Borrelia burgdorferi bba66 gene inactivation results in attenuated mouse infection by tick transmission

Abstract

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb(ΔA66), remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb(ΔA66) following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb(ΔA66)-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb(ΔA66) with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.

Fig 1

bba66 expression in feeding nymphal ticks by qRT-PCR. (A) RNA was isolated from pooled ticks from independent feedings (denoted by the open circles, filled circles, and triangles) at the designated times postattachment. Absolute bba66 transcript copies were measured as a ratio of copies per flaB. Horizontal bars represent the means of the samples. (B) Agarose gel image of bba66 amplicons following qRT-PCR. Lanes: 1, unfed nymphs; 2 to 5, nymphs at 33, 48, 57, and 72 to 80 h postinfestation, respectively; 6, replete nymphs; 7, no template DNA control; 8, Bb^WT genomic DNA positive control.

Fig 2

IFA of tick midgut approximately 72 h postattachment. (A) Field stained with anti-B. burgdorferi; (B) same field as in panel A, stained with anti-BBA66; (C) merged image of panels A and B. Arrows point to BBA66-stained organisms.

Fig 3

Insertional inactivation of bba66 in infectious B. burgdorferi, complementation, and confirmation in vitro. (A) Schematic depicting the mutagenesis of Bb^WT bba66 by insertion of the pflg-kan cassette on lp54 by homologous recombination with pBADbba66::Kan. N and B indicate NheI and BglII restriction endonuclease sites utilized for insertion of the resistance marker. (B) Plasmid construct pBSV2G-bba66comp, used to complement the loss of bba66 in Bb^ΔA66. The intact bba66 gene with its promoter (bba66 + prom) provides trans-complementation under gentamicin selection (Gent^R). Open reading frame 1 (orf 1), orf 2, orf 3, and origin of replication (ori) of pBSV2 are depicted. Numbers with arrowheads in panels A and B indicate primers (defined in panel B) and direction of amplification. (C) Agarose gel of PCR amplicons using the 3 primer sets indicated to verify kanamycin resistance marker insertion in Bb^ΔA66. Reactions a, b, and c on the right depict control PCR with plasmid templates to verify correct amplicon sizes. Markers are indicated to the left in kilobases (kb). (D) SDS-PAGE separation and silver staining of equal amounts of cell protein lysates from Bb^WT, Bb^ΔA66, and Bb^ΔA66comp grown at pH 7 or pH 8. BBA66 is not visible by silver staining, but OspA and OspC are indicated by arrows for reference. Below the stained gel is an immunoblot of these cell lysates with anti-BBA66 demonstrating the loss of BBA66 in Bb^ΔA66 and the pH regulation of bba66 in Bb^WT and Bb^ΔA66comp. The immunoblot with anti-OppA1, which is not pH regulated in vitro, served as a loading control.

Fig 4

Phenotypic analyses of C3H/HeJ mice needle inoculated with Bb^WT, Bb^ΔA66, and Bb^ΔA66comp. (A) Change in hind-limb joint diameter in millimeters (mm) was monitored 18, 21, 25, 28, 32, and 35 days postinfection. Reported changes are relative to age-matched, mock-challenged mice. One-way ANOVA with Dunnett's posttest was performed: *, P = 0.01 to 0.05; **, P = 0.001 to 0.01; ***, P = <0.001 relative to Bb^WT samples. (B) Joint tissues from mice needle inoculated with Bb^WT, Bb^ΔA66, or Bb^ΔA66comp were analyzed by qPCR to determine the relative differences in spirochete burden during infection. Borreliae were quantified by copies of ospC/10⁶ copies of the mouse β-actin gene. *, P = 0.01 to 0.05. (C) ELISA results demonstrating immunoreactivity of infected mice to recombinant BBA66. Endpoint titers are presented as the reciprocal in log₂. A two-way t test was performed to compare Bb^WT and Bb^ΔA66comp at either 21 dpi or 35 dpi; ***, P = <0.001. Data points in panels B and C denote individual mice, with the mean denoted by a horizontal line and vertical lines with bars indicating the standard error of the mean.

Fig 5

Flow chart of the experimental design to assess borrelial phenotypes through the mouse-tick-mouse natural cycle of infection.

Fig 6

qPCR enumerating Bb^WT and Bb^ΔA66 isolates in individual ticks at times during mouse feeding. Bars indicate copies of flaB in unfed (flat) nymphs, in nymphs collected at 48 and 72 h postattachment, and in replete nymphs collected between 4 and 20 h after dropoff.

Fig 7

Number of Bb^ΔA66-infected ticks feeding to repletion as a correlation to mouse infectivity. Solid circles represent individual mice that became infected following feeding by Bb^ΔA66-infected ticks; solid squares represent individual mice that did not become infected following feeding by Bb^ΔA66-infected ticks. The mean value for each sample set is shown by the horizontal lines with the standard error of the mean denoted by the vertical lines. P value (0.017) determined by one-way ANOVA.