Candidicidal Effect of Arginine-based Surfactants

doi:10.21203/rs.3.rs-2743675/v1

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Candidicidal Effect of Arginine-based Surfactants

https://doi.org/10.21203/rs.3.rs-2743675/v1

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published 29 Jun, 2023

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Amino-acid–based surfactants are a group of compounds that resemble natural amphiphiles and thus are expected to have a low impact on the environment, owing to either the mode of surfactant production or its means of disposal. Within this context, arginine-based tensioactives have gained particular interest since their cationic nature—in combination with their amphiphilic character—enables them to act as broad-spectrum biocides. This capability is based mainly on their interactive affinity for the microbial envelope that alters the latter’s structure and ultimately its function. In the work reported here, we investigated the efficiency of N^α-benzoyl arginine decyl- and dodecylamide against Candida spp. to further our understanding of the antifungal mechanism involved. For the assays, both a Candida albicans and a Candida tropicalis clinical isolates along with a C. albicans–collection strain were used as references. As expected, both arginine-based compounds proved to be effective against the strains tested through inhibiting both the planktonic and the sessile growth. Furthermore, atomic-force–microscopy techniques and lipid-monolayer experiments enabled us to gain insight into the effect of the surfactant on the cellular envelope. The results demonstrated that all the yeasts treated exhibited changes in their exomorphologic structure, with respect to alterations in both roughness and stiffness, relative to the nontreated ones. This finding—in addition to the amphiphiles’ proven ability to insert themselves within this model fungal membrane—could explain the changes in the yeast-membrane permeability that could be linked to viability loss and mixed-vesicle release.

synthetic amphiphiles

antifungal activity

amino-acid–based surfactants

Candida

arginine

mechanism of action

Fungal infections in humans are mainly produced in an immunocompromised host by opportunistic fungi that are usually found within its normal mycobiome (Nash et al. 2017). In most instances, this situation takes place during nosocomial stays in intensive-care units, including other circumstances such as central-venous-catheter use and the administration of broad-spectrum antibiotics. The prevailing agents for this kind of mycosis belong to the Candida genus (Saccharomycetaceae, Ascomycota, Saccharomycetales), with Candida albicans being the most common (d’Enfert and Bougnoux 2014; Ricotta et al. 2021). Other Candida species—such as C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii (d’Enfert and Bougnoux 2014; Mataraci-Kara et al. 2020) along with the emerging multiresistant-yeast pathogen Candida auris (Fu et al. 2020)—are gaining clinical significance, causing ca. 30% of the Candida infections in certain hospitals (Ricotta et al. 2021).

A proper disinfection of the nosocomial environment and medical devices plays a central role in preventing the spread of hospital-acquired infections, thus reducing the number of deaths and minimizing the related costs (Walker 2014). Within this context, the benefits of surface disinfection, the interruption of the transfer of pathogens to the human body, and the reduction of infection have been demonstrated by several studies (Fu et al. 2007). The chemical-disinfection formulations that are commercially available include agents such as sodium hypochlorite, peracetic acid, hydrogen peroxide, acetic acid, ethanol, and quaternary ammonium salts as the most commonly used active ingredients (Cadnum et al. 2017).

Surfactants, also known as amphiphiles, are compounds that have in common two main characteristics. Structurally, they contain two distinct portions: one moiety has a strong affinity for water or for polar environments—with that hydrophilic or polar constituent usually being referred to as the head—whereas the other portion is lipophilic—with the hydrophobic or apolar region usually being called the tail. This particular feature gives rise to the most remarkable physicochemical property of surfactants, namely their ability to lower surface tension. Other features and applications could be considered as consequences of that characteristic: for example, surfactants can be used as soaps, detergents, emulsifiers, wetting agents, and anticorrosives (Joshi 2017). Among this plethora of applications, certain kinds of surfactants, especially those bearing a cationic charge in the polar moiety, possess a remarkable biocidal activity. Cationic surfactants, such as quaternary ammonium salts (QACs), have been used as biocidal agents against bacteria, fungi, and viruses for almost a hundred years (Al-Adham et al. 2013, Hora et al. 2020). Simultaneous electrostatic and hydrophobic interaction with cell surfaces is thought to be their principal mode of action: surfactant molecules would intercalate into the cell-membrane lipid bilayer to increase its permeability thus causing cell lysis (Zhang et al. 2015b). Furthermore, cationic surfactants would diffuse to the cytoplasm to inactivate energy-producing enzymes and denature cell proteins (McKeen 2012). Consequently, the mechanism of action is quite unspecific and therefore, unlike antibiotics, the emergence of resistances against these kinds of compounds should be considerably less frequent. Nevertheless, efflux-mediated resistance to QACs has been reported, mainly due to the overuse of sublethal concentrations in preventing infections (Bjorland et al. 2005; Morrison et al. 2019). In this regard, the increase in multiresistant microorganisms has become a major concern: As of some time ago, the World Health Organization has been warning that, if no action is taken, drug-resistant diseases could cause 10 million deaths worldwide by as early as 2050 (Murray et al. 2022). Moreover, the COVID-19 pandemic created an ideal scenario through the unnecessary administration of antibiotics such as azithromycin to patients that developed pneumonia, as well as the overuse of QACs in disinfection to limit the spread of SARS-CoV-2, thus accelerating the emergence of antimicrobial resistance (Ghosh et al. 2021; CDC 2022).

In this complex scenario, the design and production of novel antimicrobial compounds is of paramount relevance. Since disinfectants are the agents in the front line of the defense against the transmission of infections, the development of alternatives can help to solve the antimicrobial-resistance problem by reducing the massive use of the existing antiseptics. A typical QAC, cetyltrimethylammonium bromide, has been found to be an effective fungicide against Candida albicans by inhibiting its growth, morphogenesis, and biofilm formation and maintenance (Yu et al. 2015). Despite this efficiency, the need for new alternative cationic surfactants is imperative because of the toxicity of QACs to mammalian cells (Zhang et al. 2015a) and the environment (Hora et al. 2020). To circumvent these disadvantages, certain strategies involve the design of surfactants that mimic the host defense peptides—also known as antimicrobial peptides or AMPs—a type of cationic amphiphilic peptide having an antimicrobial activity that is part of the innate immune system of almost all organisms (Zhou and Wang 2020). Among this type of compound, lipoaminoacids are distinguished as being natural molecules with simple architecture that serves as a model for synthetic surfactants (amino-acid–based surfactants) that are easy to design and produce (Pinazo et al. 2019). This group of compounds has two key advantages over other cationic surfactants: a production through sustainable processes and a lower ecotoxicity (Pinazo et al. 2016), both of which features turn such compounds into very attractive alternatives to QACs.

In particular, arginine-based surfactants have been demonstrated to possess both characteristics, as well as, in general, a low cytotoxicity. A representative of this type of compound is ethyl lauroyl arginate (also known as LAE or E243), an antimicrobial used as a preservative in food and cosmetics (Younes et al. 2019). The continuous development of related surfactants led to the production of new compounds with promising antimicrobial properties against, for example, resistant Candida spp. (Serpa Sampaio Moreno et al. 2021). Following this precedent, our research group obtained two arginine-based surfactants with substantial biocidal activity towards bacteria and fungi and a lower cytotoxicity than Cetrimide, a commercial QAC (Fait et al. 2015, 2019b). In the present work, we investigated the effect of N^α-benzoyl-L-arginine decyl- and dodecylamide (Bz-Arg-NHC₁₀ and Bz-Arg-NHC₁₂, respectively) on C. albicans and C. tropicalis—using a collection strain for the former and clinical isolates —by measuring their fungal-control action as well as the possible mechanism involved.

