Pavel Dibrov,a Elena Dibrov,bc Thane G. Maddaford,bc Melissa Kenneth,b Jordan Nelson,b Craig Resch,a Grant N. Piercebc

aDepartment of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.

bInstitute of Cardiovascular Sciences, Albrechtsen Research Centre, St. Boniface Hospital, Winnipeg, MB R2H 2A6, Canada.

cDepartment of Physiology and Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.

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Received September 9, 2016. Accepted October 20, 2016.

Canadian Journal of Physiology and Pharmacology, 2017, 95(5): 595-603, https://doi.org/10.1139/cjpp-2016-0505

The search for new nontraditional targets is a high priority in antibiotic design today. Bacterial membrane energetics based on sodium ion circulation offers potential alternative targets. The present work identifies the Na + -translocating NADH:ubiquinone oxidoreductase (Na + -NQR), a key respiratory enzyme in many microbial pathogens, as indispensible for the Chlamydia trachomatis infectious process. Infection by Chlamydia trachomatis significantly increased first H + and then Na + levels within the host mammalian cell. A newly designed furanone Na + -NQR inhibitor, PEG-2S, blocked the changes in both H + and Na + levels induced by Chlamydia trachomatis infection. It also inhibited intracellular proliferation of Chlamydia trachomatis with a half-minimal inhibitory concentration in the submicromolar range but did not affect the viability of mammalian cells or bacterial species representing benign intestinal microflora. At low nanomolar concentrations (IC 50 value = 1.76 nmol/L), PEG-2S inhibited the Na + -NQR activity in sub-bacterial membrane vesicles isolated from Vibrio cholerae . Taken together, these results show, for the first time, that Na + -NQR is critical for the bacterial infectious process and is susceptible to a precisely targeted bactericidal compound in situ. The obtained data have immediate relevance for many different diseases caused by pathogenic bacteria that rely on Na + -NQR activity for growth, including sexually transmitted, pulmonary, oral, gum, and ocular infections.

If the preceding line of reasoning is correct, inhibition of Na + -NQR should arrest bacterial energization and proliferation and, ultimately, disrupt the infectious process. Korormicin is a naturally occurring antibiotic and the most specific and potent Na + -NQR inhibitor known ( Nakayama et al. 1999 ). In this work, korormicin and a novel synthetic furanone inhibitor of Na + -NQR were used to test the hypothesis that Na + -NQR is a valid therapeutic target in the infection caused by Chlamydia trachomatis. The objectives of the present study were, therefore, 2-fold: ( i ) to assess Na + -NQR as a novel antibiotic target and ( ii ) to test the efficacy of a newly synthesized furanone Na + -NQR inhibitor to arrest Chlamydia trachomatis infection.

Bacterial energetics represents an attractive alternative target. Although the majority of modern bacteria exploit the transmembrane circulation of protons (H + cycle) in their membrane energetics, a number of species rely on the sodium ion circulation (Na + cycle) rather than the H + cycle ( Skulachev 1985 ). In these bacteria, primary Na + pumps generate the transmembrane electrochemical gradient of Na + that can serve as a direct source of energy for vital functions including substrate accumulation, motility, and ATP synthesis ( Skulachev 1985 ). Importantly, the Na + cycle is widespread among bacterial pathogens ( Häse et al. 2001 ).

Despite the increasing incidence of antibiotic resistance today, the number of new antibiotics grows at a distressingly slow rate ( Russell 2002 ). The essential part of the problem is that the vast majority of antibiotics target the same 3 general prokaryotic pathways: protein synthesis, DNA replication, and bacterial cell wall assembly. The identification of new targets outside of these traditional “over-targeted” pathways is, therefore, critical if the problem of antibiotic resistance is to be solved.

In this paper Top of page Introduction Materials and methods « Results Discussion References

Materials and methods

Materials Non-stereospecific synthesis of the furanone inhibitor, named PEG-2 here, was done by Enamine Ltd. (Kiev, Ukraine). According to the manufacturer, racemic PEG-2 mixture contained approximately 5%–10% of a desired enantiomer, PEG-2S. Stereospecific synthesis of PEG-2S was accomplished by Canam Bioresearch (Winnipeg, Manitoba, Canada).

