Furthermore, the XTT assay showed that ENOblock has relatively low toxicity to human umbilical vein endothelial cells. (B) Effects of different concentrations of ENOblock alone and in combination with 8 μg/ml FLC on the maintenance of mature biofilms. doi: 10.1128/AAC.00436-17. doi: 10.1128/AAC.02259-16, Keywords: Candida albicans, ENOblock, fluconazole, synergism, enolase1, transglutaminase activity, Citation: Li L, Zhang T, Xu J, Wu J, Wang Y, Qiu X, Zhang Y, Hou W, Yan L, An M and Jiang Y (2019) The Synergism of the Small Molecule ENOblock and Fluconazole Against Fluconazole-Resistant Candida albicans. As a homolog of human enolase, enolase 1 (Eno1) encoded by the only one ENO1 gene in C. albicans is a multifunctional protein that is essential for the growth and virulence of C. albicans (Yang et al., 2006; Ko et al., 2013).
The in vitro toxicity evaluation of ENOblock. FIGURE S5 | Expression and purification of recombinant C. albicans enolase 1 (rCaEno1). The plates were incubated for another 24 h. At the end of incubation, 50 μl of PMS-XTT solution (final concentration, 50 μg of XTT, and 0.38 μg of PMS per well) was added to each well and incubated at 37°C for 4 h. The absorbance at 450 nm was measured using an ELISA Plate Reader. doi: 10.1007/s11373-005-9054-6, Zhanel, G. G., Saunders, D. G., Hoban, D. J., and Karlowsky, J. rE, rCaEno1; PC, positive control (transglutaminase from guinea pig liver, catalog number T5398). In Clinical Microbiology Procedures Handbook, Fourth Edition. Transglutaminases (TGases) are widely distributed in plants, fungi and animals and are known to catalyze the formation of N-ε-(γ-glutamyl)-lysine amide bonds, resulting in covalent cross-links between proteins (Ruiz-Herrera et al., 1995). Representative photomicrographs of indicated C. albicans 0304103 grown in liquid RPMI 1640 medium for 3.5 h at 37°C, as observed with an inverted phase contrast microscope with a 40 × objective. ENOblock is the first reported non-substrate inhibitor of enolase. Annu. Front. ∗P < 0.05 (P-values are from ANOVA). 40, 45–52. Influence of human serum on antifungal pharmacodynamics with Candida albicans. Interactions of rCaEno1 with ENOblock measured by surface plasmon resonance. (2015). Biol. For protein expression, cultures were grown in LB medium containing 0.1 mg/ml ampicillin in a rotary shaker at 37°C to an OD600 nm of 0.6–0.8 before induced using 0.5 mM IPTG at 37°C for 5 h. After harvesting by centrifugation, cells were lysed by sonication in lysis buffer, and then centrifuged at more than 12,000 rpm for 20 min to collect the supernatant containing recombinant protein. (2015b).
doi: 10.1111/j.1439-0507.2009.01690.x, Vyas, V. K., Bushkin, G. G., Bernstein, D. A., Getz, M. A., Sewastianik, M., Barrasa, M. I., et al. Biomimetic ApoE-reconstituted high density lipoprotein nanocarrier for blood-brain barrier penetration and amyloid beta-targeting drug delivery. Female C57BL/6 mice aged 8–10 weeks were infected via the tail vein with 200 μl of a 2.5 × 106 CFU/ml PBS suspension, and then randomly placed into four groups (five mice per group): an untreated control group, FLC alone group, ENOblock alone group and FLC + ENOblock group. Figures . In a murine model of systemic candidiasis with C. albicans 0304103, treatment of mice with ENOblock (12 mg/kg) alone did not show significant reduction in fungal burden when compared to the untreated counterparts. Potent in vitro synergism of fluconazole and osthole against fluconazole-resistant Candida albicans. Compare: synergism. In contrast, FLC alone was not enough for drug-resistant strains, so the synergism was discovered, which is consistent with the hypothesis proposed by Li et al.
