Surfactin and Capric Acid Affect the Posaconazole Susceptibility of Candida albicans Strains with Altered Sterols and Sphingolipids Biosynthesis

Infections caused by Candida spp. pose a continuing challenge for modern medicine, due to widespread resistance to commonly used antifungal agents (e.g., azoles). Thus, there is considerable interest in discovering new, natural compounds that can be used in combination therapy with conventional antibiotics. Here, we investigate whether the natural compounds surfactin and capric acid, in combination with posaconazole, enhance the growth inhibition of C. albicans strains with alterations in sterols and the sphingolipids biosynthesis pathway. We demonstrate that combinations of posaconazole with surfactin or capric acid correspond with the decreased growth of C. albicans strains. Moreover, surfactin and capric acid can independently contribute to the reduced adhesion of C. albicans strains with altered ergosterol biosynthesis to abiotic surfaces (up to 90% reduction in adhesion). A microscopic study of the C. albicans plasma membrane revealed that combinations of those compounds do not correspond with the increased permeabilization of the plasma membrane when compared to cells treated with posaconazole alone. This suggests that the fungistatic effect of posaconazole in combination with surfactin or capric acid is related to the reduction in adhesion of C. albicans.


Introduction
Posaconazole (PSZ) is a third-generation triazole antifungal agent, which was approved by the Food and Drug Administration in 2006.It has been used in severely immunocompromised patients, and for the treatment of oropharyngeal candidiasis [1].As has been observed for other azole derivatives, the mode of action of PSZ is based on the inhibition of activity of lanosterol 14α-demethylase (Erg11p, CYP51), leading to the accumulation of toxic methylated sterol precursors, and, thereby, the depletion of ergosterol from the plasma membrane (PM) [2].This is associated with alterations in the fungal PM structure and the inhibited growth of the pathogen.
The chemical structure of PSZ consists of a triazole ring, a furan ring and two fluorine atoms (Figure 1).PSZ is characterized by the presence of an extended side chain that provides additional contact points with Erg11p [3].Multiple hydrophobic contacts of the side-chain of PSZ may result in an increased binding affinity to the enzyme, thereby inhibiting its activity more effectively than first-generation azole drugs.It has been established that the 1,2,4-triazole ring, together with the 2,4-difluorophenyl substituent, are necessary to obtain the greatest antifungal activity, and the strongest potential of the drug in inhibiting Erg11p [4].
In contrast to most azole drugs, PSZ appears to have fungistatic or fungicidal activity, depending on the species [5,6].For example, it has been reported that PSZ can exhibit activity against fluconazole-resistant Candida spp.(FLC) [7,8].In contrast to most azole drugs, PSZ appears to have fungistatic or fungicidal activity, depending on the species [5,6].For example, it has been reported that PSZ can exhibit activity against fluconazole-resistant Candida spp.(FLC) [7,8].
The common usage of azoles has contributed to azole-resistance among Candida spp., which complicates the treatment of patients suffering from candidiasis [9,10].Therefore, searching for natural compounds that are active against fungal pathogens or that improve the activity of commonly used azoles remains a key research aim of modern medicine.
We previously reported that the simultaneous treatment of C. albicans with both FLC and surfactin (SU) in the mixture corresponds with decreased MIC50 values for FLC, along with increased PM permeabilization and the remodeling of the pathogen cell wall [11].SU is a natural lipopeptide with an amphiphilic structure, produced by Bacillus subtilis.It possesses broad-spectrum antifungal and antibacterial activity [12,13].Similar to other known natural surfactants (e.g., pseudofactin II), SU has an ability to reduce the adhesion of pathogens to surfaces, which results in a reduced ability to form biofilms, and decreases the hydrophobicity of the fungi [14].
Another promising compound that could be used as an additive in azoles treatments is capric acid (CA), which is naturally produced by the probiotic yeasts Saccharomyces boulardii [15].It has been proven that CA independently impacts the growth, filamentation and biofilm formation of C. albicans [16].Additionally, CA increases the susceptibility of C. albicans to both FLC and amphotericin B [17].The chemical structures of SU and CA are presented in Figure 2. Based on prior data, we aimed to determine if SU and CA will affect the susceptibility of C. albicans to another azole drug, PSZ.Since azoles influence the structure and functioning of the fungal PM, we considered how a combination of PSZ and SU or CA would impact the growth of C. albicans mutants with alterations in ergosterol (C.albicans KS058 strain, lacking ergosterol in the PM due to the full deletion of ERG11, and three C. albicans strains, 10C1B1I1, 27A5A33A and 9B4B34A, with amino-acid substitutions in Erg11p) or mutations impacting the sphingolipids (SLs) biosynthesis pathway (C.albicans strains lacking FEN1, FEN12 or both genes).We also investigated whether perturbations The common usage of azoles has contributed to azole-resistance among Candida spp., which complicates the treatment of patients suffering from candidiasis [9,10].Therefore, searching for natural compounds that are active against fungal pathogens or that improve the activity of commonly used azoles remains a key research aim of modern medicine.
