The influence of N and S poles of static magnetic field (SMF) on Candida albicans hyphal formation and antifungal activity of amphotericin B
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Due to the increasing number of Candida albicans’ infections and the resistance of this pathogenic fungus to drugs, new therapeutic strategies are sought. One of such strategies may be the use of static magnetic field (SMF). C. albicans cultures were subjected to static magnetic field of the induction 0.5 T in the presence of fluconazole and amphotericin B. We identified a reduction of C. albicans hyphal length. Also, a statistically significant additional effect on the viability of C. albicans was revealed when SMF was combined with the antimycotic drug amphotericin B. The synergistic effect of this antimycotic and SMF may be due to the fact that amphotericin B binds to ergosterol in plasma membrane and SMF similarly to MF could influence domain orientation in plasma membrane (PM).
Candida albicans is a microorganism forming part of human microflora, which under immunosuppression causes opportunistic infections (Dadar et al. 2018). Chronic mucocutaneous candidiasis (CMC) is characterized by infections of the skin, nails, and oral and genital mucosae (Puel et al. 2011). However, under high immunodeficiency of the host, C. albicans enters the bloodstream and induces systemic infections with a mortality rate ranging from 30 to 80% (Gunsalus and Kumamoto 2016; Whaley et al. 2017). After the transition from yeast to hyphal form, C. albicans penetrates the host’s physiological barriers (Richardson et al. 2018). C. albicans infections are characterized by increasing resistance to traditional antifungal agents, such as fluconazole and amphotericin B (Pfaller 1996; Mah and O’Toole 2001). The mechanisms of resistance include overproduction of membrane drug efflux transporters (mainly Cdr1p belonging to ATP-binding cassette (ABC) family) (Hernáez et al. 1998), or changes in the expression of genes involved in ergosterol biosynthesis (mainly ERG11 gene encoding lanosterol 14α-demethylase) (Martel et al. 2010). The increasing resistance of C. albicans to drugs is associated with the need to develop new treatment strategies; one of them may be the use of SFM.
Living organisms are permanently exposed to constant Earth’s magnetic field (MF) (Zhang et al. 2018; Cao and Pan 2018). The number of MF applications in medical therapies has been increasing over the last decades and now includes magnetotherapy, magnetic stimulation (MS), and transcranial magnetic stimulation (TMS) (Sztafrowski et al. 2018).
Biological processes are currently being monitored under the influence of static magnetic field (SMF) and alternating MF, the value of which is several orders larger than the Earth’s MF (Sztafrowski et al. 2017). In vitro, SMF exposure can reduce the number of viable cells in melanoma, ovarian carcinoma, and lymphoma cell lines (Raylman et al. 1996). In clinical trials, SMF induces analgesic benefits in patients with: symptomatic diabetic peripheral neuropathy (DPN) (Weintraub et al. 2003), fibromyalgia (Alfano et al. 2004), rheumatoid arthritis (RA) (Segal et al. 2001), and postpolio (Vallbona et al. 1997).
Unlike a large number of publications about the influence of SMF on human cells, information about its effect and mechanism of toxicity on microorganisms is less known. SMF has no significant effect on the growth of pathogenic microorganisms such as Escherichia coli or Staphylococcus aureus (Grosman et al. 1992) but it induces antibiotic resistance in E. coli (Stansell et al. 2001). In phytopathogenic fungi, SMF was shown either to stimulate (Alternaria alternata and Coelophora inaequalis) or reduce conidia development (Fusarium oxysporum and Fusarium culmorum) (Albertini et al. 2003; Nagy and Fischl 2004).
Since there are limited data on the influence of SMF on microorganisms, especially on yeast and pathogenic yeast-like fungi, the aim of this study was to check whether SMF has an impact on general viability of C. albicans hyphal transition and its susceptibility to fluconazole and amphotericin B.
Materials and methods
Chemicals, strains, and growth conditions
Chemicals and reagents used in this study were purchased from the following sources: fluconazole and conventional amphotericin B (Sigma-Aldrich; Poznań, Poland); d-glucose and bacteriological agar (Lab Empire; Rzeszów, Poland); peptone and yeast extract (YE) (Diag-med; Warszawa, Poland); and fetal bovine serum (FBS) (Thermo Fisher; Warszawa, Poland).
