Cloning and sequencing of a Bordetella pertussis serum resistance locus

Abstract

We have characterized a new virulence factor in Bordetella pertussis: serum resistance. Compared with Escherichia coli HB101, wild-type B. pertussis was relatively resistant to classical-pathway, complement-dependent killing by normal human serum. However, a mutant of B. pertussis (BPM2041) which is less virulent in mice and which has Tn5 lac inserted in a previously uncharacterized bvg-regulated gene was found to be at least 10-fold more susceptible to serum killing than the wild type. We have named this locus brk, for Bordetella resistance to killing. We have cloned and sequenced the brk locus, and it encodes two divergently transcribed open reading frames (ORFs), termed BrkA and BrkB. Both ORFs are necessary for serum resistance. Within the 300 bases which separate the two ORFs and upstream of each ORF are putative sites for BvgA binding. BrkA shows 29% identity to pertactin and has two RGD motifs in addition to a conserved proteolytic processing site and an outer membrane targeting signal. Like pertactin, BrkA is involved in adherence and invasion. Despite the similarities, a pertactin mutant was found to be not as sensitive to serum killing as the BrkA or BrkB mutants. BrkB is similar to ORFs in E. coli and Mycobacterium leprae and displays domains of homology to various transporters. On the basis of its hydropathy profile, BrkB is predicted to be a cytoplasmic membrane protein. By Southern blot, brk sequences were found in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium.

Serum resistance in bvg-regulated mutants of Bordetella pertussis

“…In a previous report, we showed that the capacity of B. pertussis to survive killing by the classical pathway of complement is regulated by the bvgAS locus with the products of the brk locus (BrkA and BrkB) contributing to a substantial amount of the resistance [2]. In this report, we examine the role of other bvg-regulated virulence factors in mediating serum resistance.…”

“…All strains and plasmids are listed in Table 1. All plasmids were mobilized from Escherichia coli into B. pertussis using the suicide vector system described previously [2]. To facilitate this process, pUW2138, containing a Gent r -oriT cassette that was surrounded by the BamHI to SalI multiple cloning sites of pBluescript, was constructed.…”

“…To facilitate this process, pUW2138, containing a Gent r -oriT cassette that was surrounded by the BamHI to SalI multiple cloning sites of pBluescript, was constructed. The Gent r -oriT cassette from UW2032 [2] was excised as a SalI fragment and ligated into the SalI site of pBluescript SK(+), and a plasmid was selected that had the duplicated restriction sites on both sides of the Gent r -oriT cassette.…”

“…Adenylate cyclase toxin and pertussis toxin inhibit PMNs, monocytes, and NK cells. The BrkA protein protects from lysis by the classical (antibodydependent) pathway of complement [2].…”

The Bordetella Bps Polysaccharide Is Required for Biofilm Formation and Enhances Survival in the Lower Respiratory Tract of Swine

“…As previously mentioned, KM22 harbors a brkA gene that is predicted to be expressed as a full-length functional protein, whereas the brkA gene in RB50 is annotated as a pseudogene (62). For B. pertussis, BrkA has been shown to confer resistance to antibody-mediated complement lysis (63,64). Similar to RB50, KM22 harbors the genes encoding lipopolysaccharide (LPS) and an O-antigen (62).…”