We previously reported that the simultaneous treatment of C. albicans with both FLC and surfactin (SU) in the mixture corresponds with decreased MIC50 values for FLC, along with increased PM permeabilization and the remodeling of the pathogen cell wall [11].SU is a natural lipopeptide with an amphiphilic structure, produced by Bacillus subtilis.It possesses broad-spectrum antifungal and antibacterial activity [12,13].Similar to other known natural surfactants (e.g., pseudofactin II), SU has an ability to reduce the adhesion of pathogens to surfaces, which results in a reduced ability to form biofilms, and decreases the hydrophobicity of the fungi [14].
Another promising compound that could be used as an additive in azoles treatments is capric acid (CA), which is naturally produced by the probiotic yeasts Saccharomyces boulardii [15].It has been proven that CA independently impacts the growth, filamentation and biofilm formation of C. albicans [16].Additionally, CA increases the susceptibility of C. albicans to both FLC and amphotericin B [17].The chemical structures of SU and CA are presented in Figure 2. In contrast to most azole drugs, PSZ appears to have fungistatic or fungicidal activity, depending on the species [5,6].For example, it has been reported that PSZ can exhibit activity against fluconazole-resistant Candida spp.(FLC) [7,8].
The common usage of azoles has contributed to azole-resistance among Candida spp., which complicates the treatment of patients suffering from candidiasis [9,10].Therefore, searching for natural compounds that are active against fungal pathogens or that improve the activity of commonly used azoles remains a key research aim of modern medicine.
We previously reported that the simultaneous treatment of C. albicans with both FLC and surfactin (SU) in the mixture corresponds with decreased MIC50 values for FLC, along with increased PM permeabilization and the remodeling of the pathogen cell wall [11].SU is a natural lipopeptide with an amphiphilic structure, produced by Bacillus subtilis.It possesses broad-spectrum antifungal and antibacterial activity [12,13].Similar to other known natural surfactants (e.g., pseudofactin II), SU has an ability to reduce the adhesion of pathogens to surfaces, which results in a reduced ability to form biofilms, and decreases the hydrophobicity of the fungi [14].
Another promising compound that could be used as an additive in azoles treatments is capric acid (CA), which is naturally produced by the probiotic yeasts Saccharomyces boulardii [15].It has been proven that CA independently impacts the growth, filamentation and biofilm formation of C. albicans [16].Additionally, CA increases the susceptibility of C. albicans to both FLC and amphotericin B [17].The chemical structures of SU and CA are presented in Figure 2. Based on prior data, we aimed to determine if SU and CA will affect the susceptibility of C. albicans to another azole drug, PSZ.Since azoles influence the structure and functioning of the fungal PM, we considered how a combination of PSZ and SU or CA would impact the growth of C. albicans mutants with alterations in ergosterol (C.albicans KS058 strain, lacking ergosterol in the PM due to the full deletion of ERG11, and three C. albicans strains, 10C1B1I1, 27A5A33A and 9B4B34A, with amino-acid substitutions in Erg11p) or mutations impacting the sphingolipids (SLs) biosynthesis pathway (C.albicans strains lacking FEN1, FEN12 or both genes).We also investigated whether perturbations Based on prior data, we aimed to determine if SU and CA will affect the susceptibility of C. albicans to another azole drug, PSZ.Since azoles influence the structure and functioning of the fungal PM, we considered how a combination of PSZ and SU or CA would impact the growth of C. albicans mutants with alterations in ergosterol (C.albicans KS058 strain, lacking ergosterol in the PM due to the full deletion of ERG11, and three C. albicans strains, 10C1B1I1, 27A5A33A and 9B4B34A, with amino-acid substitutions in Erg11p) or mutations impacting the sphingolipids (SLs) biosynthesis pathway (C.albicans strains lacking FEN1, FEN12 or both genes).We also investigated whether perturbations in SLs content in the PM correspond with the altered susceptibility of these strains.This research is the continuation of our previous investigation into the synergistic activity of fungistatic azoles with SU or CA [11,17].Considering that our earlier studies have already proven that alterations in ergosterol levels affect the susceptibility of C. albicans to those compounds, we wanted to determine whether the level of SLs could also impact the response to synergistic therapy.This research is an attempt at an explanation of the mechanism of action of the combination of PSZ with SU and CA.Such research can potentially impact the perception of combined therapies used in the treatment of Candida-related infections.In the case of C. albicans SC5314 (WT), 10C1B1I1, and 9B4B34A, the MIC50 value for PSZ was 0.0156 µg/mL (Table S1), although the 10C1B1I1 strain was more susceptible to PSZ at a concentration of 0.0156 µg/mL than the WT strain (Figure 3A,C).The MIC50 for PSZ alone could not be determined for the C. albicans 27A5A33A strain over the tested range of PSZ concentrations (Table S1), but a drop in growth (%) was observed at a higher concentration of PSZ (0.0313-0.125 µg/mL) when compared to the control condition (0 µg/mL PSZ).

