Characterization of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidases from Borrelia burgdorferi: Antibiotic targets for Lyme disease

Abstract

Background.-Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome.Methods.-Here we examine three unique 5'-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay.Results.-The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5'-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5'-alkylthio substitution increased. Non-hydrolysable nucleoside

A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

“…The BacTiter Glo microbial cell viability assay provides a method for determining the number of viable microbial cells in culture based on quantitation of the ATP present by measuring luminescence. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells in culture [21][22][23][24][25]. To test the borreliacidal activity of human PGLYRP1-His 8 (0-66.7 ng/μl), we incubated the protein with 1x10 5 spirochetes in microaerophilic conditions at 33˚C for 48 h in a final volume of 300 μL, and calculated the percent viable spirochetes using BacTiter-Glo (Promega) as described previously [21][22][23][24][25].…”

Evaluation of Nucleoside Analogs as Antimicrobials Targeting Unique Enzymes in Borrelia burgdorferi

“…Recombinant MTANs were expressed and purified as previously described [18]. Briefly, overnight 10 mL cultures of recombinant E. coli BL21(DE3) pLysS cells containing engineered pET30a expression plasmids for Bgp, MtnN or Pfs were grown at 37 • C with shaking (150 rpm) in LB broth supplemented with 50 µg/mL kanamycin and 0.5% glucose.…”

“…Eluted materials were analyzed by SDS-PAGE and Coomassie blue staining and found to be >95% homogeneous. Enzyme concentrations were determined using UV scans on a Cary100 spectrophotometer (Agilent) to determine the absorbance at 280 nm and applying extinction coefficients (0.1%) of 0.594 (Bgp), 0.637 (MtnN), and 0.404 (Pfs) [18]. Enzyme stocks (1 mg/mL) were prepared in 20% glycerol and stored at −80 • C.…”

“…Enzyme specific activities were determined using previously reported optimal pH conditions (50 mM sodium phosphate, pH = 7, for Bgp, MtnN; 50 mM sodium phosphate-citrate, pH = 5, for Pfs) and a spectrophotometric activity assay that measures the decrease in UV absorbance at 275 nm that occurs when MTA is cleaved to 5-methylthioribose and adenine (ε 275 = 1.6 mM −1 cm −1 ) [18,46]. The extinction coefficients for cleavage of other nucleosides were experimentally validated and found to be the same for MSeA, FSBA, and IADO (ε 275 = 1.6 mM −1 cm −1 ).…”

“…Bacterial 5 -methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) has four known substrates: (i) S-adenosylhomocysteine (SAH), which is cleaved to produce S-ribosylhomocysteine and adenine, (ii) 5 -methylthioadenosine (MTA) that is hydrolyzed to 5-methylthioribose (MTR) and adenine, (iii) 5 -deoxyadenosine (5 dADO) that is degraded to 5-deoxyribose and adenine, and (iv) 6-amino-6-deoxy-futalosine, which is produced in some organisms expressing the menaquinone pathway [11][12][13][14][15][16][17][18]. Although detoxification of these growth inhibitory nucleosides is a major role of MTAN in bacteria, its contribution is also important in autoinducer-1 and autoinducer-2 mediated quorum sensing mechanisms that trigger the expression of virulence factors during infection in numerous Gram-negative bacterial pathogens [19].…”