2.1. Chemicals and culture media

Dimethyl sulfoxide (DMSO), ergosterol (ERG), and poly-L-lysine were provided by Sigma-Aldrich and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (PC) and soy phosphatidylethanolamine (PE) by Avanti Polar Lipids. The rest of the chemicals used in this work were of the highest analytical purity commercially available.

Sabouraud dextrose broth (SDB), Mueller Hinton broth (MHB), and agar-agar were purchased from Britania (Argentina). Culture media were prepared according to the manufacturer’s instructions and sterilized with an autoclave for 15 min at 121 °C. Solid media were prepared by adding 1.5% (w/w) of agar-agar to the corresponding broth formulations.

2.2. Fungal isolates

Candida albicans ATCC 10231 from the American Type Culture Collection (ATCC, Manassas, USA) and clinical isolates of C. albicans and Candida tropicalis obtained from the local collection of the Mycology Laboratory of the Facultad de Ciencias Exactas at the Universidad Nacional de La Plata, were the species employed. All the isolates were maintained on Sabouraud dextrose agar (SDA) at 30 °C and 70% relative humidity.

2.3. Preparation of yeast suspensions

Yeast suspensions were obtained from 24-h–old cultures grown in SDB at 30 °C. Dilutions were made in sterile saline solution and the samples homogenized by vigorous agitation in vortex. The optical densities at 600 nm (OD₆₀₀) of the resulting suspensions were adjusted in the corresponding media indicated for each assay.

2.4. Surfactant synthesis

The hydrochloride salts of the arginine-based cationic surfactants (Bz-Arg-NHC_n·HCl, n = 10 or 12) were obtained in our laboratory as previously described with papain, an endopeptidase obtained from Carica papaya latex, adsorbed onto polyamide as a biocatalyst, and purified by means of cationic exchange chromatography (Fait et al. 2015).

2.5. Preparation of the surfactant solutions

Stock solutions of the surfactants (50 mM) were prepared in DMSO, sterilized by filtration, and stored at –20 °C. Dilutions of these stock solutions were prepared in sterile nanopure water, buffer, or culture medium as indicated in each assay.

2.6. Antifungal activity against planktonic cultures

2.6.1. Susceptibility testing: determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)

Candida’s susceptibility to the surfactants was studied according to the methodology described by the Subcommittee on Antifungal Susceptibility Testing of the European Committee for Antimicrobial Susceptibility Testing (AFST-EUCAST), with certain adaptations (Arendrup et al. 2012). Assays were carried out in 96-well flat-bottom microtiter plates (Extragene, Taiwan). Serial dilutions of the surfactant stock solutions were prepared over a range of concentrations from 15 to 1,000 µM, in sterile twofold-concentrated (2X) SDB as the solvent. In each well, 100 µL of the corresponding surfactant solution were added together with 100 µL of the yeast suspension prepared in sterile saline solution adjusted to OD₆₀₀ = 0.15 (1–5 × 10⁶ CFU/mL) as described previously. Sterility and growth controls only in the presence of SDB or SDB with different DMSO concentrations added were also included. All plates were sealed and incubated at 30 °C and 70 % of relative humidity for 24 h. Changes in the OD₆₀₀ were quantified using a multimode microplate reader (Infinite M200 Pro, Tecan). The presence of DMSO did not affect the growth of the isolates. For each surfactant concentration, the percent inhibition of growth, expressed as colony-forming units (CFU), with respect to that of control wells (GI%)—with the mean optical density of untreated cells established as 100% viability—was calculated for 24 h of incubation. With these data, the MIC values were established, upon consideration that the MIC was the lowest surfactant concentration able to completely inhibit yeast growth (GI% ≥99%). Samples of those wells that did not evidence visible growth were transferred to SDA plates. The presence of colonies was analyzed after incubating the plates at 30 ºC for 24–48 h. The MFC was defined as the lowest concentration of surfactant that completely inhibited yeast growth (absence of colonies). In all instances, the data were expressed as the mean ± the standard deviation (SD) of three independent replicate determinations.

2.6.2. Effect of Bz-Arg-NHC_n on pathogen survival

We first studied the pathogen survival in SDB supplemented with Bz-Arg-NHC₁₀ or Bz-Arg-NHC₁₂ at different concentrations. For both compounds, the corresponding MICs plus a twofold MIC (2 MIC) were tested. Overnight cultures of the pathogenic yeasts were diluted in sterile saline solution to a final concentration of approximately 1 × 10⁶ CFU/mL. Surfactant solutions prepared in sterile 2X SDB (2.5 mL) containing 2 mL of added sterile saline solution were inoculated with 500 µL of the yeast suspension. At preestablished time intervals (0, 3, 6, and 24 h), aliquots were removed, serially diluted in 0.1% (v/v) tryptone, and plated on SDA plates. Colony counts were performed after incubation at 30 ºC for 24 h. In all the experiments, the data were expressed as the mean ± SD of three independent replicate determinations.

2.7. Antibiofilm activity

2.7.1. Biofilm-formation assay

Different culture media were tested in order to optimize biofilm formation. The yeast suspensions used for these assays were prepared as indicated in section 2.3 after adjusting the OD₆₀₀to 0.2 with SDB, MHB, SDB supplemented with 8% (w/v) glucose (SDB 8% glu) and MHB supplemented with 2% (w/v) glucose (MHB 2% glu). For biofilm formation, 100 µL of the yeast suspensions were inoculated per well in a polystyrene flat-bottomed 96-well microtiter plate (JetBiofil, China). The plates were then incubated for 24 h at 37 °C in a humid chamber. Wells filled with sterile media (100 µL) served as negative controls.

2.7.2. Crystal-violet assay

Biofilm quantification was determined through the use of a crystal-violet (CV) assay (Dusane et al. 2011). After incubation, the biofilms were rinsed twice with phosphate-buffered saline (PBS) in order to eliminate planktonic cells. Once rinsed, the microplates were inverted onto paper and gently tapped to remove excess buffer. The biofilms were heat-fixed by incubating the plates for 30 min at 60 °C. The adherent biomass was then stained by adding a 0.1% (w/v) CV solution (200 µL) and then incubating for 10 min at room temperature. The staining solution was discarded and the biofilms lightly rinsed twice with distilled water (200 µL) to avoid detaching the biofilms. The excess water was drained off upon inverting the plate onto paper. Next, the CV bound to the biofilms was solubilized by adding 200 µL of 30% (w/v) acetic acid followed by incubation for another 15 min at room temperature. Upon solubilization of the CV, 150 µL of each well were transferred to a new plate and the absorbance at 595 nm was quantified with a multimode microplate reader (Infinite M200 Pro, Tecan). The data were expressed as the means ± the SDs of six independent replicate determinations.