Susceptibility of Candida albicans
The lack of production of Erg11p (a target for azoles) by the C. albicans KS058 strain resulted in the resistance of that strain to PSZ over all tested concentrations.The addition of SU at 4 and 8 µg/mL resulted in the increased growth of the strains.The presence of 32 µg/mL of SU resulted in the significant growth inhibition of C. albicans KS058 (Figure 3B).
One interesting result is that in the presence of PSZ (0.0156 µg/mL) with SU (4 µg/mL), we observed the increased growth of C. albicans WT, 10C1B1I1, 27A5A33A and 9B4B34A, compared to the growth of those strains in the presence of PSZ (0.0156 µg/mL) alone.Increasing the PSZ and SU concentrations resulted in a successive decrease in the growth of all tested strains.
Figure 4 represents the growth of C. albicans strains with deletions of genes involved in SLs biosynthesis in the presence of PSZ, with or without SU.For C. albicans SN95 (WT), fen1∆/∆ and fen12∆/∆, the MIC50 for PSZ was 0.0156 µg/mL, while for the fen1∆/∆;fen12∆/∆ strain, the MIC50 was 0.0078 µg/mL (Table S1).According to the data presented in Figure 2, C. albicans fen1∆/∆ and fen12∆/∆ were more resistant to a higher concentration of PSZ alone (0.0078-0.125 µg/mL) or in combination with SU (4-32 µg/mL) than the WT strain.The double deletant at the FEN1 and FEN12 genes was less susceptible to treatment with a combination of PSZ and SU than the C. albicans WT strain.
Posaconazole in Combination with Capric Acid  Compared to C. albicans WT, the 10C1B1I1, 27A5A33A, and 9B4B34A strains were more susceptible to PSZ (0.0078 µg/mL) in combination with CA at both concentrations: 11.35 and 22.7 µg/mL.In the presence of PSZ and CA (0.0078 and 45.3 µg/mL, respectively), C. albicans 10C1B1I1 and 27A5A33A, but not 9B4B34A, were more resistant than the WT strain.Compared to C. albicans WT, the 10C1B1I1, 27A5A33A, and 9B4B34A strains were more susceptible to PSZ (0.0078 µg/mL) in combination with CA at both concentrations: 11.35 and 22.7 µg/mL.In the presence of PSZ and CA (0.0078 and 45.3 µg/mL, respectively), C. albicans 10C1B1I1 and 27A5A33A, but not 9B4B34A, were more resistant than the WT strain.
The growth of C. albicans KS058 in the presence of PSZ and CA was diminished by about 50% in comparison with growth under the control conditions (0 µg/mL of CA).Only the highest concentration of CA (90.6 µg/mL) resulted in almost complete growth inhibition of the strain.
Figure 6 depicts the growth of C. albicans strains with deletions of the genes involved in SLs biosynthesis, in the presence of PSZ, with or without CA.Only the highest concentration of CA (90.6 µg/mL) resulted in almost complete growth inhibition of the strain.Figure 6 depicts the growth of C. albicans strains with deletions of the genes involved in SLs biosynthesis, in the presence of PSZ, with or without CA.C. albicans fen1Δ/Δ and fen12Δ/Δ strains were more resistant than the WT strain to combinations of PSZ and CA (0.0156 and 11.35, 22.7 µg/mL, respectively), but the increasing addition of CA reduced the difference in growth among C. albicans strains.The double deletion of the FEN1 and FEN12 genes resulted in the significant growth inhibition of C. albicans in the presence of 11.35 and 22.7 µg/mL of CA, while 45.30 and 90.60 µg/mL concentrations of CA led to complete growth inhibition of the C. albicans fen1Δ/Δ;fen12Δ/Δ strain.This suggests that for this strain, CA is more toxic than SU (Figure 4D).

Growth of Candida albicans Strains in the Presence of Posaconazole in Combination with Surfactin or Capric Acid
The growth curves of C. albicans strains with the deletion of the ERG11 gene or with amino acid substitutions in Erg11p, under control conditions (YPD) in the presence of PSZ (0.0156 µg/mL) and PSZ with either SU (4 µg/mL) or CA (45.3 µg/mL), are presented in Figure 7. C. albicans fen1∆/∆ and fen12∆/∆ strains were more resistant than the WT strain to combinations of PSZ and CA (0.0156 and 11.35, 22.7 µg/mL, respectively), but the increasing addition of CA reduced the difference in growth among C. albicans strains.The double deletion of the FEN1 and FEN12 genes resulted in the significant growth inhibition of C. albicans in the presence of 11.35 and 22.7 µg/mL of CA, while 45.30 and 90.60 µg/mL concentrations of CA led to complete growth inhibition of the C. albicans fen1∆/∆;fen12∆/∆ strain.This suggests that for this strain, CA is more toxic than SU (Figure 4D).

