Molecular dissection of a Borrelia burgdorferi in vivo essential purine transport system

Abstract

The Lyme disease spirochete Borrelia burgdorferi is dependent on purine salvage from the host environment for survival. The genes bbb22 and bbb23 encode purine permeases that are essential for B. burgdorferi mouse infectivity. We now demonstrate the unique contributions of each of these genes to purine transport and murine infection. The affinities of spirochetes carrying bbb22 alone for hypoxanthine and adenine were similar to those of spirochetes carrying both genes. Spirochetes carrying bbb22 alone were able to achieve wild-type levels of adenine saturation but not hypoxanthine saturation, suggesting that maximal hypoxanthine uptake requires the presence of bbb23. Moreover, the purine transport activity conferred by bbb22 was dependent on an additional distal transcriptional start site located within the bbb23 open reading frame. The initial rates of uptake of hypoxanthine and adenine by spirochetes carrying bbb23 alone were below the level of detection. However, these spirochetes demonstrated a measurable increase in hypoxanthine uptake over a 30-min time course. Our findings indicate that bbb22-dependent adenine transport is essential for B. burgdorferi survival in mice. The bbb23 gene was dispensable for B. burgdorferi mouse infectivity, yet its presence was required along with that of bbb22 for B. burgdorferi to achieve maximal spirochete loads in infected mouse tissues. These data demonstrate that both genes, bbb22 and bbb23, are critical for B. burgdorferi to achieve wild-type infection of mice and that the differences in the capabilities of the two transporters may reflect distinct purine salvage needs that the spirochete encounters throughout its natural infectious cycle.

FIG 1

Genes bbb22 and bbb23 each harbor their own promoters. Rapid amplification of cDNA ends (5′ RACE) analysis of the transcription start sites for the bbb23, bbb22prox, and bbb22dist transcripts was performed using RNA extracted from B31 clone A3 B. burgdorferi. (A) Transcript-specific 5′ anchored nested PCR products generated from the 5′ RACE analysis. Data shown are representative of two replicate experiments. The DNA ladder is shown in base pairs. (B) DNA sequence chromatographs of the DNA sequence of the 5′ RACE products shown in panel A. Both sequences detected for bbb22dist are shown. The 5′ nucleotide of each transcript is indicated with a star. (C) Schematic diagram of the genetic organization of genes bbb22 and bbb23. Transcription start sites identified in panel B are shown as bent arrows and labeled with the corresponding transcripts names. The B. burgdorferi IVET in vivo active promoter sequence within the BBB23 ORF is shown as a light gray box. The locations and numbers of the transcript-specific primers used to generate the cDNA for the 5′ RACE analysis are indicated. A ruler for the nucleotide coordinates on cp26 is shown.

FIG 2

In vitro gene expression of bbb22 and bbb23 in B. burgdorferi clones. RNA was extracted from bbb22-23⁺, 23_p-bbb23 ⁺, flaB_p-bbb23⁺, 22prox_p-bbb22⁺, and 22dist_p-bbb22⁺ B. burgdorferi clones at late stationary phase (2 × 10⁸ spirochetes/ml) grown at 37°C. Gene expression for bbb22, bbb23, and flaB was quantified by reverse transcriptase qPCR. (A) bbb22 expression was analyzed for clones bbb22-23⁺, 22prox_p-bbb22⁺, and 22dist_p-bbb22⁺. bbb22 mRNA transcripts were normalized to the number of flaB mRNA copies. (B) bbb23 expression was analyzed for clones bbb22-23⁺, 23_p-bbb23⁺, and flaB_p-bbb23⁺. bbb23 mRNA transcripts were normalized to the number of flaB mRNA copies. Data represent the average values of three biological replicates. Error bars represent the standard deviations from the means. The y axis is depicted in a log₁₀ scale. Data sets were compared by one-way ANOVA using GraphPad Prism, version 5.0. *, P < 0.05; **, P < 0.01; NS, not significant.

FIG 3

bbb22 and bbb23 alone provide spirochetes with distinct abilities to take up purines. Radioactive purine uptake by B. burgdorferi bbb22-23⁺, 23_p-bbb23⁺, flaB_p-bbb23⁺, 22prox_p-bbb22⁺, 22dist_p-bbb22⁺, Δbbb22-23, and bbb22-23⁺ heat-killed spirochetes was measured over a 30-min time course following the addition of 2.5 μM [³H]hypoxanthine (A) or 0.396 μM [2,8-³H]adenine (B).The specific activity of the labeled hypoxanthine used was 20 Ci/mmol. The specific activity of the labeled adenine used was 15 Ci/mmol. Error bars represent the standard deviation from the mean for at least two biological replicates.

FIG 4

Adenine and guanine compete with hypoxanthine for transport by both BBB23 and BBB22 individually. [³H]hypoxanthine (0.25 μM, 20μCi) uptake by 22dist_p-bbb22⁺ and flaB_p-bbb23⁺ spirochetes was determined at 15 min following the addition of radiolabeled purine in the absence (no competitor) or presence of 20-fold excess (5 μM) unlabeled competitor as indicated. [³H]hypoxanthine uptake by 22dist_p-bbb22⁺ or flaB_p-bbb23⁺ spirochetes in the absence of competitor was taken as 100%. All other data are represented as the percent uptake relative to that of 22dist_p-bbb22⁺ or flaB_p-bbb23⁺ spirochetes, respectively, in the absence of competitor. Heat-killed spirochetes served as the negative controls. Data sets were compared by an unpaired t test using GraphPad Prism, version 5.0, and relevant P values are shown.

FIG 5

The bbb22 gene alone is not sufficient to maintain wild-type levels of spirochete loads in infected mouse tissues. DNA was isolated from ear, heart, and joint tissues of C3H/HeN mice inoculated with 1 × 10⁴ bbb22-23⁺ or 22dist_p-bbb22⁺ spirochetes. Samples were analyzed for B. burgdorferi (Bb) flaB and murine nidogen DNA copies by qPCR. Each data point represents the average of triplicate measures from the DNA of an individual mouse. The mean value for each group is indicated by a horizontal line. Data sets were compared by one-way ANOVA using GraphPad Prism, version 5.0. *, P < 0.05; NS, not significant.