2.7.3. Biofilm treatment and viability quantification

A C. tropicalis clinical isolate was chosen for these experiments on the basis of its strong biofilm-forming ability. Biofilms were obtained as described in section 2.8.1 in MHB 2% glu as the medium. After biofilm formation, the supernatant was removed and the biofilms rinsed twice with 150 µL of PBS in order to remove nonadherent cells. Bz-Arg-NHC₁₀ and Bz-Arg-NHC₁₂ solutions were prepared in MHB 2% glu at concentrations ranging from 1 to 12 times the corresponding MIC value. The biofilms formed were then treated with 150 µL of the surfactant solutions for 24 h at 37 °C. Biofilms incubated in the presence of 150 µL of sterile medium (MHB 2% glu) were included as negative inhibition controls. After incubation, the supernatants were removed and the biofilms rinsed twice with PBS. The biofilm viability was evaluated by a resazurin-based assay (Mahmoudabadi et al. 2014). To that end, 150 µL of fresh MHB 2% glu were added plus 15 µL of a resazurin solution (0.15 mg/mL) to each well. Resorufin formation was quantified by measuring the fluorescence emission at 590 nm (l_ex = 560 nm) after 1 h of incubation at 37 °C with a multimode microplate reader (Infinite M200 Pro, Tecan). For each surfactant concentration, the percent inhibition of biofilm viability was calculated relative to control wells (with the fluorescence emission of untreated biofilms having been established as 100% viability). The minimum biofilm-inhibitory concentration (MBIC) was established as the lowest surfactant concentration needed for a 95% reduction in biofilm viability (Macià et al. 2014). Furthermore, half-maximal–inhibitory-concentration (IC₅₀) values were determined after fitting each data set to a Boltzmann-type sigmoid curve by means of the OriginPro8^® software. In all instances, the data were expressed as the means ± the SDs from four independent replicate determinations.

2.8. Atomic-force–microscopy (AFM) experiments

2.8.1. Sample preparation

The planktonic-cell imaging was performed with C. albicans ATCC 10231 and the clinical isolate of C. tropicalis as described by Gonçalves et al. (2017). In these assays, 200 µL of each yeast suspension (10⁶ CFU/mL) were incubated with 200 µL of the surfactant solutions at the corresponding MFC prepared in 0.85% (w/v) NaCl for 30 min at 30 °C. Cells without any antifungal treatment, incubated with the sterile saline solution, were used as controls. After treatment, the cells were harvested by centrifugation (11000×g, 15 min), concentrated by removing 370 µL of the supernatant, and resuspended in the remaining volume. A 10-µL droplet of each test sample was applied onto a poly-L-lysine-coated coverslip and left at room temperature for 30 min. After deposition, the samples were rinsed with filtered deionized water and air-dried.

Untreated and treated samples were imaged by means of a MultiMode Scanning-Probe Microscope (Veeco, Santa Barbara, CA, USA) equipped with a Nanoscope V controller (Veeco, SantaBarbara, CA). Measurements were carried out in the contact mode, at room temperature, with cantilevers used having typical resonance frequencies of 145–230 kHz and nominal spring constants of 0.06 N/m. The scan rate was set to less than 1 Hz for imaging and the image resolution set to 512 × 512 pixels for all images. The height, the error signal, and phase-contrast images were recorded and line-fitted as required. From the images recorded, the height and size information was obtained by Nanoscope analysis version 5.2. Roughness analysis: The effect of the treatments on surface roughness was analyzed through the use of AFM and upon taking into consideration the surface roughness plus the surface-area difference as quantitative parameters. The AFM image and data analysis were performed by means of the Nanoscope 7.30 and the Nanoscope Analysis 1.5 software package. The results of this processing were analyzed statistically by the analysis of variances (ANOVA) and Bonferroni post-hoc tests.

2.8.2. AFM-Based Cell-Stiffness Measurements

To investigate the effect of arginine-based compounds on yeast-surface stiffness, 10 µL of cell culture were plated onto a poly-L-lysine–coated glass coverslip and left at room temperature for 30 min. Then the sample was treated with the corresponding amphiphile solution for 30 min followed by rinsing with deionized water to remove nonadherent cells and a final drying in an air stream for 5 min before removal to the AFM chamber for analysis.

All the measurements were performed with a multimode scanning-probe microscope (Veeco, Santa Barbara, CA) equipped with a Nanoscope V controller through the use of contact-sharpened silicon nitride probes (DNP-10, Veeco) having nominal tip radii between 20 and 60 nm. The cantilever spring constant (K_c) was determined for each probe before starting measurements by the thermal-tune method with planar muscovite mica (SPI grade V-1) as a flat, rigid surface for photodetector-sensitivity calibration, with the experimental values of K_c being between 0.08 and 0.13 N/m. To acquire the force-vs.-distance curves on the surface of the treated yeasts, the Force Volume (FV) tool was used with a routine of 32-×-32 force curves at a scan rate of 1 Hz. Note that different scanning rates within 0.1 and 2 produced similar results. A limit was set on the highest applied force (17 nN) in order to not damage the samples during FV acquisition.

2.8.3. Force-curve analysis, and Young’s-modulus determination, and nanoindentation analysis

This analysis was performed by means of the Nanoscope-Analysis software (version 1.5). The force-vs.-indentation curves obtained indicated a nonlinear response at low force. For the Young’s-modulus determination, curves were fitted by means of a Hertz model (Eq. 1) upon assumption of a conical point shape since a cone was the model that best fit our experimental data (Morilla and Romero 2015).

F = 2E tanα δ²/π (1-ν²) Eq. (1)

where F is the force, E is the Young's modulus, α is half the opening angle of the conical indenter (45°; based on the geometric characteristics of the tip and scanning-electron-microscopy observations), δ is the indentation, and ν is the Poisson's ratio, which is assumed to be 0.5 for soft biologic samples.

2.9. Monolayer experiments

Surface pressure (π) measurements were carried out with a NIMA Langmuir-trough Model 102M (NIMA Technology, Coventry, UK) with a Wilhelmy platinum plate as the surface-pressure sensor. The aqueous subphase consisted of PBS buffer prepared in ultrapure Milli-Q water (resistivity 18.2 MΩ.cm, Merck Millipore, Burlington, WI, USA). For insertion measurements, a ternary lipid mixture of PC/PE/ERG (2:1:1 molar ratio, based on the average fungal-membrane composition) prepared in chloroform was gently spread over the subphase surface until the desired initial surface pressure (π_o) was attained (Miñones et al. 2009). After waiting 15 min for complete solvent evaporation and π_o stabilization, a stock solution of Bz-Arg-NHC₁₀ or Bz-Arg-NHC₁₂ prepared in PBS was injected into the subphase bulk beneath the monolayers with a Hamilton microsyringe to give a final surfactant concentration of 30 µM. After the injection, the increment in surface pressure (∆π) was recorded as a function of time until a stable signal was obtained. The maximum ∆π produced (∆π_max) by the surfactants was measured for different π_o values of the lipid films, which enabled a determination of the maximum insertion pressure (MIP) from the ∆π-vs.-π_o plots by extrapolating the regression curves to the x-axis (Calvez et al. 2009). All the experiments were performed at 23 ± 1 ºC.