Growth of Candida albicans Strains in the Presence of Posaconazole in Combination with Surfactin or Capric Acid
The growth curves of C. albicans strains with the deletion of the ERG11 gene or with amino acid substitutions in Erg11p, under control conditions (YPD) in the presence of PSZ (0.0156 µg/mL) and PSZ with either SU (4 µg/mL) or CA (45.3 µg/mL), are presented in Figure 7.The addition of PSZ to C. albicans KS058 and 27A5A33A did not affect the growth of those strains.In the case of the C. albicans 10C1B1I1 strain, we observed diminished growth compared to control conditions.A shift in the logarithmic phase of growth of C. albicans 9B4B34A was observed in the presence of PSZ, and a similar trend in the growth curve was observed for the culture with the addition of PSZ and CA.Additionally, we noted that the presence of SU in the culture resulted in barely any growth of all the in- The addition of PSZ to C. albicans KS058 and 27A5A33A did not affect the growth of those strains.In the case of the C. albicans 10C1B1I1 strain, we observed diminished growth compared to control conditions.A shift in the logarithmic phase of growth of C. albicans 9B4B34A was observed in the presence of PSZ, and a similar trend in the growth curve was observed for the culture with the addition of PSZ and CA.Additionally, we noted that the presence of SU in the culture resulted in barely any growth of all the investigated strains.For C. albicans KS058, which lacks Erg11p, a target for azoles, we detected a significant decrease in growth in the presence of SU.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 9 of 19 vestigated strains.For C. albicans KS058, which lacks Erg11p, a target for azoles, we detected a significant decrease in growth in the presence of SU.
The growth curves of C. albicans strains with deleted genes involved in SLs biosynthesis, under control conditions (YPD) and in the presence of PSZ (0.0156 or 0.0078 µg/mL for fen1Δ/Δ;fen12Δ/Δ strain) and PSZ with SU (4 µg/mL) or CA (22.7 or 11.35 µg/mL for fen1Δ/Δ;fen12Δ/Δ strain), are presented in Figure 8.The presence of PSZ in cultures of C. albicans fen1Δ/Δ, fen12Δ/Δ and fen1Δ/Δ;fen12Δ/Δ did not result in the decreased growth of those strains, in contrast to the WT strain.The SN95 (WT) strain, in the presence of PSZ, entered the logarithmic phase after 9-10 h of culture, while under control conditions, logarithmic growth started in the 7th hour of culture.In the presence of PSZ and CA, the growth curves of the C. albicans fen1Δ/Δ and fen12Δ/Δ strains remained similar to those obtained under control conditions, except for a shift in the timing of the logarithmic phase of growth by 2-3 h.The growth of SN95 (WT) and fen1Δ/Δ;fen12Δ/Δ was significantly reduced in the presence of PSZ and CA.
The combination of PSZ and SU resulted in barely any growth of the C. albicans SN95 (WT) strain.An interesting observation is that, for fen1Δ/Δ, fen12Δ/Δ and fen1Δ/Δ;fen12Δ/Δ, the growths of these strains were significantly reduced compared to The presence of PSZ in cultures of C. albicans fen1∆/∆, fen12∆/∆ and fen1∆/∆;fen12∆/∆ did not result in the decreased growth of those strains, in contrast to the WT strain.The SN95 (WT) strain, in the presence of PSZ, entered the logarithmic phase after 9-10 h of culture, while under control conditions, logarithmic growth started in the 7th hour of culture.In the presence of PSZ and CA, the growth curves of the C. albicans fen1∆/∆ and fen12∆/∆ strains remained similar to those obtained under control conditions, except for a shift in the timing of the logarithmic phase of growth by 2-3 h.The growth of SN95 (WT) and fen1∆/∆;fen12∆/∆ was significantly reduced in the presence of PSZ and CA.
The combination of PSZ and SU resulted in barely any growth of the C. albicans SN95 (WT) strain.An interesting observation is that, for fen1∆/∆, fen12∆/∆ and fen1∆/∆;fen12∆/∆, the growths of these strains were significantly reduced compared to those under control conditions, and after a short stationary phase of growth, we observed a decrease in OD 600 value, which suggests that cells entered the death phase.those under control conditions, and after a short stationary phase of growth, we observed a decrease in OD600 value, which suggests that cells entered the death phase.

Candida albicans Strains with Altered Ergosterol or Sphingolipids Biosynthesis Exhibit Diminished Adhesion to Abiotic Surfaces in the Presence of Surfactin or Capric Acid
The adhesion to abiotic surfaces of all investigated C. albicans strains under control conditions, in the presence of SU (4 µg/mL) or CA (45.3; 22.7 or 11.35 µg/mL depending on C. albicans strain), is presented in Figure 9.The presence of SU or CA resulted in the significantly decreased adhesion to abiotic surfaces of all C. albicans strains with the deletion of the ERG11 gene, or with amino acid substitutions in Erg11p (Figure 9A), compared to results under control conditions (PBS alone).However, all of those strains were characterized by better adhesion to abiotic surfaces in the presence of CA, in comparison to SU.The highest reduction in adhesion was observed for C. albicans KS058 in the presence of SU, but it is worth mentioning that KS058 also exhibited reduced adhesion under control conditions compared to other strains, and the addition of SU enhanced that phenotype.
The C. albicans strains with deletions of genes involved in SLs biosynthesis exhibited reduced adhesion in the presence of SU, compared to under control conditions (Figure 9B).The C. albicans fen1Δ/Δ;fen12Δ strain, acting similarly to C. albicans KS058, exhibited decreased adhesion to abiotic surfaces under control conditions in comparison to the WT strain.The presence of SU significantly decreased the adhesion of the fen1Δ/Δ;fen12Δ strain.The addition of CA reduced adhesion of the strain to a limited extent.
In contrast to the C. albicans strains with the deletion of the ERG11 gene or with amino acid substitutions in Erg11p (Figure 9A), the presence of CA did not reduce the adhesion of the C. albicans strains with deletions of one or more of the genes involved in SLs biosynthesis, except for the fen1Δ/Δ;fen12Δ/Δ strain (Figure 9B).The presence of SU or CA resulted in the significantly decreased adhesion to abiotic surfaces of all C. albicans strains with the deletion of the ERG11 gene, or with amino acid substitutions in Erg11p (Figure 9A), compared to results under control conditions (PBS alone).However, all of those strains were characterized by better adhesion to abiotic surfaces in the presence of CA, in comparison to SU.The highest reduction in adhesion was observed for C. albicans KS058 in the presence of SU, but it is worth mentioning that KS058 also exhibited reduced adhesion under control conditions compared to other strains, and the addition of SU enhanced that phenotype.
The C. albicans strains with deletions of genes involved in SLs biosynthesis exhibited reduced adhesion in the presence of SU, compared to under control conditions (Figure 9B).The C. albicans fen1∆/∆;fen12∆ strain, acting similarly to C. albicans KS058, exhibited decreased adhesion to abiotic surfaces under control conditions in comparison to the WT strain.The presence of SU significantly decreased the adhesion of the fen1∆/∆;fen12∆ strain.The addition of CA reduced adhesion of the strain to a limited extent.
In contrast to the C. albicans strains with the deletion of the ERG11 gene or with amino acid substitutions in Erg11p (Figure 9A), the presence of CA did not reduce the adhesion of the C. albicans strains with deletions of one or more of the genes involved in SLs biosynthesis, except for the fen1∆/∆;fen12∆/∆ strain (Figure 9B).The treatment of all investigated C. albicans strains with PSZ resulted in the formation of cell clumps (Figure 10).This effect was heightened in the case of the C. albicans SC5314 (WT) strain after the addition of SU to the culture.We observed that most of those cells lost their integrity, which is associated with the increased PM permeabilization.An interesting observation is that the C. albicans 10C1B1I1 strain (ERG11 K143R ), after treatment with PSZ in combination with SU, formed filaments, which could be a stress response to the presence of SU in the culture medium.

