Porin-Mediated transport of fluoroquinolones in Mycobacterium Tuberculosis

Ph.DDOCTOR OF PHILOSOPH

Polyamines inhibit porin-mediated fluoroquinolone uptake in mycobacteria.

Polyamines decrease the permeability of the outer membrane of Escherichia coli to fluoroquinolones and β-lactams. In this study, we tested the effect of four polyamines (spermidine, spermine, cadaverine and putrescine) on fluoroquinolone uptake in Mycobacterium bovis BCG. Our results show that polyamines are also capable of reducing the permeability of the mycobacterial outer membrane to fluoroquinolones. Spermidine was most effective and demonstrated reversible dose- and pH-dependent inhibition of ciprofloxacin accumulation. The extent of this inhibition was demonstrated across the fluoroquinolone compound class to varying degrees. Furthermore, we have shown that the addition of spermidine increases the survival of M. bovis BCG after a 5-day exposure to ciprofloxacin by up to 25 times. The treatment of actively-replicating Mycobacterium tuberculosis with spermidine reduced ciprofloxacin accumulation by half while non-replicating nutrient-starved M. tuberculosis cultures lacked similar sensitivity to polyamines. Gene expression studies showed that several outer membrane proteins are significantly down-regulated during the shift to non-replication. Collectively, these characteristics of fluoroquinolone uptake in M. bovis BCG are consistent with facilitated transport by porin-like proteins and suggest that a reduction in intracellular uptake contributes to the phenotypic drug resistance demonstrated by M. tuberculosis in the non-replicating state

The Role of Transport Mechanisms in Mycobacterium Tuberculosis Drug Resistance and Tolerance

In the fight against tuberculosis, cell wall permeation of chemotherapeutic agents remains a critical but largely unsolved question. Here we review the major mechanisms of small molecule penetration into and efflux from Mycobacterium tuberculosis and other mycobacteria, and outline how these mechanisms may contribute to the development of phenotypic drug tolerance and induction of drug resistance. M. tuberculosis is intrinsically recalcitrant to small molecule permeation thanks to its thick lipid-rich cell wall. Passive diffusion appears to account for only a fraction of total drug permeation. As in other bacterial species, influx of hydrophilic compounds is facilitated by water-filled open channels, or porins, spanning the cell wall. However, the diversity and density of M. tuberculosis porins appears lower than in enterobacteria. Besides, physiological adaptations brought about by unfavorable conditions are thought to reduce the efficacy of porins. While intracellular accumulation of selected drug classes supports the existence of hypothesized active drug influx transporters, efflux pumps contribute to the drug resistant phenotype through their natural abundance and diversity, as well as their highly inducible expression. Modulation of efflux transporter expression has been observed in phagocytosed, non-replicating persistent and multi-drug resistant bacilli. Altogether, M. tuberculosis has evolved both intrinsic properties and acquired mechanisms to increase its level of tolerance towards xenobiotic substances, by preventing or minimizing their entry. Understanding these adaptation mechanisms is critical to counteract the natural mechanisms of defense against toxic compounds and develop new classes of chemotherapeutic agents that positively exploit the influx and efflux pathways of mycobacteria

Characteristics of fluoroquinolone accumulation in <i>M. bovis</i> BCG.

<p>(A) Effect of PBS washes on ciprofloxacin (CPX) accumulation in BCG that has been pre-incubating with spermidine (SPM). Relative CPX accumulation was expressed as percentages of uninhibited accumulation. (B) Effect of increasing pH on the inhibitory effects of SPM. CPX accumulation in the presence of SPM was normalized against uninhibited accumulation for the each pH condition respectively. (C) Effect of efflux pump inhibitors, verapamil (75 µM) and reserpine (20 µM), on CPX, moxifloxacin (MXF) and gatifloxacin (GFX) accumulation in BCG. Relative accumulation of each drug is expressed as the percentage of uninhibited accumulation respectively. Experiments were done in biological triplicates. Standard deviations are shown as error bars. Asterisks denote significant differences between inhibited and uninhibited reactions (**, p <0.001; *, p <0.01).</p

Differential expression of OMP genes in <i>M. tuberculosis</i> during non-replication.

<p>Relative expression of each gene was calculated according to the 2^−ΔΔCt method using <i>16s rRNA</i> as the internal control and the expression levels of actively-replicating cultures as the calibrator. Significant fold-changes (≤−2.5 or ≥2.5) were bolded.</p

Effects of polyamines on ciprofloxacin accumulation in <i>M. bovis</i> BCG.

<p>(A) Inhibition of ciprofloxacin (CPX) accumulation following treatment with 4 different polyamines. Relative accumulation of CPX is expressed as a percentage of average uninhibited accumulation. Experiments were done in biological triplicates. Asterisks denote data points that differed significantly between polyamine-treated and -untreated BCG. (**, p <0.001; *, p <0.01). (B) Time-course of CPX accumulation by spermidine-treated and -untreated BCG. Data was normalized against bacterial counts for each experiment. Results are expressed as the amount of CPX (nmol) per CFU. Experiments were done in biological duplicates. Standard deviations are shown as error bars.</p

Primer pairs used for qRT-PCR analysis of OMP gene expression in <i>M. tuberculosis</i>.

<p>Primer pairs used for qRT-PCR analysis of OMP gene expression in <i>M. tuberculosis</i>.</p

Dose-dependency of spermidine-induced inhibition of intracellular accumulation.

<p>(A) Plot of percentage inhibition of ciprofloxacin (CPX) accumulation by <i>M. bovis</i> BCG in response to increasing concentrations of spermidine (SPM). This experiment was done in biological triplicates. (B) Kill -kinetics of 10 mM of SPM against <i>M. bovis</i> BCG for the first 60 min of incubation. This experiment was done in biological duplicates. Standard deviations are shown as error bars.</p

Effects of spermidine on the accumulation of fluoroquinolone and non-fluoroquinolones in <i>M. bovis</i> BCG.

<p>(A) Inhibition of moxifloxacin (MXF), ofloxacin (OFX) and gatifloxacin (GFX), and ciprofloxacin (CPX) accumulation in BCG by spermidine (SPM). (B) Inhibition of linezolid (LNZ), rifampicin (RIF) and ethambutol (EMB) accumulation in BCG by SPM. Relative accumulation of each drug is expressed as a percentage of uninhibited drug accumulation respectively. Experiments were done in biological triplicates. Standard deviations are shown as error bars. Asterisks denote data points that differed significantly between SPM-treated and -untreated BCG. (**, p <0.001; *, p <0.01).</p