Numerous clinically relevant organisms have acquired resistance to a wide variety of drugs and biocides owing to the widespread misuse or abuse of antibiotics and other antimicrobial agents (Irfan et al. 2022). The example of Candida resistance is of particular concern, since the treatment of infections produced by these pathogenic yeasts relies on only a few antifungal drugs, such as fluconazole, amphotericin, and caspofungin (Meade et al. 2021). Within this context, the design of novel biocidal compounds having a nonspecific target, such as cationic surfactants—which compounds are supposed to alter mainly membrane integrity and its electrochemical potential (Serpa Sampaio Moreno et al. 2021)—could help minimize the spread of antimicrobial resistance.

Amino-acid–based surfactants have been demonstrated to be an attractive alternative to QACs because of their low environmental impact and moderate cytotoxicity (Pinazo et al. 2016). The Bz-Arg-NHC_n series, as representatives of this group, also exhibit substantial bactericidal and/or bacteriostatic and fugicidal and/or fungistatic activity (Fait et al. 2015, 2019b). In the experiments described below, the antifungal activity of these compounds against Candida isolates was tested and demonstrated.

3.1. Candida-susceptibility testing

Different types of cationic surfactants have evidenced significant yeasticidal effects, either alone or as active ingredients in different formulations (Obłąk et al. 2013; Paluch et al. 2018; Faustova et al. 2019; Mataraci-Kara et al. 2020). Despite the growing interest in the development of novel antimicrobial compounds demonstrated mainly by academia and industry (Guo et al. 2022), the investigation of the biocidal characteristics of amino-acid–based cationic surfactants against yeasts is scarcer. Both arginine-based cationic surfactants studied in the present work (Bz-Arg-NHC₁₀ and Bz-Arg-NHC₁₂) proved to have antifungal activity against the Candida isolates tested. Table 1 reveals that MIC and MFC values did not exhibit significant differences when both compounds were tested against the both the ATCC strain and a clinical isolate of C. albicans. As expected, the concentrations needed to kill the latter were slightly higher than those calculated for the ATCC strain. This result can be attributed to virulence differences among the two isolates, i.e., intraspecies heterogeneity: Whereas the ATCC strain is cataloged as a Biosafety-Level-1 microorganism—which are known to present a minimal potential hazard to laboratory personnel and the environment, but can consistently cause disease in immunocompetent adult humans—C. albicans clinical isolates are considered as one of the most pathogenic species of Candida (Sardi et al. 2013). A larger number of isolates should be tested, however, to prove this hypothesis. Finally, in the example of C. tropicalis, the MIC values were considerably lower and equal to the corresponding MFCs, evidencing that this isolate was more susceptible to both compounds.

Table 1. Inhibition of the growth of Candida isolates by the surfactants, expressed as the minimum inhibitory and fungicidal concentrations (µM)

Surfactant	*C. albicans*				*C. tropicalis*
	ATCC 10231		Clinical isolate		Clinical isolate
	MIC^a	MFC^b	MIC^a	MFC^b	MIC^a	MFC^b
Bz-Arg-NHC₁₀	62.5	62.5	62.5	125.0	31.3	31.3
Bz-Arg-NHC₁₂	31.3	125.0	31.3	250.0	31.3	31.3

^aLowest surfactant concentration that completely inhibited yeast growth (GI% ≥99%) after 24 h of exposure to the surfactants

^bLowest surfactant concentration able to kill yeast (absence of colonies) after 24 h of exposure to the surfactants

The antimicrobial properties of amino-acid–based lipopeptides have been reported to be significantly affected by parameters such as the amino-acid sequence; the cationic-charge density; and the overall physicochemical properties of the molecules, including the relationship between the hydrophobicity and the polarity (Pinazo et al. 2016). These parameters affect both of the following stages proposed for the interaction of cationic surfactants with the envelope of the microorganism: the initial penetration of the outer envelope by the surfactant molecules to establish electrostatic interactions between the negatively charged molecules of the microbial membrane and the amphiphile’s positively charged polar head plus the subsequent penetration of the hydrophobic alkyl chain into the hydrophobic core of lipid bilayers to modify membrane architecture and promote the leakage of intracellular constituents (Fait et al. 2019a). In view of these stages, the observation that the composition of the membrane directly influences the antimicrobial activity of this type of molecules is hardly surprising: Certain authors have reported a reduced antifungal activity for cationic surfactants compared to their antibacterial effect, which difference was attributed to the lower negative-charge density at the fungal-cell membrane (Pinazo et al. 2016). In this regard, whereas the Gram-positive bacterium S. aureus contains around 58 mol% phosphatidylglycerol and 42 mol% cardiolipin, E. coli (Gram-negative) has anionic components in its inner membrane at only 26 mol%; where this figure is around 30 mol% for C. albicans (Fa et al. 2022). Nonetheless, we found no significant differences between the antifungal efficiency and the previously reported antibacterial activity of our amino-acid–based compounds (Fait et al. 2015).

As to the anticandidal activity of arginine-based surfactants, certain examples found in the literature were reviewed and compared with our results. The MIC values reported for the N^α-dodecylarginine methyl ester hydrochloride and the arginine N-dodecylamide dihydrochloride against C. albicans isolates were slightly higher than those obtained for Bz-Arg-NHC₁₂; whereas Bz-Arg-NHC₁₀ exhibited an enhanced anticandida activity compared to its analogues—i.e., ACA and CAM (Morán et al. 2001). In addition, an MIC value of 15.6 µM was found for C₃(LA)₂—the gemini surfactant bis-(N^α-caproyl-L-arginine)-1,3-propanediamide dihydrochloride (Coronel-León et al. 2017), a value comparable to those found for the Bz-Arg-NHC_n series. C₃(CA)₂ evidenced an MIC value of 166 µM against C. albicans. Of relevance was that, in the same study, the authors found that chlorhexidine—the reference biguanide disinfectant with no surface activity—had an MIC 55 µM for the same microorganism (Castillo et al. 2006).

Recent investigations have described the antifungal activity of N-acyl arginine methyl ester hydrochloride (AAM) and arginine N-alkyl amides di- (AAA) surfactants against different Candida strains (Pérez et al. 2021). Those investigations demonstrate that these amphiphiles exerted a high inhibition of all the yeast strains tested, including both C. albicans and C. tropicalis. Regarding the previously reported influence of the length of the surfactant’s alkyl moiety (Tot et al. 2022)—i.e., the longer the alkyl moiety, the more biocidal the effect—twelve-carbon analogues were more efficient against C. albicans than those bearing alkyl moieties with ten carbons among the AAM and AAA series of surfactants (Morán et al. 2001). No clear tendency had been found, however, for Bz-Arg-NHC_n in this regard.

According to Pérez et al. (2021), compounds with MFC values equal to or twice their MIC values can be listed as fungicidal agents. Therefore, those authors classified the arginine-based surfactants studied in their work as fungicidal compounds; on the basis of this concept, Bz-Arg-NHC₁₀ can be included within this group. The results reported by those authors also suggested an antagonistic effect of the amino and guanidinium groups on the antifungal behavior of the arginine-based tensioactives studied: therefore, the presence of the amino group may partially neutralize the anticandida action of the guanidinium group (Pérez et al. 2021). This effect seemed to be compensated in the example of our compounds, since the benzoyl group attached to the amino-acid moiety increases the molecule’s hydrophobicity (Hermet et al. 2021). This greater hydrophobicity becomes relevant in view of the previously discussed mode of action of cationic surfactants: hydrophobicity enhances the molecule’s affinity for the membrane phase, thus improving ability of the former to insert itself within the lipidic regions (Fonseca et al. 2010).