The Combination of Posaconazole and Surfactin or Capric Acid Results in Plasma Membrane Permeabilization in Candida albicans Strains with Altered Ergosterol or Sphingolipids Biosynthesis
In the case of the C. albicans KS058 strain (erg11Δ/Δ) treated with a combination of PSZ and CA, we observed cells with disrupted PM integrity.It is worth mentioning that, during the preparation of the staining, KS058 cells treated with PSZ and CA exhibited a reduced capacity for adhesion during centrifugation.
Figure 11 presents representative microphotographs of C. albicans strains with deletions of genes involved in SLs biosynthesis cultures stained with propidium iodide (PI).The treatment of all investigated C. albicans strains with PSZ resulted in the formation of cell clumps (Figure 10).This effect was heightened in the case of the C. albicans SC5314 (WT) strain after the addition of SU to the culture.We observed that most of those cells lost their integrity, which is associated with the increased PM permeabilization.An interesting observation is that the C. albicans 10C1B1I1 strain (ERG11 K143R ), after treatment with PSZ in combination with SU, formed filaments, which could be a stress response to the presence of SU in the culture medium.
In the case of the C. albicans KS058 strain (erg11∆/∆) treated with a combination of PSZ and CA, we observed cells with disrupted PM integrity.It is worth mentioning that, during the preparation of the staining, KS058 cells treated with PSZ and CA exhibited a reduced capacity for adhesion during centrifugation.
Figure 11 presents representative microphotographs of C. albicans strains with deletions of genes involved in SLs biosynthesis cultures stained with propidium iodide (PI).The treatment of C. albicans SN95 (WT), fen12Δ/Δ and fen1Δ/Δ;fen12Δ/Δ with PSZ alone resulted in the formation of cell clumps, similar to those observed in the case of C. albicans strains with alterations in ergosterol biosynthesis (Figure 10).In contrast, the fen12Δ/Δ strain exhibited resistance to all combinations of antifungal agents, suggesting that those compounds did not affect the integrity of the PM.
In this group of investigated strains, only C. albicans SN95 (WT) produced filaments after treatment with a combination of PSZ and SU (Figure 11).The C. albicans fen12Δ/Δ strain treated with PSZ alone, or PSZ in combination with SU, exhibited increased permeabilization of the PM, but not when CA was added to the culture medium.
The C. albicans fen1Δ/Δ;fen12Δ/Δ exhibited a tendency to form cell clumps in the presence of PSZ alone, or PSZ in combination with SU or CA, and the permeabilization of the PM was increased in all cases of treated cells in comparison to control conditions.
The summary of the percentages of cells with a permeabilized PM after treatment with PSZ alone, and with a combination of PSZ with SU or CA, is presented in Table 1.The treatment of C. albicans SN95 (WT), fen12∆/∆ and fen1∆/∆;fen12∆/∆ with PSZ alone resulted in the formation of cell clumps, similar to those observed in the case of C. albicans strains with alterations in ergosterol biosynthesis (Figure 10).In contrast, the fen12∆/∆ strain exhibited resistance to all combinations of antifungal agents, suggesting that those compounds did not affect the integrity of the PM.
In this group of investigated strains, only C. albicans SN95 (WT) produced filaments after treatment with a combination of PSZ and SU (Figure 11).The C. albicans fen12∆/∆ strain treated with PSZ alone, or PSZ in combination with SU, exhibited increased permeabilization of the PM, but not when CA was added to the culture medium.
The C. albicans fen1∆/∆;fen12∆/∆ exhibited a tendency to form cell clumps in the presence of PSZ alone, or PSZ in combination with SU or CA, and the permeabilization of the PM was increased in all cases of treated cells in comparison to control conditions.
The summary of the percentages of cells with a permeabilized PM after treatment with PSZ alone, and with a combination of PSZ with SU or CA, is presented in Table 1.
According to these calculations, the most significant increase in the percentage of permeabilization of the PM of cells occurred with a combination of PSZ and SU, compared to control conditions.The only exception to this was the fen1∆/∆ strain, which displayed resistance to all combinations of compounds at the tested concentrations.In comparison to cultures supplemented with PSZ alone, only the SC5314 strain treated with PSZ and SU exhibited an elevated level of PM permeabilization.This suggests that the presence of SU affects C. albicans cells predominantly through a reduction in adhesion, rather than by disrupting the PM integrity.
Table 1.The permeabilization (%) of the plasma membrane (PM) of C. albicans strains with altered ergosterol (SC5314 (WT), KS058, 10C1B1I1, 27A5A33A and 9B4B34A) or sphingolipids biosynthesis (SN95 (WT), fen1∆/∆, fen12∆/∆ and fen1∆/∆;fen12∆/∆) pathways.The cells were stained with propidium iodide (PI, 4 µg/mL) for 5 min and then analyzed for PM permeabilization.For every strain under each condition, at least 150 cells were analyzed in three independent biological repetitions.The data represent mean permeabilization (%) ± SE from the population (*, p < 0.05; **, p < 0.01).Obtained data were analyzed using Student's t-test.The combination of PSZ and CA did not induce the increased permeabilization of the PM in most of the investigated strains of C. albicans when compared to cells treated with PSZ alone.The most vulnerable strains to this treatment were the ERG11 gene deletant KS058 and a double deletant of the FEN1 and FEN12 genes.This result is also supported by the decreased growth and adhesion of those strains in the presence of PSZ and CA (Figures 7-9).