Bacterial strains and cultivation Vibrio cholerae O395N1 strain (O1 classical biotype; SmR, ΔctxA1) (Mekalanos et al. 1983), and its isogenic ΔnqrA-F mutant derivative (Dibrov et al. 2002), were used for isolation of sub-bacterial vesicles. To maximize the expression of Na+-NQR, V. cholerae cells were grown with vigorous aeration at 30 °C in FM medium (30 g/L NaCl, 0.75 g/L KCl, 1.2 g/L MgSO 4 , 1 g/L (NH 4 ) 2 SO 4 , 0.5 mmol/L KH 2 PO 4 , 0.4% glycerol, 50 mmol/L Tris, pH 8.0) (Fadeeva et al. 2007) containing 0.4% succinate. As representatives of benign gastrointestinal microflora, Escherichia coli DH5α (supE44 hsdR17 recA1 endA1, gyrA96 thi-1 relA1) (U.S. Biochemical Corp.), Lactococcus lactis (Boreal Northwest-VWR, Cat. No. 851150), and Enterococcus faecalis (NCTC 775) were used. Cells were maintained/propagated at 37 °C in standard tryptic soy broth (Difco) and on agar plates prepared on the same broth. Chlamydia trachomatis serovar L 2 was propagated as described previously (Hirono et al. 2003). The purified organism was stored in SPG buffer (0.22 mol/L sucrose, 8.6 mmol/L Na 2 HPO 4 , 3.8 mmol/L KH 2 PO 4 , 5 mmol/L glutamic acid, 0.2 μm filtered, pH 7.4) at –80 °C until use. The titer of Chlamydia trachomatis was determined in HeLa cells in the presence of cycloheximide (1.0 μg/mL), and concentrations used were expressed as inclusion-forming units (IFU) per millilitre.

Mammalian cells HeLa 229 cells (ATCC, CCL-2.1), HEK-293 cells (ATCC, CRL-1573), and primary rabbit vascular smooth muscle cells (VSMCs) were used. VSMCs were harvested as previously described (Massaeli et al. 1999). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with 5% fetal bovine serum (Invitrogen) at 37 °C, 5% CO 2 .

Assays of the growth of free-living bacteria Overnight starter cultures of Escherichia coli, Lactococcus lactis, and Enterococcus faecalis were grown aerobically in tryptic soy broth and used to inoculate 200 μL tryptic soy broth medium in 96-deep-well plates (Whatman) at an initial OD 600 of 0.05. The obtained cultures were supplemented with 0.5–50.0 μmol/L PEG-2S (or pure DMSO in “zero” controls) and grown at 37 °C for 24 h with vigorous aeration. At 2, 5, and 24 h, growth was measured as OD 600 by scanning the plates on a Bio-Rad iMark microplate reader. For samples taken at 5 and 24 h, serial dilutions with the pre-warmed growth medium were prepared using the aliquots of bacterial cultures. The experiments were repeated at least 3 times. Evaluation of PEG-2 efficiency against Clostridium difficile (Clostridium difficile ATCC 700057) was performed by the Drop method on pre-reduced BAK plates (Brucella agar with 5% laked sheep blood, hemin, and vitamin K) according to Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria (CLSI 2012).