C. albicans strain CAF2-1 (genotype: ura3Δ::imm434/URA3) was a kind gift of Prof. D. Sanglard (Lausanne, Switzerland) (Fonzi and Irwin 1993). It was routinely grown at 28 °C on YPD medium (2% glucose, 1% peptone, 1% YE) with agitation (120 rpm). Agar in a final concentration of 2% was used for medium solidification.
Exposure of biological material to SMF
The impact of SMF on general C. albicans viability
Twenty-four-hour cultures of C. albicans (YPD medium; 120 rpm; 28 °C) were centrifuged (5 min, 4.5 k rpm), washed with fresh YPD medium, and resuspended in YPD medium of A600 = 0.1 (corresponding to cell concentration of 1.4 × 106 cfu/mL). Eight-well culture chambers were inoculated as described in “Exposure of biological material to SMF” section, to a final volume of 300 μL and cultured for 24 h at 28 °C. The material was then transferred to a 96-well plate and A600 was measured using ASYS UVM 340 (Biogenet) microplate reader.
The impact of SMF on yeast to yeast-to-hyphae transition
Twenty-four-hour cultures of C. albicans (YPD medium; 120 rpm; 28 °C) were centrifuged (5 min, 4.5 k rpm), washed with fresh YPD medium, and resuspended in YPD medium of A600 = 0.4 (corresponding to a cell concentration of 5.9 × 106 cfu/mL). At this point, control microscopic preparation was made (negative control). The exposure to SMF was performed in 8-well culture chambers, as described in “Exposure of biological material to SMF” section (positive control: induction of hyphal transition with no exposure to SMF). To induce hyphal transition, the suspensions were treated with FBS (final conc. = 10%) for 2 h at 37 °C. The samples were observed under Zeiss Axio Imager A2 microscope equipped with Zeiss Axiocam 503 mono microscope camera for the assessment of cell morphology (n = 50–100 cells in four repetitions). The length (μm) of straight hyphae was measured using Zeiss ZEN 2 Blue software.
The impact of SMF on drug susceptibility of C. albicans
Twenty-four-hour culture of C. albicans (YPD medium; 120 rpm; 28 °C) was centrifuged (5 min, 4.5 k rpm), washed with fresh YPD medium, and resuspended in YPD medium of A600 = 0.1 (corresponding to cell concentration of 1.4 × 106 cfu/mL). Eight-well culture chambers were inoculated, as described in “Exposure of biological material to SMF” section to a final volume of 300 μL. Each well was treated with fluconazole (final conc. = 2 or 4 μg/mL) or amphotericin B (final conc. = 0.063 or 0.125 μg/mL) and cultured for 24 h at 28 °C. Such concentrations of antibiotics have been selected that lower the A600, but do not kill the cells. Thereafter, the material was transferred to a 96-well plate and A600 was measured using ASYS UVM 340 (Biogenet) microplate reader.
Each experiment was performed at least in triplicate. Statistical significance was determined using the Tukey-Kramer HSD post hoc test after the one-way ANOVA (α = 0.05).
In each experiment, C. albicans CAF2-1 cells were divided into four groups. Control cells were not subjected to the influence of SMF. Other cell groups were subjected to different conditions in the SMF magnet zones: at the north pole (N), at the south pole (S), or between the north and south poles (N/S) (Fig. 1). Most of the data were presented in a twofold manner for comprehensive interpretation: box-and-whiskers plot (minimal and maximal data, median, first and third quartiles (Q1; Q3)) and histograms (average ± standard deviation (SD)).
Data obtained for various drug concentrations are shown in separate graphs for a clearer presentation.
C. albicans cells exposed to SMF in the presence of 2 μg/mL fluconazole displayed no significant changes in median, Q1 (Fig. 4A) and average (Fig. 4B) of A600. Unexposed cells display lower Q3 of A600, and cells exposed to S pole show lower minimal A600. Cells exposed to N/S pole display higher maximum A600. In the presence of 4 μg/mL fluconazole, the most noticeable is the higher susceptibility of C. albicans cells exposed to S pole, reflected as lower parameters of A600: minimum, maximum, median, and Q1 and Q3 (Fig. 4C), as well as the average (Fig. 4D). The result is significant at p = 0.021.
