Apparent role for Borrelia burgdorferi LuxS during mammalian infection

Abstract

The Lyme disease spirochete, Borrelia burgdorferi, controls protein expression patterns during its tick-mammal infection cycle. Earlier studies demonstrated that B. burgdorferi synthesizes 4,5-dihydroxy-2,3-pentanedione (autoinducer-2 [AI-2]) and responds to AI-2 by measurably changing production of several infection-associated proteins. luxS mutants, which are unable to produce AI-2, exhibit altered production of several proteins. B. burgdorferi cannot utilize the other product of LuxS, homocysteine, indicating that phenotypes of luxS mutants are not due to the absence of that molecule. Although a previous study found that a luxS mutant was capable of infecting mice, a critical caveat to those results is that bacterial loads were not quantified. To more precisely determine whether LuxS serves a role in mammalian infection, mice were simultaneously inoculated with congenic wild-type and luxS strains, and bacterial numbers were assessed using quantitative PCR. The wild-type bacteria substantially outcompeted the mutants, suggesting that LuxS performs a significant function during mammalian infection. These data also provide further evidence that nonquantitative infection studies do not necessarily provide conclusive results and that regulatory factors may not make all-or-none, black-or-white contributions to infectivity.

FIG 1

Activated methyl pathway of B. burgdorferi. MetK synthesizes S-adenosylmethionine (SAM) from ATP and methionine. SAM then acts as a methyl donor for many metabolic steps, also producing the by-product S-adenosylhomocysteine (SAH). SAH is toxic, so Pfs cleaves that molecule to produce adenine and S-ribosylhomocysteine (SRH). SRH is nontoxic, and some bacterial species, such as the spirochete Treponema pallidum, end the pathway at this step. B. burgdorferi instead uses LuxS to cleave SRH into homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD). Biochemical and genetic analyses demonstrated that B. burgdorferi lacks the ability to further metabolize homocysteine (7, 10). DPD, also known as autoinducer-2 (AI-2), is secreted by B. burgdorferi into the environment (7).

FIG 2

Specificity of PCR oligonucleotide primer pairs to detect wild-type (297) and luxS mutant (AH309) B. burgdorferi strains. (A) Schematic representation of locations of sequences complementary to PCR primers. Primers ermC-F and ermC-R both correspond to sequences within the ermC gene that is inserted into luxS of AH309 (22). Oligonucleotide luxS-R overlaps the ermC insertion site of the AH309 locus and thus cannot serve to prime PCR for that strain. (B) Purified genomic DNAs from strains 297 and AH309 were subjected to PCRs using each primer pair and then subjected to agarose gel electrophoresis and ethidium bromide staining. Lane 1, 297 with primers luxS-F and luxS-R; lane 2, 297 with primers ermC-F and ermC-R; lane S, molecular size markers; lane 3, AH309 with primers luxS-F and luxS-R; lane 4, AH309 with primers ermC-F and ermC-R. Sizes of markers are indicated to the right of the gel.

FIG 3

qPCR analyses of tissues from mice that were singly infected with either strain 297 or AH309. For each tissue, chromosomal loci of B. burgdorferi and mice (flaB and nidogen, respectively) were quantified, and data were plotted as numbers of B. burgdorferi genome equivalents per mouse genome equivalent. Differences in bacterial loads of heart tissues were equivocal (P = 0.049), while there were no significant differences in bacterial loads of ear tissues (P > 0.05).

FIG 4

qPCR analyses of tissues from mice that were simultaneously injected with 10⁴ bacteria (each) of both strains 297 and AH309. Total DNAs were extracted from the hearts, urinary bladders, and ear pinnae of eight mice. Ratios of wild-type (strain 297) to luxS mutant (strain AH309) bacteria were calculated from the ΔC_T values for each strain in each tissue. Competition indexes were calculated for every mouse tissue.