Other arginine-based surfactants, such as the double-chained N-lauroyl arginine N-alkyl amide hydrochloride series (LANHC_n, n = 10, 12, 14, or 18), were also tested against C. albicans (Pinazo et al. 2016). Whereas LANHC₁₂ and LANHC₁₈ had no activity, the MIC values obtained for LANHC₁₀ and LANHC₁₄ were 68 and 243 µM, respectively, with LANHC₁₀ manifesting an antifungal activity similar to that of the Bz-Arg-NHC_n.

According to the literature, the MIC/MFC values reported for the standard QAC cetyl trimethyl ammonium bromide were comparable to those found for our compounds (Nicoletti et al. 1993). Furthermore, benzalkonium chlorides (BACs)—another family of QACs widely used as disinfectants in many pharmaceutical formulations—also exhibited an inhibitory activity similar to that of Bz-Arg-NHC_n towards C. albicans upon comparison of those BACs of the same alkyl length—i.e., C₁₀ and C₁₂(Brycki et al. 2017). Of particular interest to us was that Bz-Arg-NHC_n proved to be more efficient than the sulfates of piperidinium- and morpholinium-based surfactants, whereas the MIC values reported for the chlorides were of the same order (Wieczorek et al. 2020). Similarly, Bz-Arg-NHC_n evidenced MIC results comparable to the most biocidal gemini QAC derivative of 1,4,3,6-dianhydro-L-iditol upon testing against Candida spp. as reported by Sikora et al. (2022); within this context, we need to remark that the most active gemini QAC obtained by the group has two alkyl chains of C₁₀.

Other amino-acid–based surfactants have been investigated for biocidal activity against different Candida species. Those compounds evidenced a comparable effect to that observed for Bz-Arg-NHC_n. In this regard, Perinelli et al. (2019) reported the synthesis of quaternary-ammonium surfactants based on Leu and Met, bearing alkyl moieties with ten, twelve, and fourteen carbon atoms (C₁₀, C₁₂, and C₁₄); all those compounds manifested a better performance than benzalkonium chloride against C. albicans. The MIC values found for the cationic histidine-based surfactants N(π) and N(t)-bis(methyl)-L-histidine n-decyl amide (DMHNHC_n) against C. albicans were 64 µM for C₁₂ and 35 µM for C₁₄(Bustelo et al. 2017). Pinazo et al. (2019) reported MIC values in the same pattern for the gemini histidine-based surfactants C₃(DMHNHC_n)₂ of 10 and 18 µM for C₁₀ and C₁₂, respectively. For the lysine derivative N-lauroyl lysine methyl ester (LKM), however, Candida inhibition was found at 169 µM (Pérez et al. 2009), with results of the same order being noted for other lysine-based surfactants with the general formula (C_n)₂-KKKK-NH₂ (n = 8, 10, 12, 14, or 16) upon testing against C. albicans and C. tropicalis (Greber et al. 2014).

We also need to point out that arginine is likewise a key amino acid in the composition of the so-called antimicrobial peptides or AMPs. As stated above, this family of peptides is present in all living organisms as part of their natural immune system (Pinazo et al. 2016). The net cationic charge seems to be a critical attribute responsible for the antimicrobial effect since the proposed mechanism of action resembles that of cationic surfactants: AMPs interact with the negatively charged molecules present in the cell envelope of pathogens—including both the lipopolysaccharides and lipotechoic acid in Gram-negative and Gram-positive bacteria, phosphatidylinositol in fungal cells, and anionic phospholipids in cell membranes in general (Makovitzki and Shai 2005)—thus penetrating down to the plasma membrane, where the AMPs promote global bilayer destabilization and/or membrane-spanning–pore formation (Wimley and Hristova 2011). Within this context, Yang and collaborators (2018) designed several AMPs by the bioinformatics optimization of a natural one. By this means, the authors obtained an arginine-rich peptide with higher biocidal activity than the natural AMP. The rationale for this advantage could be related to the guanidinium group present in the arginine residue—which is positively charged at a wide range of conditions and facilitates electrostatic interactions in three possible directions, representing a clear advantage over other basic–amino-acid residues such as lysine (Yang et al. 2018). These examples would explain why arginine-based surfactants are the most widely studied amino acid-based compounds.

The antifungal-activity results obtained for Bz-Arg-NHC_n in the present experiments encouraged us to evaluate the killing kinetics (Fig. 1) in order to monitor the antimicrobial effect over time. Since biocidal activity is defined as a 99.9% killing of the initial inoculum—a reduction greater than 3 log₁₀-fold in colony-forming units—we assumed from the results depicted in Fig. 1 that after 1 h of contact, Bz-Arg-NHC₁₀ would produce the appropriate reduction in the initial inoculum when a concentration of 2 MIC for C. albicans ATCC 10231 and C. tropicalis was tested. With clinical isolate of C. albicans, reductions of higher than 3 log₁₀ were reached between 4 and 8 h of exposure to 2 MIC. A notable result was that Bz-Arg-NHC₁₂ proved to be not as effective as the C₁₀ homolog, especially when assayed against both isolates of C. albicans. The reason for this lesser activity could be explained by the concentration of the surfactant chosen for this test being 2 MIC: That level was insufficient to kill either strain of C. albicans since the MFCs obtained for C. albicans ATCC 10231 was 4 times and for the clinical isolate of C. albicans 8 times this MIC. Nevertheless, C. tropicalis was more susceptible to Bz-Arg-NHC₁₂: this compound produced the 99.9% reduction in the initial inoculum after approximately 3 h of contact at both concentrations tested. The susceptibility of C. tropicalis to Bz-Arg-NHC_n can be clearly seen for all the concentrations tested, an expected sensitivity since the MFC values matched those of the MIC.

In this regard, the commercial cationic disinfectant benzalkonium chloride (BAC)—with a chemical structure comparable to that of our compounds—demonstrated a yeasticidal variability. The study performed by Mataraci-Kara et al. (2020) involving Candida species isolated from hospital wastewater revealed that a 0.01% (w/v) BAC solution (280 µM) was unable to kill 99.9% of the initial inoculum after 30 min contact for almost all the isolates tested. Formerly, Ohta et al. (1996) had analyzed the fungicidal activity of BAC solutions and found yeasticidal effects on C. albicans and C. tropicalis isolates after 15 min of exposure at concentrations equal to 0.2 and 0.4% (w/v)—6 and 12 mM, respectively (Ohta et al. 1996). Of relevance was that a comparison of the biocidal activity of 0.5% (w/v) solutions of BAC and Cetrimide against clinical isolates of three Candida species revealed that both QAC solutions were fungicidal for C. parapsilopsis and C. krusei after 60 min of contact; whereas BAC was able to kill C. albicans, where Cetrimide was inefficient (Gupta et al. 2002). In view of these results—though the treatment times were different—our compounds evidenced a most promising performance.

