Borrelia burgdorferi lacking DbpBA exhibits an early survival defect during experimental infection

Abstract

Several Borrelia burgdorferi genes induced under mammalian host conditions have been purported to be important in Lyme disease pathogenesis based on their binding to host structures. These genes include the dbpBA locus, whose products bind host decorin and glycosoaminoglycans. Recently, the dbpBA genes were reported to be involved in borrelial infectivity. Here we extended the previous observations by using culture and quantitative PCR to evaluate low- and high-dose murine infection by a Delta dbpBA::Gent(r) derivative of B. burgdorferi strain B31. The results indicate that the Delta dbpBA::Gent(r) mutant is attenuated in the ability to initially colonize and then persist in multiple tissues. The mutant exhibited a colonization defect as early as 3 days postinfection, before the development of an adaptive immune response, and after low-dose infection of SCID mice, which are deficient in adaptive immunity. These findings suggest that the inability to adhere to host decorin may promote clearance of B. burgdorferi, presumably via innate immune mechanisms. In a high-dose infection, the mutant disseminated to several tissues, particularly joint tissue, but it was generally cleared from these tissues by 3 weeks postinfection. Finally, following high-dose infection of SCID mice, the dbpBA mutant exhibited only a mild colonization defect, suggesting that the adaptive response is involved in the clearance of the mutant in immunocompetent mice. Taken together, these results suggest that the DbpBA proteins facilitate the colonization of multiple tissues by B. burgdorferi and are required for optimal resistance to both innate and adaptive immune mechanisms following needle inoculation.

FIG. 1.

Isolation and confirmation of dbpBA deletion mutants of infectious B. burgdorferi. (A) Strategy for deleting dbpBA. Plasmid pNP3, in which the BBA24 (dbpA) and BBA25 (dbpB) genes from lp54 were replaced by the gentamicin resistance gene aacC1, was used to transform B. burgdorferi strain B31 isolate ML23. Transformants were screened by PCR using primers shown to confirm the dbpBA deletion. Arrows 1 through 4 represent oligonucleotide primers 5ABMut, 3ABMut, 3pflgGentSal, and 5pflgGentSal, respectively (Table 2). One isolate that retained all plasmids (except cp9 and lp25) was obtained and designated JF105. (B) PCR analysis of the dbpBA deletion mutant and complemented strain. Lane 1, ML23/pBBE22 (parental strain); lane 2, JF105/pBBE22 (ΔdbpBA::Gent^r); lane 3, JF105/pJBF17 (ΔdbpBA::Gent^r with intact dbpBA). The primers used are indicated on the right (see panel A). (C) Western blot analysis of the ΔdbpBA::Gent^r mutant and the genetically complemented strain. Samples were immunoblotted using antiserum specific for DbpA. Lane 1, ML23/pBBE22 (parental strain); lane 2, JF105/pBBE22 (ΔdbpBA::Gent^r); lanes 3 and 4, JF105/pJBF17 (ΔdbpBA::Gent^r with intact dbpBA). Lanes 1 and 2 contained protein from 1 × 10⁷ borrelial cells, while lanes 3 and 4 contained whole-cell equivalents from 1.25 × 10⁶ and 2.5 × 10⁶ organisms, respectively. The arrow indicates the location of DbpA.

FIG. 2.

Quantitative real-time PCR analysis of the B. burgdorferi parental (ML23/pBBE22), ΔdbpBA::Gent^r (JF105/pBBE22), and genetically complemented (JF105/pJBF17) strains in joint tissue following 21 days of infection in C3H mice. The absolute numbers of B. burgdorferi cells obtained from joint tissue were determined using mice infected with all three strains at a dose of 10³ organisms. To normalize for mouse tissue in each sample tested, the number of β-actin copies was also determined. The results are expressed as the number of B. burgdorferi genome copies per 10⁶ mouse β-actin copies. P values are indicated above the data.

FIG. 3.

Quantitative PCR to determine the absolute numbers of spirochetes in joint tissue (A) and skin tissue (B) after 21 days of infection of C3H-SCID mice by the B. burgdorferi parental, ΔdbpBA::Gent^r, and genetically complemented strains at the inocula indicated. The results are expressed as the number of B. burgdorferi genome copies per 10⁶ mouse β-actin copies. P values are indicated above the data sets that are being compared.

FIG. 4.

Kinetics of B. burgdorferi dissemination in C3H mice. C3H mice were infected with the B. burgdorferi parental, ΔdbpBA::Gent^r, and genetically complemented strains using 10³ spirochetes, and the ΔdbpBA::Gent^r and genetically complemented strains were also infected using 10⁵ organisms. Mice were sacrificed on days 3, 5, 7, 14, and 21, and the tissues were cultured to determine B. burgdorferi growth. The strains and doses tested are indicated below the x axis. The percentages of culture-positive samples are indicated on the y axis. The results are expressed as percentages of culture-positive samples for the strains and concentrations of spirochetes tested. The values represent data for three to six mice depending on the strain and time point evaluated. An asterisk indicates that all samples tested were culture negative. LN, lymph nodes.