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. 2004 Apr;72(4):2035-44.
doi: 10.1128/IAI.72.4.2035-2044.2004.

Whole-genome DNA array analysis of the response of Borrelia burgdorferi to a bactericidal monoclonal antibody

Julie M Anderton  1 , Rafal Tokarz, Charles D Thill, Christopher J Kuhlow, Chad S Brooks, Darrin R Akins, Laura I Katona, Jorge L Benach
Affiliations

Whole-genome DNA array analysis of the response of Borrelia burgdorferi to a bactericidal monoclonal antibody

Julie M Anderton et al. Infect Immun. 2004 Apr.

Abstract

Identification and characterization of genes that contribute to infection with Borrelia burgdorferi and, of those, genes that are targets of host responses is important for understanding the pathogenesis of Lyme disease. The complement-independent bactericidal monoclonal antibody (MAb) CB2 recognizes a carboxy-terminal, hydrophilic epitope of the outer surface protein B (OspB). CB2 kills B. burgdorferi by an unknown bactericidal mechanism. Upon binding of CB2 to OspB, differentially expressed gene products may be responsible for, or associated with, the death of the organism. A time course of the response of B. burgdorferi to CB2 was completed to analyze the differential gene expression in the bacteria over a period of visual morphological changes. Bacteria were treated with a sublethal concentration in which spirochetes were visibly distressed by the antibody but not lysed. Preliminary whole-genome DNA arrays at various time points within 1 h of incubation of B. burgdorferi with the antibody showed that most significant changes occurred at 25 min. Circular plasmid 32 (cp32)-encoded genes were active in this period of time, including the blyA homologs, phage holin system genes. DNA array data show that three blyA homologs were upregulated significantly, >/==" BORDER="0">2 standard deviations from the mean of the log ratios, and a P value of </=0.01. Quantitative real-time PCR analysis verified blyA and blyB upregulation over an 18- to 35-min time course. The hypothesis to test is whether the killing mechanism of CB2 is through uncontrolled expression of the blyA and blyB phage holin system.

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Figures

FIG. 1.
FIG. 1.
Verification of 20 μg of CB2/ml as the optimal sublethal concentration. (A) Borrelia RNA concentration after HS or FT conditions. Error bars represent the mean of triplicate values ± the standard error. (B) Borrelia RNA concentration after treatment with various CB2 concentrations for 60 min. (C) 1% agarose gel electrophoresis of RNA extracts shown in panel B. Lane 1, RNA ladder; lanes 2 and 3, untreated; lanes 4 and 5, 2 μg of CB2/ml; lanes 6 and 7, 20 μg of CB2/ml; lanes 8 and 9, treatment with 200 μg of CB2/ml. Arrows indicate rRNA subunits. (D) Recovery of Borrelia after treatment with 2 (□), 20 (▴), or 200 (×) μg of CB2/ml or no treatment (♦).
FIG. 2.
FIG. 2.
Spot graph log ratio plot analysis of Borrelia gene expression from array raw data of untreated versus CB2-treated (20 μg/ml) condition at 25 min. Dashed horizontal lines represent 2 SD above or below the mean of the log ratio plot. Solid vertical lines separate chromosomal (left) from plasmid (right) genes. Highlighted spots (○) represent genes that are significant by both criteria (see also Table 2).
FIG. 3.
FIG. 3.
Real-time PCR analysis of blyA (A to C) and blyB (D to F) in two different time courses. A, B, D, and E are ratios relative to the 16S ribosomal subunit; C and F are ratios relative to flaB. Black bars (U) represent untreated samples; gray bars (C) represent samples treated with 20 μg of CB2/ml. Error bars represent the mean of three replicates ± the standard error.
FIG. 4.
FIG. 4.
Concordance of differential gene expression between DNA array and quantitative real time-PCR Analysis. Fold change values for negative controls and differentially expressed genes were generated by comparing untreated to CB2-treated samples. In all cases, 16S ribosomal subunit was used as the internal control for real-time PCR (RT-PCR). Correlation coefficient = 0.90; trend line was generated with linear regression analysis.
FIG. 5.
FIG. 5.
Representative SDS-PAGE gels of samples were collected at 5, 27, 35, or 60 min; the 35-min time point is shown. Silver stain (A) and anti-BlyA Western blot (B) of untreated samples and samples treated with 20 μg of CB2/ml. S, supernatant; P, pellet. Lane 1, MM294-pTG3, positive BlyA control; lanes 2 and 3, untreated Borrelia; lanes 4 and 5, CB2-treated Borrelia samples.
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