial development of the biofilm. These results suggest that B. bronchiseptica biofilm formation is growth phase dependent in addition to being regulated by the Bvg virulence system.     

Molecular aspects of Bordetella pertussis pathogenesis.

The molecular mechanisms of Bordetella virulence are now well understood, and many virulence factors have been identified and characterized at the molecular level. These virulence factors can be grouped into two major categories: adhesins, such as filamentous hemagglutinin, pertactin and fimbriae, and toxins, such as pertussis toxin, adenylate cyclase, dermonecrotic toxin and tracheal cytotoxin. The production of most virulence factors is coordinately regulated by a two-component signal transduction system composed of the regulator BvgA and the sensor protein BvgS. The adhesins and toxins act in concert to establish infection. Some adhesins exert their effects synergically or are redundant functioning only in the absence of another adhesin, illustrating the importance of adhesion in infection. Most virulence factors are secreted into the culture supernatant or exposed at the surface of the bacterial cell. A notable exception is dermonecrotic toxin, which remains in the cytoplasmic compartment of bacterial cells. Most virulence factors are produced by all of the three major Bordetella species, B. pertussis, B. parapertussis and B. bronchiseptica. However, some, such as pertussis toxin and the tracheal colonization factor, are only produced by B. pertussis. Our understanding of Bordetella virulence at the molecular level has led to the development of new acellular vaccines against whooping cough, and of genetically attenuated B. pertussis strains to be used as recombinant live bacterial vaccine vectors for homologous and heterologous protection.     

Alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is not repressed by the BvgAS virulence control system

A previous study found that alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is Bvg repressed. In contrast, we report that alcaligin production by RB50 does not require Bvg phenotypic phase modulation and that isogenic Bvg(Con) and Bvg(-) phase-locked mutants both produce alcaligin in response to iron starvation.