C. albicans
We observed an altered morphology of the cells after treatment with a combination of PSZ and SU (different shapes of cells, elongation of cells, formation of cell clumps), but this did not always correspond with a significantly increased permeability of the PM.In the case of the fen12∆/∆ strain cultured with PSZ alone, and PSZ in combination with CA, we observed a mixed culture of intact cells and cells with disrupted PM integrity.The percent of permeabilized cells increased compared to under control conditions, but this change was not statistically significant.

Discussion
Discovering natural compounds that exhibit potential in azole combination therapy against fungal pathogens poses a challenge for modern medicine.One example of such compound is surfactin (SU), which is examined in this study.Tested concentrations of SU (4-32 µg/mL) did not inhibit growth in any of the tested C. albicans strains as an isolated treatment, with the exception of strain KS058 (erg11∆/∆) at 16 and 32 µg/mL of SU.There are several other azole potentiators that lead to the decreased growth of Candida spp.only when in combination with azoles, including 1,4-benzodiazepines and pitavastatin [18,19].Even supplementing fungal cultures with copper can lead to increased FLC activity, because azoles form a complexes with copper [20].On the other hand, most of the known azole potentiators exhibit fungistatic effects of their own (e.g., catechol, propolis and derivatives of berberine) [21].Nevertheless, it is worth investigating the mechanisms of action of these kinds of combinations on fungal cells.
In this study, we investigated whether changes in the C. albicans plasma membrane's (PM) sterol and sphingolipids (SLs) content would result in altered susceptibility to combination treatment with posaconazole (PSZ) and either SU or capric acid (CA).We have previously reported that SU and CA could be promising additives to conventional antifungals such as azoles [11,12,15,16].We used C. albicans strains with amino acid substitutions in Erg11p, which have previously been observed in azole-resistant Candida spp.[22,23].We also employed the C. albicans erg11∆/∆ (KS058) strain, which lacks ergosterol in the PM.Another group of strains key to our investigation consisted of C. albicans strains with the deletion of one or more genes involved in SLs biosynthesis.We elected to test combinations of PSZ with SU and CA on these specific C. albicans mutants due to the fact that sterols and SLs biosynthesis functionally interact, which can impact the physiology of the PM and fungal cells [24].
Our study indicates that a double substitution in the ERG11 Y132F,F145L in C. albicans leads to increased resistance to PSZ, compared to the WT strain (Figure 3).An increased concentration of SU resulted in a successive decrease in growth in all strains except for C. albicans KS058.This is due to the lack of Erg11p, which makes this strain resistant to PSZ.Only the highest concentration of SU (32 µg/mL) resulted in the complete growth inhibition of this strain.On the other hand, the C. albicans fen1∆/∆ and fen12∆/∆ strains exhibited increased resistance to PSZ alone at higher concentrations (0.0156-0.125 µg/mL), and to PSZ in combination with SU when compared to the WT strain (Figure 4).The FEN1 and FEN12 genes are involved in the elongation of the fatty acids of SLs in fungi; therefore, the deletion of those genes could affect PM structure, thereby reducing the effect of a combination treatment of PSZ and SU on cells.Moreover, C. albicans fen1∆/∆ was found to have an elevated level of ergosterol, which could contribute to its decreased susceptibility to azoles [21].
A combination of PSZ (0.0156-0.125 µg/mL) and an increasing concentration of CA resulted in the decreased growth of C. albicans strains 10C1B1I1, 27A5A33A and 9B4B34A compared to the WT strain (Figure 5).This proves that CA induces the synergistic activity with not only fluconazole, but potentially also other azole drugs (e.g., PSZ) [15].Similar observations were made in relation to the C. albicans strains with the deletion of one or more of the genes involved in SLs biosynthesis, despite their higher tolerance to PSZ (Figure 6).The addition of CA successively decreases the growth of those strains, and a combination of PSZ and CA at respective concentrations of 0.0078 µg/mL and 90.6 µg/mL corresponds with significantly reduced growth when compared to the WT strain.For the C. albicans strain fen1∆/∆;fen12∆/∆, treatment with 45.30 µg/mL CA resulted in total growth inhibition.
Interestingly, a combination treatment of PSZ and SU resulted in a significant reduction in the growth of C. albicans strains SC5314 (WT), 10C1B1I1, 27A5A33A and 9B4B34A (Figure 7).We can assume that this is the result of synergistic activity between these two compounds, because in the case of a C. albicans strain lacking Erg11p (target for azoles), we observed a decrease in growth rate, but the characteristic growth curve was maintained.The addition of both PSZ with CA resulted in a shift in the timing of the logarithmic phase of growth for all tested strains, with the exception of strain 9B4B34A, but they reached similar OD 600 at the stationary phase of growth with or without the treatment.Based on the collected data, we can assume that PSZ in combination with SU or CA has a fungistatic character, but that the effect of SU in enhancing this activity is more dramatic than that of CA.
The growth curves determined for C. albicans strains with deletions of genes involved in SLs biosynthesis reveal that fen1∆/∆, fen12∆/∆ and fen1∆/∆;fen12∆/∆ strains are more resistant to a combination of PSZ and SU, while strain SN95 (WT) exhibited no growth in the presence of these compounds (Figure 8).The growth curves for these strains are notably shifted in comparison to that of SN95, with a prolonged lag phase, a shifted logarithmic phase regarding control condition and a short stationary phase.The decrease in OD 600 in the late stages of incubation suggests the eventual death of cells treated with PSZ and SU.On the other hand, similar to C. albicans strains with altered sterols biosynthesis (Figure 7), a combination treatment with PSZ and CA corresponds with a shift in the logarithmic phase of growth of the investigated strains, which suggests fungistatic activity.The dramatic growth inhibition in the presence of PSZ and SU, which we recorded for strains SC5314, 10C1B1I1 and 27A5A33A (Figure 8) does not fully correspond with the initial data obtained during susceptibility testing (Figure 1).We assume that this is due to the agitation of cultures during growth curve monitoring, while for susceptibility tests the cultures were incubated stationary.SU, due to its amphiphilic structure, is known for reducing adhesion among pathogenic bacteria and fungi [25,26].Fatty acids with short alkyl chains such as CA also exhibit the ability to reduce adhesion and biofilm formation, and to kill pathogenic fungi [12,27].Moreover, the treatment of C. albicans with CA results in the decreased expression of a gene encoding key proteins involved in the adhesion of Hwp1p [16].Our findings confirm that SU or CA alone can lead to a significant decrease in the adhesion of C. albicans with altered sterols biosynthesis (Figure 9A).Taking into consideration that both SU and CA also reduce biofilm formation, this has broad applications for preventing these processes on abiotic surfaces (e.g., medical devices) [28,29].
Our investigations also indicate that combined therapy with PSZ and SU results in PM permeabilization in a broad range of C. albicans strains (Table 1), while simultaneously, culturing with PSZ and CA did not correspond with an increased level of PM permeabilization.The addition of CA to cultures did not result in greater PM permeabilization in comparison to cultures treated with PSZ alone, with the exception of the KS058 strain.In relation to PM permeabilization, we can assume that the combination of PSZ and CA has an antagonistic effect.On the other hand, the microscopic analysis revealed that the combination of PSZ and SU led to the formation of cell clumps.This was particularly pronounced in C. albicans strains 27A5A33A and 9B4B34A (Figure 10).Moreover, the addition of SU to cultures of C. albicans 10C1B1I1 culture resulted in filaments formation, which may be a stress response to the presence of this compound.
The synergistic activity of PSZ with SU or CA can be associated with a reduction in adhesion, which corresponds to interrupted cell proliferation and division.Moreover, the altered cell morphology linked to PM perturbations can lead to a significant reduction in biofilm formation, and, ultimately, a fungistatic effect.Therefore, SU and CA are promising compounds that could be used in combined therapy with azoles to combat fungal pathogens.