3.2. Antibiofilm assays

Candida spp. can proliferate as adherent biofilms, which property is considered a major virulence factor since those fungi exhibit an enhanced resistance to antifungal drugs and host immune responses (Eix and Nett 2020). The biofilm morphology and density may vary among different Candida species; but the structure is uniformly conserved, being characterized by a dense polymeric extracellular matrix—with a distinctly different composition from that of the cell wall—that encases and protects the microbial cells, thus acting as a physical barrier for antifungal therapeutics (Cavalheiro and Teixeira 2018). These fungal communities can lead to the emergence of metabolically dormant cells—called persisters—which exhibit a substantial tolerance to high doses of antimicrobials and therefore become a real problem for candidiasis treatment since those cells are capable of producing recurring infections (Galdiero et al. 2020). Considering this scenario, we measured the biofilm-forming capability of the three Candida strains tested in this study by means of a crystal-violet–staining assay (Fig. 2). A quantification of the biomass indicated that all the isolates were able to form biofilms in vitro in all the media tested, though the addition of glucose was found to enhance biofilm formation in some instances. The clinical isolate of C. tropicalis evidenced a strong biofilm-producing ability, and therefore this isolate was chosen for further experiments aimed at evaluating the performance of Bz-Arg-NHC_n in disrupting preestablished biofilms.

After a 24-h treatment of the biofilms with the compounds, the viability was quantified by a resazurin-staining assay (Fig. 3). The results indicated that both arginine-based surfactants exerted an antibiofilm effect, with IC₅₀ values of 124 µM (almost 4 × MIC) and 49.1 µM (1.57 × MIC) for the 10- and 12-carbon derivatives. The experiments also enabled us to establish minimum–biofilm-inhibition–concentration (MBIC) values—i.e., the lowest surfactant concentration needed for a 95% reduction in biofilm viability after a 24-h treatment—for C. tropicalis sessile growth (Table 2). As expected, all the parameters calculated were significantly higher than the corresponding MIC values obtained for the planktonic cultures: The MBICs were established at 5 and 7 times the corresponding MICs for Bz-Arg-NHC₁₀ and Bz-Arg-NHC₁₂, respectively. These results are consistent with the observation that Candida biofilms are usually 30- to 2,000-fold more resistant to antifungal agents than planktonic cells (da Silva et al. 2020). Despite the lack of any certainty about the mechanisms of Candida’s biofilm resistance, the literature suggested that the latter could involve restrictions in drug penetration and diffusion through the biofilm matrix (Gilbert et al. 2002). Notwithstanding, the high surface activity and the positive charge present in our cationic surfactants can contribute to overcoming this barrier through a destabilization of the biofilm’s architecture: Amphiphilic cationic molecules are generally accepted as being able to penetrate the biofilms’ structure more easily than noncharged compounds and thus dislodge the biofilm to expose sessile cells to the toxicity of the antimicrobial agent (Carrillo et al. 2003).

Table 2. Effect of Bz-Arg-NHC_n on C. tropicalis biofilms expressed as the minimum biofilm-inhibitory concentrations* (MBIC) and the corresponding IC₅₀ values in µM

Surfactant	*C. tropicalis* clinical isolate
Surfactant	MBIC^a	IC₅₀^b
Bz-Arg-NHC₁₀	156.5 (5×MIC)	124.3 (3.97×MIC)
Bz-Arg-NHC₁₂	219.1 (7×MIC)	49.1 (1.57×MIC)

^aThe lowest surfactant concentration needed for a 95% reduction in biofilm viability after 24 h of exposure to the surfactants

^bHalf-maximal inhibitory concentration after 24 h of exposure to the surfactants

Although potential biofilm disruptors exist, little background information is available regarding the study of the effect of amino-acid–based surfactants on the ontogeny of fungal and bacterial biofilms. On this subject, Obłąk et al. (2013) reported that the gemini quaternary ammonium salt TMPG-10 Cl exhibited an optimal antibiofilm activity against C. albicans—about a 60% disruption—at its MIC (80 µM). This 10 carbon-homolog was also found to exert the most potent antifungal activity for planktonic growth also. Nevertheless, an additional increase in the concentration of the compound did not cause any further eradication of the biofilm.

Furthermore, da Silva and coworkers (2020) have recently studied the antibiofilm activity of arginine and lysine-based mono- and dirhamnolipids with alkyl chains of 10 carbon atoms against resistant Candida spp. (da Silva et al. 2020). They observed biofilm disruption by the arginine derivatives at doses greater than the correspoding MIC values in a concentration-dependent fashion. Monorhamnolipid arginine derivatives (monoRL_Arg) reduced C. albicans- and C. parapsilosis–biofilm viability by about 50% at an approximate concentration of 2 × MIC. This effect was enhanced at a concentration of 10 × MIC, with percent–viability-inhibition values higher than 70%. Candida tropicalis biofilms were also affected by these compounds: a 50% inhibition was found at a concentration equal to the corresponding MIC, increasing up to almost 70% at 2 × MIC. A similar pattern was noted for Bz-Arg-NHC₁₂ at twice its MIC. By comparison, both of our compounds proved to be more effective than monoRL_Arg: an 80% inhibition was reached at 3 and 4 × MIC for Bz-Arg-NHC₁₂ and Bz-Arg-NHC₁₀, respectively, whereas an almost complete lethal effect occurred when biofilms were treated with a 7- or 5-x-MIC dose, respectively. In contrast, monoRL_Arg evidenced an almost complete inhibition only when a 10-x-MIC dose was tested.

Moreover, Luczynski et al. (2012) studied the antibiofilm activity of two gemini bis-quaternary surfactants based on bis-alanine esters (TMPAL-nBr, n = 10 or 12). Between those two compounds, the one with alkyl moieties of twelve carbon atoms demonstrated a higher antiadhesive activity and a stronger dislodging capability of C. albicans biofilms, but the ten-carbon homolog exhibited an enhanced antifungal activity in planktonic cultures. The authors used C. albicans strains with deletions of the genes encoding ABC transporters as model systems. Those loci encode a family of transporter proteins that are responsible for drug resistance and low biopenetration by pumping a variety of drugs out of cells with ATP hydrolysis as an energy source (Sipos and Kuchler 2006). Those findings suggested that, in contrast to the observations for TMPAL-12Br, TMPAL-10Br, antifungal activity did not depend on the presence or absence of the ABC transporters.

3.3. Fungicidal mechanism

3.3.1. AFM measurements

Antimicrobial treatments usually can induce morphologic changes in treated fungal cells. Taking this likely symptom into account, we used AFM imaging to evaluate the changes occurring in the exomorphology of the planktonic cells of C. albicans ATCC 10231 (Fig. 4) and C. tropicalis (Fig. 5) after 30 min of incubation with Bz-Arg-NHC_n at the corresponding MFC. Figs. 4 and 5 illustrate representative images. Untreated control cells (Figs. 4 and 5, panels a, d and g), contained a smooth surface and regular shapes After a 30-min treatment, some small irregularities on the cell surface were found, and additional material was deposited over and around the cells. This exudate could result from a release of intracellular content (Gonçalves et al. 2017) or via a redeposition of lipid-detergent mixed micelles shedded from the plasma membrane after the bilayer became saturated by the surfactant-monomers that had been inserted (Fait et al. 2017).