Assessment of antichlamydial properties of antibiotics For minimal concentration of the antibiotic that inhibits formation of chlamydial inclusions by 50% (MIC 50 ) determination, HeLa cells were grown in 24-well or 96-well plates overnight prior to chlamydial inoculation. Elementary bodies (105 IFU/mL) were applied to the cells in SPG buffer (as identified above) and, after 2 h of incubation, unattached elementary bodies were removed and infected cells were treated with different concentrations of antibiotics in DMEM with 5% fetal bovine serum in the presence of cycloheximide (1.0 μg/mL). After 42 h, inclusions were visualized by immunocytochemistry. To determine the chlamydicidal effect of korormicin and PEG-2, HeLa cells were plated on 35 mm dishes, infected with Chlamydia trachomatis, and treated with different concentrations of the antibiotic of choice. After 48 h, Chlamydia trachomatis was collected (P1) and used to infect fresh cells. Subsequent collections of Chlamydia trachomatis were used to obtain P2, P3, P4, and P5 stocks of Chlamydia trachomatis. Dilution of Chlamydia trachomatis used to infect cells after treatment with PEG-2 had to be minimized to visualize inclusions. The last passage collection of Chlamydia trachomatis (P5) after korormicin treatment and all collected passages of Chlamydia trachomatis treated with PEG-2 were used to establish infectivity of treated Chlamydia trachomatis. The minimal chlamydicidal concentration (MCC 2 ) for korormicin, PEG-2, and PEG-2S was calculated after 2 passages of infection with treatment and reflect inhibition of infection by more than 90%. In all experiments, the inclusions were visualized by immunocytochemistry.

Immunocytochemistry Cells were seeded on glass coverslips in 24-well plates (2–4 × 104 cells/well) or on glass-bottomed 96-well plates (104 cells/well) overnight and then inoculated with Chlamydia trachomatis. After 42 h, cells were fixed with 90% acetone (or 100% methanol in the case of 96-well plates), probed with anti-Chlamydia specific antibody (Thermo Fisher Scientific) and inclusions were visualized by Alexa Fluor 488–conjugated secondary antibody (Molecular Probes, Invitrogen) with DAPI (300 nmol/L) as a counter-staining. Inclusion bodies and cell nuclei were identified using a fluorescent inverted microscope (TE- 2000s; Nikon). Cells and inclusions were counted using Adobe Photoshop CS3.Ink software.

Cell proliferation assay Cells were seeded at 5 × 103 cells/well in 96-well plates in growth media and treated with different concentration of antibiotics. After 48 h, the number of living cells was determined by colorimetric enzyme assay (CellTiter 96 Cell Proliferation Assay; Promega Corporation, Madison, Wisconsin, USA) (Cory et al. 1991).

Preparation of sub-bacterial membrane vesicles from V. cholerae cells Membrane vesicles from V. cholerae strains were prepared as described previously (Dibrov et al. 2002). The membrane pellet was suspended in Buffer A containing 100 mmol/L KCl, 50 mmol/L NaCl, 5 mmol/L MgSO 4 , 20 mmol/L HEPES-Tris, pH 8.0 at 20–30 mg protein/mL, snap-frozen in liquid nitrogen and stored at –80 °C.

Na+-NQR activity assays in sub-bacterial vesicles The activity of Na+-NQR in sub-bacterial V. cholerae vesicles was measured at 25 °C as oxidation of dNADH (ε 340 = 6.22 L/(mmol·cm)) by following the changes in its fluorescence at 440 nm (excitation light λ = 340 nm) using Shimadzu RF-1501 spectrofluorometer. The assays were conducted in Buffer A supplemented with 15 μmol/L Na+-dNADH with constant stirring. The reaction was initiated by the addition of vesicles (aliquots of 50 μg of protein). Fluorescence at 440 nm as a function of [dNADH] in the experimental buffer was linear up to 20 μmol/L of added dNADH (data not shown).

Measurements of intracellular pH and sodium in cells pHrodo Green AM and CoroNa Green Sodium Indicator (both Molecular Probes, Invitrogen) were used to measure intracellular pH (pH i ) and intracellular sodium (Na+ i ) concentration, respectively. HEK293 cells were seeded on 24-well plates and infected with Chlamydia trachomatis after overnight incubation in DMEM with 5% fetal bovine serum. After 2 h of incubation with Chlamydia trachomatis, cells were treated with different concentrations of PEG-2S and subjected to pH i and Na+ i measurements at different time points according to the manufacturer’s protocols. In all assessments, calibration curves were performed prior to the experiments. A fluorescent inverted microscope (TE- 2000s; Nikon) was used to obtain images and mean intensity was quantified using NIS Elements imaging software (Nikon, Mississauga, Ontario, Canada).