Role of Vomp Adhesins in Bartonella quintana Pathogenesis
Joanna MacKichan, UC-San Francisco
Biomedical and Clinical Sciences
2005
Background: Bartonella quintana (BQ) is a louse-borne pathogen that can persist for months in the human bloodstream. BQ causes opportunisticinfections in HIV-infectedpatients, producing relapsing fever (trench fever),endocarditis, bacillary angiomatosis, and chronic bacteremia. Relapsing and/or chronic bloodstream infection occurs in patients at all stages of HIV infection and can last for months to more than a year, causing debilitating and even fatal sequelae. When BQ gains access to the human bloodstream, some bacteria remain free and some invade and reside within red blood cells. An additional persistence strategy is suggested by the relapsing pattern of fever observed during BQ infection: phase variation of antigenic surface proteins, enabling escape from the host immune system. Recently, our lab demonstrated that a novel family of BQ outer membrane protein (OMP) adhesins, designated Vomp (Variably-expressed OMP), undergoes phase variation. The four Vomp, homologous to the adhesin YadA from Yersinia, were expressed in the strain (a clinical isolate, JK-31) used to inoculate our animal model, but were no longer expressed in isolates from the bloodstream 70 days post-inoculation. Preliminary data suggest that the Vomp are major virulence factors, with collagen binding and autoaggregation functions.
Methods: Using a negative selection method, we constructed a vomp null mutant in BQ. A mutagenic plasmid, containing sequences flanking the vomp locus, was conjugated into JK-31, and colonies containing the integrated plasmid were selected. A subsequent selection, for strains that had lost the integrated plasmid, was performed. Approximately one third of the strains had lost the vomp locus along with the plasmid. The vomp null mutant was verified by colony PCR, Western, and Southern blot. The mutant is currently being complemented, both with each of the genes individually (with their native promoters), and with the entire vomp locus. In each case, the complementing gene or region is being amplified by PCR and cloned into the replicating plasmid pANT3. An autoaggregation assay, to determine the rate at which BQ falls out of suspension, was done for the mutant as well. The vomp null mutant was then tested for virulence in an animal model in which BQ wild type inoculation results in bloodstream infection 100% of the time.
Results: The vomp null mutant was confirmed by PCR, Western blot, and Southern blotting techniques. Complementation of the vomp null mutant with each of the individual vomp genes, or with the entire vomp locus, is currently ongoing. The vomp null mutant has been found to be defective for autoaggregation, suggesting that the Vomp function as autoadhesins. In addition, while every strain of wild type BQ that has been used to infect an animal model can be recovered from the bloodstream, the mutant could not be recovered. This confirms that the Vomp are important virulence factors, and are essential for infection.
Conclusion: This is the first time that targeted mutagenesis has been achieved in BQ. The lack of genetic tools has hindered the study of this emerging opportunistic pathogen. The vomp mutant has been found to be deficient for autoagglutination, a virulence phenotype, as well as for infection in an animal model. This suggests that the Vomp are important virulence factors for BQ. The vomp null mutant and complemented strains will be used to further address the role of the individual Vomp in BQ pathogenesis.