Of all the treatments, C. albicans ATCC 10231 exposed to Bz-Arg-NHC₁₂ evidenced the most extensive cell deformation (Fig. 4, panels c, f, and i) since the cells appeared swollen with increased diameters and heights (Fig. 6) and their surfaces containing irregularities, as evidenced from the roughness measurements (Fig. 7, Panel a). In the example of the clinical isolate of C. tropicalis, the cell size seemed to be less affected by the Bz-Arg-NHC_n at the concentrations tested, and the cell exomorphology exhibited only some slight changes after 30 min of incubation in the presence of the twelve-carbon homologs (Fig. 5 panels c, f and i). Although an increase in cell size is apparent in the AFM images, the results were not statistically significant (Fig. 6).

Webb and coworkers (2012) proposed the use of statistical parameters as a standard for describing the nanoarchitecture of a surface. In particular, surface roughness and surface-area differences can give an overall description of both dimensions of a surface—i.e., the vertical and the horizontal (Zilli et al. 2022). Taking these parameters into account, we analyzed the surfaces of the treated yeasts calculating both values in order to assess the effect of the surfactants on the cell envelope. While the surface roughness and the surface-area difference increased on C. albicans ATCC 10231 treated with Bz-Arg-NHC₁₂ (Fig. 7, Panel a), the opposite effect occurred with the clinical isolate of C. tropicalis treated with both surfactants, with the cell surface appearing to become smoother (Fig. 7, Panel b).

Furthermore, changes in the cell stiffness were assessed through determination of the Young’s modulus by means of AFM-based force spectroscopy (Fig. 8). Only Bz-Arg-NHC₁₂ evidenced a significant effect on cell stiffness after treatment of both isolates tested. According to these observations, the measurements suggested that changes in this parameter under these conditions depended on the Candida strain analyzed: whereas only a 73% reduction in the cell initial stiffness was obtained for C. tropicalis, an almost 3-fold increment was observed with C. albicans.

On the basis of these results, even though the ten-carbon homolog proved to be more effective when the kinetics of lethality were analyzed (Fig. 1), the AFM experiments documented that Bz-Arg-NHC₁₂ induced greater exomorphological alterations in the yeasts after treatment under the conditions tested.

As previously described, cationic agents can produce membrane distortion and protoplast lysis under osmotic stress, also affecting the regulation and normal functioning of membrane proteins. The membrane of yeasts, though, is additionally surrounded by a glucan-enriched hydrophobic cell wall, also composed of chitin and mannoproteins (Chaffin 2008). While the outer layer of that envelope acts as an impermeable barrier to macromolecules, the inner layer is more permeable and provides mechanical strength to the cell wall (Walker et al. 2018). The envelope, as such, can easily enable binding, but may interfere with the penetration of the antimicrobial amphiphiles by blocking them from reaching the cytoplasmic membrane (Fa et al. 2022). Nevertheless, the cell wall exhibits viscoelastic properties that facilitate the transit of intact vesicular structures—such as AmBisomes—through the chemically diverse matrices that compose its structure (Walker et al. 2018). That these vesicles are larger than the theoretical cell wall pores suggests that the cell wall can undergo an instantaneous remodeling, which alteration may also be a mechanism for the release of extracellular vesicles. Furthermore, lipid-surfactant mixed microvesicles might become released from the plasma membrane, as has been previously discussed (Fait et al. 2017). A transient accumulation of these vesicles beneath and outside the cell wall could also explain the irregularities and protuberances observed to be present in the cell’s surface, such as those observed by means of AFM imaging. This phenomenon could be pictured by comparing the cell wall to a tight and elastic carpet covering these zones of the damaged plasma membrane, including the shedded micelles and reproducing its topographic irregularities. Furthermore, apoptosis can induce the production of extracellular vesicles; while, under such stressful conditions, the pore size in the yeast cell walls could increase, thus enabling the shedded microvesicles to traverse the cell wall (Brown et al. 2015). Nevertheless, further studies would be necessary to confirm whether the proposed yeasticidal mechanisms definitely involve mixed-vesicle shedding.

Differences in the structure and composition of the cell wall have been described among different Candida species. In fact, C. tropicalis was reported to have a higher content of phosphomannan within its cell wall than that found for C. albicans as well as a significantly higher porosity (Navarro-Arias et al. 2019). These differences in the composition may lead to distinctive responses upon surfactant transit through the cell-wall matrix, therefore contributing to the differences observed in the parameters tested in both strains after treatment with Bz-Arg-NHC₁₂, since no clear tendency was found.

Studies involving QACs evidenced that those compounds can cause cell death by protein denaturation plus disruption of cell-wall permeability and membrane transport (Simões et al. 2005). These effects may also alter the regulation and functioning of membrane-bound enzymes, including those involved in the synthesis of the cell-wall–polysaccharide components previously mentioned (Tyagi and Malik 2010). Paluch and collaborators (2021) used transmission electron microscopy (TEM) imaging to study the changes in the morphology of C. albicans cells that are influenced by the cationic dicephalic surfactants: C_n(DAPACl)₂ and C_n(TAPABr)₂ respectively, with n = 14 or 16. Those authors reported that the QAS derivatives induced a significant cell-wall thickening as well as membrane deformations along with lipid-droplet formation in the cytoplasm, acting also as oxidative-stress stimulants. Bz-Arg-NHC_n was previously found to induce both membrane permeabilization and induction of oxidative stress as part of their antifungal mechanisms (Fait et al. 2018). The combination of these effects may also alter the cell wall, as evidenced in the cell-roughness and stiffness changes in the Bz-Arg-NHC_n–treated yeasts. These results are in agreement with those observed for the action of LAE against both fungi and bacteria. Recent investigations have used TEM to document that the cell wall of spores of Penicillium digitatum treated with LAE became rough and contained cavities (Xu et al. 2018). In addition, organelles were affected, and the cells underwent plasmolysis as the protoplast shrank. This response also suggested a leakage of the intracellular components—as empty spaces appeared within the treated spores—indicating changes in membrane permeability. By contrast, Pectobacterium carotovorum subsp. carotovorum (a Gram-negative bacterium) exhibited a thinning in its cell wall upon treatment with LAE in addition to the previously described effects observed for P. digitatum. Published evidence therefore indicates that the biocidal mechanism of arginine-based surfactants, in addition to a disruption of the plasma membrane, may involve changes in the cell-wall composition and structure (Tyagi and Malik 2010). Nevertheless, further studies would helpful in validating that those alterations do take place in the treated yeasts.

3.3.2. Monolayer-penetration experiments

The adsorption of cationic surfactants on the microbial-cell surface would be expected to be enhanced by electrostatic interactions involving the negative charge of the cell’s envelope (Pérez et al. 2022). Once surfactants have penetrated the cell wall, they can interact with the cytoplasmic membrane to exert their fungicidal effect and, eventually, reach the cytoplasm.Previously published results (Fait et al. 2019b) and preliminary experiments (data not shown) indicate that, although cellular lysis is not evident, membrane integrity is compromised in fungi and yeasts exposed to Bz-Arg-NHC_n—as demonstrated by the uptake of propidium iodide. This evidence encouraged us to study the interaction of our arginine-based surfactants with lipid monolayers as yeast model membranes with respect to to their antifungal mechanism against Candida sp.