The plates were further incubated statically for 48 h at 37°C. Following purity determination by SDS-PAGE, aliquots (less than 100 μl) of purified recombinant proteins were flash-frozen with liquid nitrogen and stored at −80°C. Rev. |, Antimicrobials, Resistance and Chemotherapy, https://www.frontiersin.org/articles/10.3389/fmicb.2019.02071/full#supplementary-material, Creative Commons Attribution License (CC BY). PLoS Pathog. Agents Chemother.
At 2 h after infection, 0.25 mg/kg FLC and 12 mg/kg ENOblock were administered intraperitoneally (i.p.). The rate of agreement of the E test and checkerboard methods was found to be 55%.
Data are shown as the means ± standard deviations for three independent experiments. This finding suggests that their synergistic antifungal activity might be relevant to FLC resistance, or that FLC alone was sufficiently effective against drug-sensitive strains at low concentrations, so the synergism could not be observed. (2006).
New Microbiol. Am. Minn, Y., Brummer, E., and Stevens, D. A. (B) Western blot of total proteins from C. albicans SC5314 (lane 2) and purified rCaEno1 protein (lane 3) using mouse anti-rCaEno1 protein antibodies and anti-His-tag polyclonal antibodies (lane 4). 71, 753–775. rE, rCaEno1; PC, enolase-positive control (catalog number MAK178F).
New CRISPR mutagenesis strategies reveal variation in repair mechanisms among fungi. Activity of sanguinarine against Candida albicans biofilms. ∗∗P < 0.01; ∗∗∗P < 0.001 compared with the value of the control biofilms.
ENOblock does not inhibit the activity of the glycolytic enzyme enolase.
ENOblock and FLC were administered at 2, 24, and 48 h post infection. Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases. The results indicated that while ENOblock showed strong interaction with rCaEno1p, it had no obvious binding with BSA.
*Correspondence: Maomao An, [email protected]; Yuanying Jiang, [email protected], Front. A checkerboard microdilution assay showed that ENOblock has a significant synergistic effect in combination with FLC against FLC-resistant C. albicans.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Future Microbiol. doi: 10.1371/journal.ppat.1002683, Iranzo, M., Aguado, C., Pallotti, C., Canizares, J. V., and Mormeneo, S. (2002).
J. Microbiol. Furthermore, our data suggest that the synergism of FLC and ENOblock was associated with the disruption of hypha and biofilm formation due to the inhibition of TGase activity of CaEno1 bound by ENOblock, and the potential of ENOblock as a new antifungal candidate. ENOblock binds recombinant C. albicans enolase 1 (rCaEno1). Sci. According to previous reports (Minn et al., 1997; Zhanel et al., 2001), it is known that in vitro testing of some compounds can be used to predict in vivo efficacy, but also may not correlate with in vivo efficacy, due to the influence of serum, formulation of compounds, and some other factors. doi: 10.1128/AAC.50.3.1096-1099.2006, Radwan, M. A., Alquadeib, B. T., Siller, L., Wright, M. C., and Horrocks, B. Briefly, Synergism was defined as a ≥ 2-log 10 decrease in CFU per milliliter for the combination compared to that by the most active agent alone after 24 h, antagonism was defined as a ≥ 2-log 10 increase in CFU per milliliter for the combination compared to that by the most active agent alone after 24 h, while a change of < 2 log 10 CFU/ml was considered indifferent. The human protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection, which is accompanied by a species-diversified vaginal microbiota named community state type IV (CST-IV). Mol.
