Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions

Supramolecular Porphyrin‐Based Metal–Organic Frameworks with Fullerenes: Crystal Structures and Preferential Intercalation of C70

The syntheses and characterization of two new porphyrin‐based metal–organic frameworks (P‐MOFs), through the complexation of 5,10,15,20‐tetra‐4‐pyridyl‐21 H,23 H‐porphine (H2TPyP) and copper(II) acetate (CuAcO) in the presence of the fullerenes C60 or C70 are reported. Complex 1 was synthesized in conjunction with C60, and this reaction produced a two‐dimensional (2D) porous structure with the composition CuAcO‐CuTPyP⊃m‐dichlorobenzene (m‐DCB), in which C60 molecules were not intercalated. Complex 2 was synthesized in the presence of C70, generating a three‐dimensional (3D) porous structure, in which C70 was intercalated, with the composition CuAcO‐CuTPyP⋅C70⊃m‐DCB⋅CHCl3. The structures of these materials were determined by X‐ray diffraction to identify the supramolecular interactions that lead to 2D and 3D crystal packing motifs. When a combination of C60 and C70 was employed, C70 was found to be preferentially intercalated between the porphyrins.How Porefessional: Solutions of copper(II) acetate (CuAcO), 5,10,15,20‐tetra‐4‐pyridyl‐21 H,23H‐porphine (H2TPyP), and fullerenes C70 or C60 produce crystalline precipitates with the compositions CuAcO‐CuTPyP⊃m‐dichlorobenzene (m‐DCB) (1) and CuAcO‐CuTPyP⋅C70⊃m‐DCB⋅CHCl3 (2). The structures of these materials have been determined by X‐ray diffraction to identify the supramolecular interactions that lead to two‐ and three‐dimensional crystal packing motifs. Complexation in the presence of both C60 and C70 leads to preferential intercalation of C70 between the porphyrins.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137276/1/asia201501422-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137276/2/asia201501422.pd

In Vitro Activity of Lascufloxacin against Streptococcus pneumoniae with Mutations in the Quinolone Resistance-Determining Regions

Lascufloxacin showed potent activity against Streptococcus pneumoniae with a GyrA or ParC mutation (first-step mutant). The frequency of selecting resistant strains tended to be lower for lascufloxacin than for levofloxacin and garenoxacin after drug exposure in first-step mutants but was similar in the comparison between lascufloxacin and moxifloxacin. The increase in MIC was smaller for lascufloxacin than for levofloxacin, garenoxacin, and moxifloxacin when clinical strains with only ParC mutations were exposed to the corresponding drug

Interaction of amphotericin B and clorgyline against <i>Candida</i> species determined by fractional inhibitory concentration index.

<p>Interaction of amphotericin B and clorgyline against <i>Candida</i> species determined by fractional inhibitory concentration index.</p

Effects of two known azole transporter inhibitors, carbonyl cyanide 3-chlorophenylhydrazone and milbemycin A4 oxime, on micafungin susceptibility in <i>C</i>. <i>glabrata</i> wild-type strain and <i>CDR1</i>, <i>CDR2</i>, and <i>PDR1</i> mutant strains.

<p>Spot dilution tests were performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g001" target="_blank">Fig 1B</a>. Final drug concentrations: carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 40 μM; milbemycin A4 oxime (Milbemycin), 4 μM; micafungin (MCFG), 0.03 μg/ml. <i>C</i>. <i>glabrata</i> strains: Wild type, CBS138; Δ<i>cdr1</i>, TG-C1; Δ<i>cdr2</i>, TG-C2; and Δ<i>pdr1</i>, TG-C3.</p

Strains used in this study.

<p>Strains used in this study.</p

Interaction of micafungin and clorgyline against <i>Candida</i> species determined by fractional inhibitory concentration index.

<p>Interaction of micafungin and clorgyline against <i>Candida</i> species determined by fractional inhibitory concentration index.</p

Effects of clorgyline on susceptibility to micafungin and amphotericin B in <i>Candida</i> wild-type strains.

<p>Spot dilution tests were performed using MIN plates as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g001" target="_blank">Fig 1B</a>. Clorgyline was added at a final concentration of 0 μg/ml or 80 μg/ml. Micafungin (MCFG) final concentrations: <i>C</i>. <i>glabrata</i>, 0.06 μg/ml; <i>C</i>. <i>albicans</i>, 0.08 μg/ml; <i>C</i>. <i>parapsilosis</i>, 0.25 μg/ml; <i>C</i>. <i>tropicalis</i>, 0.06 μg/ml; and <i>C</i>. <i>krusei</i>, 0.08 μg/ml. Amphotericin B (AMPH-B) final concentrations: <i>C</i>. <i>glabrata</i>, 0.5 μg/ml; <i>C</i>. <i>albicans</i>, 0.5 μg/ml; <i>C</i>. <i>parapsilosis</i>, 1 μg/ml; <i>C</i>. <i>tropicalis</i>, 0.5 μg/ml; and <i>C</i>. <i>krusei</i>, 2 μg/ml. <i>Candida</i> wild-type strains: <i>C</i>. <i>glabrata</i>, CBS138; <i>C</i>. <i>albicans</i>, SC5314; <i>C</i>. <i>parapsilosis</i>, ATCC 90018; <i>C</i>. <i>tropicalis</i>, ATCC 750; and <i>C</i>. <i>krusei</i>, ATCC 6258.</p

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in <i>Candida glabrata</i> and other pathogenic <i>Candida</i> species - Fig 4

<p>(A) Time-course analysis of <i>FMS1</i> expression in <i>C</i>. <i>glabrata</i> wild-type strain. Cell culture, RNA extraction, real-time qRT-PCR, and data presentation were performed as described in the Materials and Methods section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g003" target="_blank">Fig 3A</a> legend. (B) Effects of clorgyline on susceptibility to micafungin and amphotericin B in the <i>C</i>. <i>glabrata</i> wild-type and <i>FMS1</i> mutant strains. Spot dilution tests were performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g001" target="_blank">Fig 1B</a>. Final drug concentrations: clorgyline, 80 μg/ml; micafungin (MCFG), 0.03 μg/ml; and amphotericin B (AMPH-B), 1.25 μg/ml. <i>C</i>. <i>glabrata</i> strains: Wild type, CBS138; and Δ<i>fms1</i>, TG-C4.</p