The Bvg accessory factor (Baf) enhances pertussis toxin expression in Escherichia coli and is essential for Bordetella pertussis viability

Pertussis toxin expression in the Gram-negative respiratory pathogen, Bordetella pertussis, is regulated by the BvgAS two-component system. Previous studies suggested that an additional gene encoding a Bvg accessory factor (Baf) was required, along with BvgAS, for expression of a ptx-lacZ fusion in Escherichia coli grown in rich medium. However, other studies showed that BvgAS is sufficient for ptx-lacZ expression in minimal medium. Here we show that Baf acts with BvgAS to further increase ptx-lacZ expression in E. coli grown in minimal media and this is concomitant with a two-fold increase in BvgA protein levels. Gene replacement experiments show that baf is essential for viability of B. pertussis, suggesting that Baf affects the expression of other genes in addition to ptx.     

Osmolarity affects Bvg-mediated virulence regulation by Bordetella pertussis

Bordetella pertussis dramatically alters its phenotype by sensing its environment via the BvgAS regulatory system. Increased concentrations of specific chemicals are used in vitro to induce modulation of the bacterium from the Bvg(+) virulent phenotype to a fully Bvg(-) phenotype. Varied expression of sets of Bvg(-)regulated molecules depends on the modulating capacity of the environment. We examined the effect of a number of chemicals on the modulating capacity of B. pertussis growth media, both alone and in combination with known modulators. It was demonstrated that under certain conditions the Bvg(-)intermediate protein, BipA, is coexpressed with the Bvg(-) antigen, VraA. This demonstrates that the patterns of molecules expressed in the different phenotypes of B. pertussis are more fluid than has previously been demonstrated. The in vitro modulator, sulfate, was found to be a relatively inefficient modulator of our Tohama I-derived B. pertussis strain. However, addition of nicotinic acid, MgCl2, or sucrose in combination with relatively low sulfate concentrations resulted in effective modulation. This suggests that multiple signals may affect modulation through the BvgAS system or possibly through other regulatory networks. In addition, the cooperative modulating effect of sucrose implicates osmolarity as an environmental stimulus that affects phenotypic modulation.     

Differential Bvg phase-dependent regulation and combinatorial role in pathogenesis of two Bordetella paralogs, BipA and BcfA

To successfully colonize their mammalian hosts, many bacteria produce multiple virulence factors that play essential roles in disease processes and pathogenesis. Some of these molecules are adhesins that allow efficient attachment to host cells, a prerequisite for successful host colonization. Bordetella spp. express a number of proteins which either play a direct role in attachment to the respiratory epithelia or exhibit similarity to known bacterial adhesins. One such recently identified protein is BipA. Despite the similarity of BipA to intimins and invasins, deletion of this protein from B. bronchiseptica did not result in any significant defect in respiratory tract colonization. In this study, we identified an open reading frame in B. bronchiseptica, designated bcfA (encoding BcfA [bordetella colonization factor A]), that is similar to bipA. In contrast to the maximal expression of bipA in the Bvg intermediate (Bvg(i)) phase, bcfA is expressed at high levels in both the Bvg(+) and Bvg(i) phases. We show here that BvgA and phosphorylated BvgA bind differentially to the bcfA promoter region. Utilizing immunoblot assays, we found that BcfA is localized to the outer membrane and that it is expressed during animal infection. While deletion of either bipA or bcfA did not significantly affect respiratory tract colonization, concomitant deletion of both genes resulted in a defect in colonization of the rat trachea. Our results indicate that the two paralogous proteins have a combinatorial role in mediating efficient respiratory tract colonization.     

A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens

The Bordetella BvgAS signal-transduction system has traditionally been viewed as mediating a transition between two distinct phenotypic phases: the Bvg⁺ phase, characterized by the expression of adhesins and toxins, and the Bvg⁻ phase, characterized by motility in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis . In B. bronchiseptica , the Bvg⁺ phase is necessary and sufficient for respiratory tract colonization whereas the Bvg⁻ phase is required for growth under nutrient-limiting conditions. This report describes the characterization of a mutant that is locked in a Bvg-intermediate (Bvgⁱ) phase. The mutation conferring this phenotype, designated bvgS -I1, results in a threonine-to-methionine substitution near the primary site of phosphorylation in BvgS. Compared to its Bvg⁺-phase-locked parent, the Bvgⁱ mutant displays increased resistance to nutrient limitation and reduced virulence. Molecular analyses indicate that the mutant has lost the ability to express a subset of Bvg⁺-phase factors and has gained the ability to express factors unique to the Bvgⁱ phase. Although identified by mutation, this work indicates that the Bvgⁱ phase is expressed by wild-type B. bronchiseptica in response to certain (semi-modulating) environmental conditions. The identification of Bvgⁱ-specific antigens suggests the existence of a new class of Bvg-regulated genes. We hypothesize that BvgAS is capable of mediating the expression of a spectrum of phenotypic phases in response to the various environments encountered as Bordetella travels within and between mammalian hosts.     

Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins.

Mannitol-specific enzyme II, or mannitol permease, of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system of Escherichia coli carries out the transport and phosphorylation of D-mannitol and is most active as a dimer in the membrane. We recently reported the importance of a glutamate residue at position 257 in the binding and transport of mannitol by this protein (C. Saraceni-Richards and G. R. Jacobson, J. Bacteriol. 179:1135-1142, 1997). Replacing Glu-257 with alanine (E257A) or glutamine (E257Q) eliminated detectable mannitol binding and transport by the permease. In contrast, an E257D mutant protein was able to bind and phosphorylate mannitol in a manner similar to that of the wild-type protein but was severely defective in mannitol uptake. In this study, we have coexpressed proteins containing mutations at position 257 with other inactive permeases containing mutations in each of the three domains of this protein. Activities of any active heterodimers resulting from this coexpression were measured. The results show that various inactive mutant permease proteins can complement proteins containing mutations at position 257. In addition, we show that both Glu at position 257 and His at position 195, both of which are in the membrane-bound C domain of the protein, must be on the same subunit of a permease dimer in order for efficient mannitol phosphorylation and uptake to occur. The results also suggest that mannitol bound to the opposite subunit within a permease heterodimer can be phosphorylated by the subunit containing the E257A mutation (which cannot bind mannitol) and support a model in which there are separate binding sites on each subunit within a permease dimer. Finally, we provide evidence from these studies that high-affinity mannitol binding is necessary for efficient transport by mannitol permease.