Strains and Growth Conditions
The C. albicans strains used in this study are listed in Table 2. Strains were pre-cultured in YPD medium (1% YE, 1% peptone, 2% glucose) for 24 h, at a temperature of 28 • C with shaking at 120 rpm.

C. albicans Susceptibility Testing
In order to determine the MIC values, a serial dilution of PSZ (0-0.125 µg/mL; original PSZ stock 1 mg/mL in DMSO) alone or in combination with SU (0-32 µg/mL; original SU stock 1 mg/mL in H 2 O dd ) or with capric acid (0-90.6 µg/mL; original CA stock 45.3 mg/mL in methanol) was prepared in YPD medium using sterile 96-well plates (Sarstedt, Nümbrecht, Germany) [17].Then, the YPD medium was inoculated with C. albicans strains at a final OD 600 = 0.01 and cultured overnight (28 • C, stationary).After this time, the OD 600 was measured using a plate reader (ASYS UVM 340 Biogenet, Cosenza, Italy).The negative control consisted of a serial dilution of the compounds in the same media, without C. albicans inoculation.The positive control (100% of growth) consisted of C. albicans cultures in YPD medium without the tested compounds.The experiment was performed in 3 biological repetitions.

C. albicans Growth Curves
To determine the growth phases in the presence of PSZ alone or in combination with SU or CA, the C. albicans strains were pre-grown in the YPD medium (28 • C; with shaking at 120 rpm) overnight.Then 100 µL of fresh YPD medium was added to the sterile 96-well plate (Sarstedt, Germany) and inoculated with C. albicans to the final OD 600 = 0.1.The OD 600 was registered for 24 h (or 48 h if indicated) in 1 h intervals using the Spark multimode microplate reader (Tecan, Männedorf, Switzerland) as described before [31].The experiment was performed with 3 biological repetitions.