The Candida cell membrane is mainly composed of zwitterionic lipids—e.g., phosphatidilcholine and PE—constituting up to 80% of the fungal phospholipid pool. The remaining 20% consists of anionic phospholipids, such as phosphatidylserines (PSs) and phosphatidylinositols (PIs). ERG is also usually present in yeast membranes, where the content may fluctuate from almost 0 to over 50% of the entire lipid pool depending on the species, the fungal-development state, and the evironmental conditions (Perczyk et al. 2020). Taking these features into account, we investigated the interaction of Bz-Arg-NHC_n with fungal model membranes using monolayers composed of PC/PE/ERG—a lipid mixture proposed to mimic the major composition of Candida’s membranes (Schneiter et al. 1999).

During the experiments, surfactant solutions (30 µM final concentration) were injected into the aqueous subphase beneath the monolayers compressed at different initial surface pressures (π_o) and the increment in π, ∆π, analyzed over time. Fig. 9illustrates representative curves obtained after the insertion of Bz-Arg-NHC_ninto the lipid monolayersat a π_o of 10 and 25 mN/m. When the surfactants were injected into the bulk subphase, an increase in the surface pressure of the films was detected, evidencing the penetration of the compounds into these model membranes. Differences in surface-pressure kinetics were detected after the insertion of each compound. In the example of Bz-Arg-NHC₁₂, the surface pressure increased until a maximum value (∆π_max) was attained (Fig. 9, Panel b), while Bz-Arg-NHC₁₀ produced a slight decrease in π after reaching the ∆π_max(Fig. 9, Panel a), thus suggesting a reorganization of the monolayers after the incorporation of this compound.

At the π_o assayed, the injection of Bz-Arg-NHC₁₀ produced a higher ∆π_max than Bz-Arg-NHC₁₂, (Fig. 9, panels a and b). In all instances, as expected, the resulting ∆π was dependent on the π_o of the films since the incorporation of the compounds into the monolayers is usually prevented when the lipid-packing density increases—i.e. at a higher π_o (Calvez et al. 2009). From the plots of ∆π_max vs. π_o, the MIP was calculated by extrapolating the regression curves to the x-axis (Fig. 9, Panel c). This parameter corresponds to the surface pressure beyond which no further incorporation of the surfactants into the monolayers would occur (i.e., a ∆π = 0) and is proportional to the penetration capability of the compounds (Calvez et al. 2011). The MIP values obtained for Bz-Arg-NHC₁₀ and Bz-Arg-NHC₁₂were 51.5 ± 6.6 and 36.6 ± 5.7 mN/m, respectively. These results indicated that both compounds were able to insert themselves within the yeast model membranes, even at high surface pressures of the lipid films. MIP values were higher than 30 mN/m—the average π usually considered representative of the lipid packing in biologic membranes—suggesting that both surfactants would be able to insert into fungal-cell membranes in vivo and that this capability would be an integral part of the antifungal mechanism to dismantle the yeast’s envelope structure (Marsh 1996). Furthermore, the results reinforce the conclusion of a stronger yeasticidal effect of Bz-Arg-NHC₁₀ observed throughout this work.

Studies involving the interaction of Bz-Arg-NHC₁₀ with dipalmitoylphosphatidylcholine (DPPC) model membranes had been carried out previously in order to explain the enhanced antibacterial activity of this compound (Hermet et al. 2021). Those experiments enabled the calculation of the MIP for DPPC monolayers at the same surfactant concentration used in the present work (30 µM). Bz-Arg-NHC₁₀also became inserted into these monolayers, at an MIP value of 59.6 mN/m, one of the same order of magnitude as the MIP found for the yeast model membranes. Studies with DPPC monolayers and bilayers had also suggested that, once incorporated into the lipid film, Bz-Arg-NHC₁₀ may reach a concentration high enough to induce the formation of mixed micelles or vesicle release, thus promoting a lipid removal from the model membranes (Nitenberg et al. 2018). This activity was also evident for the same compound when the PC/PE/ERG mixture was employed (Fig. 9, panel a), in support of the hypothesis proposed above involving microvesicle shedding as part of the antifungal mechanism.

The antifungal activity of Bz-Arg-NHC_n was demonstrated against different Candida isolates. Arginine-based surfactants proved to have a biocidal effect against both planktonic and sessile growth, with an efficacity similar to that of other cationic surfactants commonly used as disinfectants. That effectiveness of our compounds against Candida biofilms is especially promising, since those communities are considered to be a fundamental virulence factor and one of the main causes of recurring infections.

The differing responses to the surfactants upon the isolates tested would arise from differences in the architecture and composition of the cellular envelopes. Nonetheless, alterations in the plasma-membrane structure and permeability before cellular lysis seem to be a common occurrence in all instances. The presence of secreted material over and around the cells might be related to the redeposition of lipid-detergent mixed vesicles shedded from the plasma membrane. The accumulation of these microvesicles beneath and outside the cell wall could also explain the irregularities observed in the cell surface (Fig. 10). These results along with exomorphologic changes observed among the treated yeasts, involving both roughness and stiffness alterations, support the antifungal mechanism we proposed for Bz-Arg-NHC_n against Candida. On the basis of the results presented here and the accompanying discussion, the rationale for the antifungal effect of arginine-based surfactants could finally be explained by two fundamental activities: a) a detergent-like action on the plasma-membrane lipids that induces the shedding of mixed micelles and microvesicles and b) the resulting apoptosis caused by the oxidative stress produced by Bz-Arg-NHC_n. Nevertheless, further experiments would be necessary to elucidate further details on this mechanism.

Acknowledgements

M.E.F. and P.G. share co–first-authorship since they have worked together on this manuscript and contributed equally. This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, FONCyT PICT 2017-815, PICT 2018-1651, PICT 2019-0207, PICT 2020-SERIE A-3223) and the Universidad Nacional de La Plata (Project X-828, Project A-344), Argentina. The funding sources were not involved in any of the following: the study design; the collection, analysis and interpretation of data; the writing of the report; or the decision to submit the article for publication. P.G. is a ANPCyT fellow; M.E.F., G.L.G., R.F.V., M.C.N.S. and S.R.M. are members of the CONICET Research Career; E.P. is a member of the CONICET Support Professional Career Program. The valuable advice and contribution of Dr. Sabina Maté (INIBIOLP, CCT-La Plata, CONICET, UNLP) in the monolayer experiments is also acknowledged. Dr. Donald F. Haggerty, a retired academic career investigator and native English speaker, edited the final version of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest. This article does contains no studies with human or animal subjects performed by any of the authors.

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Candidicidal Effect of Arginine-based Surfactants

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Abstract

Figures

1. Introduction

2. Materials And Methods

F = 2E tanα δ²/π (1-ν²) Eq. (1)

3. Results And Discussion

4. Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

Candidicidal Effect of Arginine-based Surfactants

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

F = 2E tanα δ2/π (1-ν2) Eq. (1)

3. Results And Discussion

4. Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

F = 2E tanα δ²/π (1-ν²) Eq. (1)