Treatment of cells with 0.0625 μg/mL amphotericin B in the presence of SMF resulted in lower median A600 (Fig. 5A), with values of 1.17 in the case of N and N/S poles and 1.14 in the case of S pole (1.2 in control). A600 of unexposed cells was between 1.19 (minimum) and 1.21 (maximum). The minimal A600 after exposure to SMF was 1.16 (N pole), 1.13 (S pole), and 1.17 (N/S pole), whereas the maximal A600 increased to 1.17 (N pole), 1.16 (S pole), and 1.17 (N/S pole). Q3 data of cells exposed to N, S, and N/S are considerably lower (1.17, 1.15, and 1.17, respectively) than Q3 and Q1 of A600 in unexposed cells (1.21 and 1.19, respectively). Average A600 of cells exposed to both N and N/S poles (both = 1.17) was lower than A600 of unexposed cells (= 1.2); the lowest average A600 (= 1.14) was obtained after exposing cells to the S pole, with statistical significance (p = 0.008).
A similar trend was observed after exposing C. albicans cells to SMF in the presence of 0.125 μg/mL amphotericin B (Fig. 5 C, D). A600 of unexposed cells was between 0.39 (minimum) and 0.45 (maximum), with a median at 0.43. The minimal A600 after exposure towards SMF was 0.35 (N pole), 0.37 (S pole), and 0.35 (N/S pole); the maximal A600 was 0.41 (N pole), 0.38 (S pole), and 0.39 (N/S pole) and the median A600 was with a value of 0.39 (N pole), 0.38 (S pole), and 0.39 (N/S pole). Q3 of A600 in cells exposed to SMF was in all cases lower than the median A600 of unexposed cells and, in the case of cells exposed to S and N/S poles, lower than Q1 of A600 in unexposed cells. The average A600 of cells exposed to both N, S, and N/S poles in the presence of 0.125 μg/mL amphotericin B (0.39, 0.38 and 0.38, respectively) was lower than A600 of unexposed cells (= 0.42). The results obtained were significant at p = 0.003.
Considering our preliminary results, it seems that the potential use of a SMF in antifungal therapy could be a new option of supporting treatment for Candidas’ infections. Previously, an inhibitory effect of SMF on cancer cell lines was identified (Raylman et al. 1996; Sabo et al. 2002; Luo et al. 2016; Sztafrowski et al. 2018) with no influence on prokaryotic bacterial spp. (Grosman et al. 1992). This led us to the conclusion that SMF may inhibit eukaryotic fungal cells. The rate of C. albicans growth inhibition is rather slight (7.2–8.6% reduction in maximal A600, Fig. 2A). The SMF inhibitory effect towards phytopathogenic fungi was at a similar rate (5–10%) (Nagy and Fischl 2004). However, the response of fungi to SMF appears to depend on the species, because, e.g., SMF inhibited the growth of Aspergillus niger (Mateescu et al. 2011).
We identified a significant reduction of C. albicans’ hyphal length (Fig. 3). SMF also inhibited myceliar growth of phytopathogenic F. culmorum (Albertini et al. 2003) and pathogenic necrotroph Syspastospora parasitica (Mazurkiewicz-Zapalowicz et al. 2015). This activity does not seem to be universal, since SMF had no impact on myceliar growth in Tuber borchii fungus (Potenza et al. 2012). In the case of C. albicans, SMF does not completely inhibit hyphal formation, but it should be taken into consideration that the median hypha length was from 34.8 μm (control, Fig. 3B) to 16.2 (N pole), 13.1 (S pole), and 20.1 (N/S pole), i.e., respectively 53, 62, and 42% length reduction. The ability of C. albicans to form hyphae at 37 °C is one of the virulence determinants and is connected with biofilm formation and further colonization of tissues (Suchodolski et al. 2017). Moreover, C. albicans deprived of the ability to form hyphae becomes avirulent in mouse models (Diez-Orejas et al. 1997; Lo et al. 1997; Calera et al. 2000; Cao et al. 2006; Ku et al. 2017).