Adhesion of C. albicans to Polystyrene
SU and CA were assessed as C. albicans adhesion inhibitors using 96-well plates (Sarstedt, Germany) using a previously described method [34].Firstly, the wells were preincubated with 100 µL of SU (4 µg/mL) and CA (45.3; 22.7 or 11.35 µg/mL, depending on the strain used) in a solution of PBS for 2 h at 37 • C (stationary).PBS buffer was used as a positive control.Next, the solutions were removed from the plate, and the wells were rinsed three times with PBS buffer.C. albicans suspensions in PBS were diluted to final OD 600 = 0.6.The prepared inoculum was transferred to a 96-well plate (100 µL) and incubated under the same conditions (2 h, 37 • C, stationary).After this time, all supernatants were removed, and the wells were washed three times with PBS to remove nonadherent cells.The remaining adhered cells were stained with 0.1% crystal violet solution (5 min, 25 • C), and then the wells were washed three times with fresh PBS.Then the PBS was removed, and dye was released by adding 200 µL of 0.05M HCl with 1% SDS to the isopropanol.Absorbance at 590 nm was registered using a plate reader (ASYS UVM 340 Biogenet, Cosenza, Italy).This experiment was performed in 6 repetitions.

Analysis of Plasma Membrane Permeabilization
To assess the effects of PSZ alone or in combination with SU or CA on PM permeability, we performed the propidium iodide (PI) staining of cells [17].C. albicans cultures treated for 24 h (YPD, 20 mL, 28 • C, 120 rpm) without tested compounds, with PSZ (0.0156 µg/mL), with PSZ (0.0078 or 0.0156 µg/mL, depending on the used strain) and SU (4 µg/mL), or with PSZ (0.0156 µg/mL) and CA (45.3; 22.7 or 11.35 µg/mL, depending on strain used), were centrifuged (5 min, 4500 rpm), washed twice with 0.9% NaCl and resuspended in fresh 0.9% NaCl, then adjusted to OD 600 = 0.3.The cells were stained with propidium iodide (4 µg/mL) and incubated at 25 • C for 5 min, in the dark, then washed twice with 0.9% NaCl (5 min, 4500 rpm), concentrated, and observed under a Zeiss Axio Imager A2 microscope equipped with a Zeiss Axiocam 503 mono microscope camera and a Zeiss HBO100 mercury lamp (Pozna ń, Poland).The percentage of permeabilization was evaluated by calculating PI-positive cells as a proportion of at least 150 cells in three independent replicates for each experiment (described as a population of cells).

Statistical Analysis
For data analysis, statistical significance was determined using a Student's t-test (binomial, unpaired).Data represent the means ± standard deviations (SD) of at least three biological replicates.In the case of the data presented in Table 1, the mean percentage of permeabilization was determined from the population (consisting of 3 independent biological replicates) and the standard error (SE) was determined according to the following formula: SE = p × q n where p = percent (%) of cells with permeabilized PM in the population; q = percent (%) of cells without permeabilized PM in the population; n = number of cells in population.
The data presented in Figures 3-6 and Figure 9 were prepared using GraphPad Prism 6.0.(GraphPad Software, San Diego, CA, USA).

Conclusions
The main conclusions of our research are as follows: • PSZ in combination with SU and CA leads to the decreased growth of C. albicans.The level of growth inhibition is dependent on the types of alterations in the ergosterol or SLs biosynthesis pathways of C. albicans strains;

•
The strain most susceptible to the combination of PSZ and SU is C. albicans that lacks the ERG11 gene.The most susceptible to the combination of PSZ and CA is the strain of C. albicans that lacks both FEN1 and FEN12 genes; • PSZ in combination with SU causes the significantly decreased adhesion of all investigated C. albicans strains.The combination of PSZ with CA does not lead to the impaired adhesion of C. albicans with the deletion of genes involved in SLs biosynthesis, and for C. albicans ERG11 gene mutants, the effect is less dramatic comparing to a combined therapy of PSZ and SU;

•
The combination of PSZ with SU and CA does not correspond with increased C. albicans PM permeabilization compared to control conditions.Nevertheless, the formation of cell clumps was observed due to the use of PSZ with SU and CA.Therefore, the synergy between PSZ and SU or CA is based on a reduction in the adhesion of C. albicans, rather than PM permeabilization.
Figure 3 represents the growth of C. albicans strains with the deletion of the ERG11 gene, or with amino acid substitutions in Erg11p, in the presence of PSZ, with or without SU.

Figure 5
Figure 5 represents the growth of C. albicans strains with the deletion of the ERG11 gene or with amino-acid substitutions in Erg11p, in the presence of PSZ, with or without SU.Int.J. Mol.Sci.2023, 24, x FOR PEER REVIEW 6 of 19

2. 2 .
Candida albicans Strains with Altered Ergosterol or Sphingolipids Biosynthesis Exhibit Diminished Adhesion to Abiotic Surfaces in the Presence of Surfactin or Capric Acid The adhesion to abiotic surfaces of all investigated C. albicans strains under control conditions, in the presence of SU (4 µg/mL) or CA (45.3; 22.7 or 11.35 µg/mL depending on C. albicans strain), is presented in Figure 9. Int.J. Mol.Sci.2023, 24, x FOR PEER REVIEW 10 of 19

Figure 10
Figure 10 presents representative microphotographs of C. albicans strains with deletions of the ERG11 gene, or with amino acid substitutions in Erg11p cultures stained with propidium iodide (PI).

Figure 10
Figure 10 presents representative microphotographs of C. albicans strains with deletions of the ERG11 gene, or with amino acid substitutions in Erg11p cultures stained with propidium iodide (PI).

Table 2 .
C. albicans strains used in this study.