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The gram-negative respiratory pathogen Legionella pneumophila infects and grows within mammalian macrophages and protozoan host cells. Upon uptake into macrophages, L. pneumophila establishes a replicative organelle that avoids fusion with endocytic vesicles. There are 24 dot/icm genes on the L. pneumophila chromosome required for biogenesis of this vacuole. Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been investigating the function of L. pneumophila dot/icm gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis. The icmX gene product falls into this category. To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an L. pneumophila icmX deletion mutant and murine bone marrow-derived macrophages. These data demonstrate that icmX is required for biogenesis of the L. pneumophila replicative organelle. Immunoblot analysis indicates that the icmX gene product is a polypeptide with an estimated molecular mass of 50 kDa. The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal sec-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-type L. pneumophila growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other Legionella species by immunoblot analysis using a monoclonal antibody specific for L. pneumophila IcmX protein. From these data, we conclude that the IcmX protein is an essential component of the dot/icm secretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the Legionellaceae family.

The virulence regulon of Bordetella pertussis includes a trans-acting regulatory locus, bvg, that is required for expression of several virulence factors. The virulence control system also responds to environmental signals. We have reconstructed a bvg-dependent regulatory system in Escherichia coli by using bacteriophage lambda vectors carrying transcriptional fusions to lacZYA. Single-copy lacZYA fusions to the B. pertussis fhaB locus, which encodes the attachment factor filamentous hemagglutinin, were activated nearly 400-fold by pBR322 replicons carrying sequences that included bvg. In contrast, bvg had no effect on the pertussis toxin operon (ptxA-E) promoter in E. coli as measured by ptxA-lacZ expression. Environmental signals that modulate expression of virulence genes in B. pertussis had a pronounced effect on bvg-mediated activation of fhaB-lacZ. MgSO4, nicotinic acid, and low temperature resulted in decreases in beta-galactosidase activities of 175-, 115-, and 45-fold respectively. Sensory transduction and transcriptional activation were tightly coupled, and both required an intact bvg locus as determined by 5' and 3' deletions that eliminated both activities.

The bvg region of the respiratory pathogen Bordetella pertussis coordinately regulates the expression of several unlinked virulence determinants in response to environmental signals. The DNA sequence of the bvg region contains three genes (bvgA, bvgB, and bvgC). Transcription of a single-copy fusion consisting of the upstream region of a bvg-activated B. pertussis gene (fhaB) attached to the promoterless lac operon in Escherichia coli requires the entire bvgABC region in trans. Activation of the fhaB::lacZYA fusion is sensitive to the same environmental stimuli in E. coli that modulate the expression of bvg-activated genes in B. pertussis. Our data show that overexpression of the bvgA gene from a strong heterologous promoter results in transcriptional activation of the fhaB::lacZYA fusion even in the absence of the bvgB and bvgC products. Activation of fhaB transcription by bvgA overexpression in E. coli is no longer repressed by environmental conditions. The bvgA product has been identified by maxicell analysis as a 23-kilodalton protein. A B. pertussis mutant containing an in-frame deletion in bvgA was constructed. This mutant was nonhemolytic and no longer produced filamentous hemagglutinin and pertussis toxin. The mutation in this strain was complemented by returning the bvgA gene in trans. Transcriptional chloramphenicol acetyltransferase fusions to the fhaB and ptx promoter regions were returned to both the B. pertussis bvgA deletion mutant and its parental wild-type strain. Analysis of these strains indicated that the deletion mutant was defective in transcription of both ptx and fhaB. We conclude from these data that bvgA, bvgB, and bvgC comprise an operon encoding the components essential for coordinate regulation and sensory transduction. The BvgA protein is a transcriptional regulatory factor. The bvgB and bvgC products may be important in regulating the activity of BvgA in response to the changing environmental stimuli that B. pertussis encounters during the diseases whooping cough.