Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host

The Fur Homologue in Borrelia burgdorferi

Borrelia burgdorferi contains a gene that codes for a Fur homologue. The function of this Fur protein is unknown; however, spirochetes grown at 23 or 35°C expressed fur as determined by reverse transcriptase PCR. The fur gene (BB0647) was cloned and overexpressed as a His-Fur fusion protein in Escherichia coli. The fusion protein was purified by zinc-chelate chromatography, and the N-terminal His tag was removed to generate recombinant Fur for use in mobility shift studies. Fur bound DNA containing the E. coli Fur box sequence (GATAATGATAATCATTATC) or Bacillus subtilis Per box sequence (TTATAAT-ATTATAA) with an apparent K(d) of ∼20 nM. Fur also bound the upstream sequences of three Borrelia genes: BB0646 (gene encoding a hydrolase of the α/β-fold family), BB0647 (fur), and BB0690 (napA). Addition of metal ions was not required. Binding activity was greatly decreased by either exposure to oxidizing agents (H(2)O(2), t-butyl hydroperoxide, cumene hydroperoxide, or diamide) or by addition of Zn(2+). B. burgdorferi NapA is a homologue of Dps. Dps functions in E. coli to protect DNA against damage during periods of redox stress. Fur may function in B. burgdorferi as a repressor and regulate oxidative stress genes. Additional genes (10 chromosomal and 15 plasmid) that may be Fur regulated were identified by in silico analysis

Whole-Genome DNA Array Analysis of the Response of Borrelia burgdorferi to a Bactericidal Monoclonal Antibody

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, ≥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

<i>lon-2</i>, but not <i>lon-1</i>, complements the filamentous phenotype of an <i>E. coli lon</i> mutant.

<p>(A) Brightfield microscopy of <i>E.coli</i> cultures grown at 37°C for 3.5 hours with or without 0.2% arabinose. Cells were applied to microslides, heat fixed, and visualized by Gram stain. (B) Western blot showing arabinose-induced Lon-1 and Lon-2 expression after culture at 37°C for 3.5 hours.</p

Phylogenetic trees based on Lon amino acid sequences of spirochetes.

<p>(A) Phylogenetic tree of spirochetes derived from the amino acid sequences of Lons from reference strains of <i>B. burgdorferi</i> sensu lato and relapsing fever <i>Borrelia</i>, <i>Leptospira</i> and <i>Treponema</i> species with <i>E. coli</i> and <i>B. subtilis</i> as an external group. (B) Phylogenetic tree of spirochetes derived from the amino acid sequences of the amino-terminal NH₂-terminal domains from reference strains of <i>B. burgdorferi</i> sensu lato and relapsing fever <i>Borrelia</i>, <i>Leptospira</i> and <i>Treponema</i> species, with <i>E. coli</i> and <i>B. subtilis</i> as outgroups. The neighbour joining trees were constructed with MEGA3 software. Bootstrap confidence levels above 50% are indicated to left of each relevant cluster. Asterisks: * Lon-1; ** Lon-2. All of the sequences were obtained from Swiss-Prot Protein knowledgebase. Accession numbers are listed next to each species. Distance marker is shown at lower left for each panel.</p

Lon-1 does not degrade λ-CI-N reporter protein carrying a <i>Borrelia</i>-SsrA tag.

<p>(A) SsrA-encoded proteolysis tags are shown. Truncated <i>Borrelia</i> SsrA tag refers to the reporter protein carrying a partial SsrA tag (determined by mass spectroscopy). (B) <i>In vitro</i> proteolysis assay was performed as described and protein samples taken at indicated time points were resolved on 15% polyacrylamide Tris-tricine gel followed by Coomassie blue staining. Protein bands labeled as tagged and trunc. tagged refer to the λ-CI-N reporter protein carrying a full-length or truncated <i>Borrelia</i> SsrA tag, respectively. Creatine kinase, present in the reaction mixture, was used as a loading control. (C) Protein gels were quantitatively analyzed using the Odyssey infrared imaging system (LI-COR) and remaining reporter proteins were calculated and presented as a percentage (average of three independent experiments). Error bars represent standard deviations. (D) Proteolysis assay of insulin was performed in a manner similar to λ-CI-N reporter <i>in vitro</i> assays.</p

SDS-PAGE and western blot analysis of purified recombinant wild-type and mutant (S714A) <i>B. burgdorferi</i> Lon-1 protein.

<p>(A) Coomassie blue-stained 12.5% gel of recombinant Lon-1 wild-type (rLon-1) and S714A mutant (rLon-1^S714A). (B) Corresponding western blot using rabbit polyclonal antiserum.</p

<i>lon-2</i>, but not <i>lon-1</i>, complements an <i>E. coli lon</i> mutant.

<p>(A) β-galactosidase activity of wild-type HDB97 (<i>cpsB</i>::<i>lacZ</i>), <i>lon</i> mutant HDB98 (HDB97<i>lon</i>⁵¹⁰), and HDB98 transformed with empty pBAD24 vector (HDB98pBAD24) or vector containing <i>B. burgdorferi lon-1</i> (HDB98pBAD<i>lon-1</i>) or <i>lon-2</i> (HDB98pBAD<i>lon-2</i>). Figure represents the combined data from two independent experiments. HDB97 and HDB98 were cultured in LB medium and HDB98pBAD24, HDB98pBAD<i>lon-1</i>, and HDBpBAD<i>lon-2</i> were cultured in LB medium supplemented with 0.1% arabinose. ***, significantly different from HDB98, HDB98pBAD24, and HDB98pBAD<i>lon-1</i> (P<0.001, Tukey-Kramer Multiple Comparisons Test). Error bars represent standard deviations. (B) β-galactosidase activity of <i>E. coli</i> grown with (black bars) and without (white bars) 0.1% arabinose. Figure is representative of two independent experiments that produced similar results. Error bars in panels A and B represent the standard deviations of triplicate samples. (C, D) Western blot analysis of culture whole cell lysates from experiment depicted in panel A showing Lon-1 and Lon-2 expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (E) Solubility of Lon-1 and Lon-2 protein produced by HDB98pBADlon-1 and HBD98pBADlon-2, respectively. WCL, whole-cell lysate; S, soluble fraction (supernatant) after centrifugation for 10 minutes at 16,000× g. For each <i>E. coli</i> strain, western blots are shown on the left. Corresponding Coomassie Blue-stained protein is shown on the right to verify that equivalent amounts of protein were loaded.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Oligonucleotide primers used in this study.

<p>All PCR primers shown here were designed for this study.</p>a<p>Restriction sites are underlined.</p