Sztafrowski et al. 2018 identified an additive effect of SMF on HL-60 cancer cell line treatment with busulfan cytostatic. In the case of candidiasis treatment, a combination of azole/polyenes with other drugs/treatment strategies is highly desirable (Fiori and Van Dijck 2012; Perlin 2015). The combination of fluconazole with SMF resulted in visible growth inhibition only with 4 μg/mL concentration and S pole (Fig. 4). The result was significant according to ANOVA – Tukey-Kramer’s test; however, it was not observed when the fluconazole concentration was increased (data not shown). On the other hand, a statistically significant additive effect can be seen when SMF was combined with amphotericin B (Fig. 5). Amphotericin B binds to ergosterol in the plasma membrane (PM) with subsequent PM permeabilization and lethal effect (Gray et al. 2012). MF was shown to influence domain orientation in PM (Beck et al. 2010). Ruzic et al. 1997 found that sinusoidal MF leads to an increase of ergosterol content in mycorrhizal fungus Pisolithus tinctorius. It is known that C. albicans hyphal formation depends on sphingolipid-ergosterol domains (Pasrija et al. 2005a, b; McCourt et al. 2016; Wu et al. 2018), so it is possible that SMF influences plasma membrane organization.
In all experiments, the S pole generated the most promising results: lowest minimal and average A600 of C. albicans (Fig. 2); hyphal length reduction, the lowest minimal length, median, and average (Fig. 3B); a possible combination with fluconazole (Fig. 4); and the highest and most statistically significant additive effect with amphotericin B (Fig. 5). Our results suggest that SMF may have a potential in C. albicans treatment by influencing hypha formation and, especially, within amphotericin B treatment. However, this technique must be further studied and improved for future research and application.
This work was supported by the National Science Centre, Poland, NCN Grants: 2016/23/B/NZ1/01928, 2017/25/N/NZ1/00050.
- Cao C, Pan Y (2018) Bioinspired magnetic nanoparticles for biomedical applications. In: Thanh NTK (ed) Clinical applications of magnetic nanoparticles. From fabrication to clinical applications, vol 4. Taylor & Francis Group, pp 53–68. https://doi.org/10.1201/9781315168258
- Ku M, Baek YU, Kwak MK, Kang SO (2017) Candida albicans glutathione reductase downregulates Efg1-mediated cyclic AMP/protein kinase A pathway and leads to defective hyphal growth and virulence upon decreased cellular methylglyoxal content accompanied by activating alcohol dehydrogenase and glycolytic enzymes. Biochim Biophys Acta 1861(4):772–788CrossRefGoogle Scholar
- Martel CM, Parker JE, Bader O, Weig M, Gross U, Warrilow AGS, Kelly DE, Kelly SL (2010) A clinical isolate of Candida albicans with mutations in ERG11 (encoding sterol 14α-demethylase) and ERG5 (encoding C22 desaturase) is cross resistant to azoles and amphotericin B. Antimicrob Agents Chemother 54:3578–3583CrossRefGoogle Scholar
- Mateescu C, Burnutea N, Stancu N (2011) Investigation of Aspergillus niger growth and activity in a static magnetic flux density field. Rom Biotech Lett 16(4):6364–6368Google Scholar
- Mazurkiewicz-Zapalowicz K, Twaruzek M, Formicki K, Korzelecka-Orkisz B, Wolska M, Tanski A, Szulc J (2015) The effect of magnetic field on in vitro development of fungus-like organisms Saprolegnia parasitica on selected microbiological media. EJPAU 18(2)Google Scholar
- Puel A, Cypowyj S, Bustamante J, Wright JF, Liu L, Kyung Lim H, Migaud M, Israel L, Chrabieh M, Audry M, Gumbleton M, Toulon A, Bodemer C, El-Baghdadi J, Whitters M, Paradis T, Brooks J, Collins M, Wolfman NM, Al-Muhsen S, Galicchio M, Abel L, Picard C, Casanova JL (2011) Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science 332:65–68CrossRefGoogle Scholar
- Weintraub MI, Wolfe GI, Barohn RA, Cole SP, Parry GJ, Hayat G, Cohen JA, Page JC, Bromberg BM, Schwartz SL, Magnetic Research Group (2003) Static magnetic field therapy for symptomatic diabetic neuropathy: a randomized, doubleblind, placebo-controlled trial. Arch Phys Med Rehabil 84:736–746CrossRefGoogle Scholar
- Wu Y, Wu M, Wang Y, Chen Y, Gao J, Ying C (2018) ERG11 couples oxidative stress adaptation, hyphal elongation and virulence in Candida albicans. FEMS Yeast Res 18(7)Google Scholar
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