Drug Deliv. Trop. Although ENOblock alone was not sufficient to treat C. albicans infection, the combination of FLC and ENOblock showed significant in vivo activity against FLC-resistant C. albicans. In addition to synergism, other terms are used to define toxicologic interactions. This effect is the most common when two chemicals are given together. To test the binding of rCaEno1 protein by ENOblock, serial dilutions of ENOblock were injected into the flow system. Med. AMB and FLC were used as controls. It is possible to observe the in vivo synergism against C. albicans SC5314 in the animal study. Data are shown as the means ± standard deviations for three independent experiments. An FICI value of > 0.5 but ≤ 4 was considered indifferent (Odds, 2003). doi: 10.1128/AAC.49.4.1593-1596.2005, Cho, H., Um, J., Lee, J. H., Kim, W. H., Kang, W. S., Kim, S. H., et al. To obtain highly purified protein, the recombinant C. albicans enolase 1 (rCaEno1) protein was purified by Ni-chelate affinity chromatography (Qiagen), followed by size exclusion chromatography using a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare). 45, 2475–2479.
We thank Professor Changzhong Wang (School of Integrated Traditional and Western Medicine, Anhui University of Traditional Chinese Medicine, Hefei, China) for providing C. krusei ATCC2340 and C. glabrata ATCC1182. Table 1. 184, 2058–2061. Specifically, the combination of ENOblock and FLC exhibited inhibitory activity against mature biofilms. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods. The results represent the means ± standard deviations from three independent experiments.
A unique small molecule inhibitor of enolase clarifies its role in fundamental biological processes. The rCaEno1 protein was coated on the CM5 sensor chip and serial dilutions of ENOblock (1250, 625, 312.5, 156.25, and 19.53 nM) were used as analytes. Briefly, rCaEno1 and ENOblock were added into the appropriate wells, and the volume was adjusted to 50 μl with ultrapure water. Denoting mutual opposition in action among structures, agents, diseases, or physiologic processes. Consistently, as shown in Supplementary Figure S3, the significant inhibitory activity of ENOblock against biofilm formation of C. albicans SC5314 was also observed and even more obvious. doi: 10.1126/scitranslmed.3004404, Calderone, R., Sun, N., Gay-Andrieu, F., Groutas, W., Weerawarna, P., Prasad, S., et al. 2. The in vitro biofilm formation assay was carried out according to methods described previously (Ramage et al., 2002; Li et al., 2017) with some modifications. Figure 6. The final concentration of ENOblock and FLC ranged from 0.125 to 64 μg/ml, and then MICs were determined.
Different concentrations of ENOblock (8 μg/ml or 16 μg/ml) and FLC (8 μg/ml) were added. Is there an emerging need for new antifungals? Finally, using surface plasmon resonance analysis as well as an inhibition assay, we determined that ENOblock directly interacted with CaEno1 and significantly inhibited the transglutaminase activity of this enzyme, which is involved in the growth and morphogenesis of C. albicans. Microbiology 163, 1145–1147. 49, 1593–1596. Briefly, Synergism was defined as a ≥ 2-log10 decrease in CFU per milliliter for the combination compared to that by the most active agent alone after 24 h, antagonism was defined as a ≥ 2-log10 increase in CFU per milliliter for the combination compared to that by the most active agent alone after 24 h, while a change of < 2 log10 CFU/ml was considered indifferent. In this study, our data confirmed that ENOblock in combination with FLC changed the fungistatic azoles into fungicidal (Figure 1), reversing antifungal drug resistance and broadening the therapeutic implications of combination therapy with azoles. doi: 10.1074/jbc.M117.810440, Robbins, N., Caplan, T., and Cowen, L. E. (2017). Role of the RAM network in cell polarity and hyphal morphogenesis in Candida albicans. FIGURE S1 | Effects of different concentrations of ENOblock in combination with FLC on hypha formation in liquid Lee medium and liquid Spider medium. To evaluate the efficacy of combination therapy with FLC and ENOblock, we compared the renal fungal burden and survival rate of C. albicans-infected mice using the well-established murine model of systemic candidiasis. Antimicrob.
Synergistic antifungal effect of amphotericin B-loaded poly(Lactic-Co-Glycolic acid) nanoparticles and ultrasound against Candida albicans biofilms.