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Brazilian Journal of Microbiology logoLink to Brazilian Journal of Microbiology
. 2023 Dec 2;55(1):365–374. doi: 10.1007/s42770-023-01198-6

Streptococcus mutans supernatant affects the virulence of Candida albicans

Maíra Terra Garcia 1,✉,#, Jéssica Diane dos Santos 1,#, Paulo Henrique Fonseca do Carmo 1, Gabriela Vieira Mendes 1, Jonatas Rafael de Oliveira 2, Luciane Dias de Oliveira 1, Juliana Campos Junqueira 1
PMCID: PMC10920551  PMID: 38040990

Abstract

Candida albicans causes a variety of clinical manifestations through multiple virulence factors that act simultaneously to overcome the immune system and invade the host tissues. Owing to the limited number of antifungal agents available, new candidiasis therapeutic strategies are required. Previous studies have demonstrated that the metabolites produced by Streptococcus mutans lead to a decrease in the number of Candida cells. Here, for the first time, we evaluated whether the C. albicans cells that survived the pretreatment with S. mutans supernatant can modify their virulence factors and their capability to infect Galleria mellonella larvae. Streptococcus mutans supernatant (SM-S) was obtained by filtering the culture supernatant of this bacterium. Then, C. albicans cells were pretreated with SM-S for 24 h, and the surviving cells were evaluated using in vitro and in vivo assays. The C. albicans pretreated with SM-S showed a significant inhibition of hyphal growth, an altered adhesion pattern, and an impaired capability to form biofilms; however, its proteolytic activity was not affected. In the in vivo assays, C. albicans cells previously exposed to SM-S exhibited a reduced ability to infect G. mellonella and a higher amount of circulating hemocytes. Thus, SM-S could inhibit important virulence factors of C. albicans, which may contribute to the development of new candidiasis therapeutic strategies.

Keywords: Antifungal metabolites, Candida albicans, Candidiasis, Galleria mellonella, Streptococcus mutans, Virulence factors

Introduction

Candida spp. are part of the normal human microbiota and coexist with the host without causing infections [1]. This fungus can mainly be found in the oral cavity, vaginal mucosa, skin, and respiratory tract [2]. However, the commensal colonization can become pathogenic due to changes in the pH and oxygen levels, an imbalance in the host microbiota (e.g., caused by antibiotic use), or changes in the immune response (caused by stress, infection, or immunosuppressive therapy) [3, 4]. Among the Candida species, Candida albicans exhibits a wide range of virulence factors that act simultaneously to overcome the immune defenses of the host and invade the host tissues [5, 6]. These factors may be related to the adhesion of the fungus to the host cells, morphogenesis (morphological change from the yeast to the filamentous form), biofilm formation, and the production of hydrolytic enzymes, such as proteinases and phospholipases [7].

Unlike antibacterial drugs, the available antifungal classes, among which azoles, polyenes, and echinocandins are the three main ones, are quite limited [8]. Moreover, their prolonged use, particularly of azoles, as prophylactic treatment may lead to the emergence of antifungal-resistant Candida strains [9]. In addition, the similarities between fungal and human eukaryotic cells considerably compromise the use of antifungal drugs without any toxicity to the host [10]. Some studies involving the identification of new antifungal agents from natural sources have been conducted to overcome these challenges.

In this context, the use of products from the secondary metabolism of bacteria for the prevention and treatment of candidiasis has received great attention. One of these strategies is investigating the products secreted by microorganisms that naturally coexist with Candida species. In the oral cavity, Candida species usually form mixed biofilms with Streptococcus mutans, a pathogenic bacterium that can secrete quorum sensing molecules with antifungal activity. Streptococcus mutans is a Gram-positive bacterium that plays an important role in the oral microbial community and in the development of dental caries, partly due to its various interactions with other bacteria [11], fungi [12], and host cells [13].

Streptococcus mutans metabolites with antifungal activity against C. albicans have been investigated. Joyner et al. identified a peptide produced by S. mutans capable of influencing the mycelial-yeast form transition of C. albicans in in vitro cultures [14]. In a previous study conducted by our research group, S. mutans secreted molecules with antifungal activity that inhibited the growth of C. albicans cells both in vitro and in vivo. In an in vivo study, Galleria mellonella was used as a study model, and the microscopic analysis showed a significant reduction in the number of C. albicans cells in the yeast and hyphal forms in the larval tissues after the treatment with the S. mutans supernatant [15]. In a subsequent study, our group extracted and fractionated the supernatants from an S. mutans cultures to identify the antifungal agents against C. albicans. The SM-F1 and SM-F2 fractions presented dose-dependent antifungal activity in vitro and the capacity to reduce the infection level of oral candidiasis in a mouse model [16].

However, all these studies were performed by the addition of the S. mutans supernatant in direct contact with the biofilms already formed or with a candidiasis episode already occurred. Thus, the objective of the present study was to evaluate whether the previous exposure of C. albicans to the supernatant of S. mutans cultures can modify the virulence factors of the surviving fungal cells. Thus, C. albicans cells were treated with the supernatant from a culture of S. mutans (namely SM-S) for 24 h. Next, the surviving C. albicans cells were quantified, and their virulence factors, such as their proteolytic activity, filamentation, adherence, and biofilm formation abilities, were evaluated. Furthermore, the pathogenic capacity of Candida cells pretreated with SM-S in infecting G. mellonella was also verified.

Materials and methods

Microorganisms and activation

C. albicans (ATCC 18804) and S. mutans UA 159 (Bratthall serotype c) were used in this study. The microorganisms were activated in Sabouraud Dextrose agar (SD; Himedia Laboratories, Mumbai, India) or Brain Heart Infusion agar (BHI; Himedia) at 37 °C for 24 h, and S. mutans was cultured at 5% CO2.

Preparation of SM-S

S. mutans was cultivated in BHI broth supplemented with 5% sucrose at 37 °C for 24 h and 5% CO2. A solution containing 1 mL of S. mutans (1 × 107 cells/mL) and 6 mL of BHI broth was prepared. After 4 h of incubation, the culture was centrifuged (5000 rpm for 10 min), and the supernatant was filtered using a 0.22-μm diameter pore membrane and a vacuum filtration system (Stericup® and Steritop® Filter Unit, Millipore, MA, USA) [15].

Preparation of the fungal suspensions and exposure to SM-S

C. albicans was grown in Yeast Extract Peptone Dextrose broth (YPD; Himedia) at 37 °C for 24 h. After centrifugation (2000 rpm for 10 min), the supernatant was discarded, and the pellet was resuspended in 6 mL of phosphate buffered saline (PBS). This procedure was repeated twice. The cell density of the C. albicans suspension was adjusted to 107 cells/mL using a hemocytometer based on the number of viable cells, which could be obtained after staining the cells with trypan blue. Next, an inoculum of 1 mL of this suspension was added to 6 mL of BHI broth (group BHI) or BHI broth with the S. mutans supernatant containing the antifungal metabolites released by this species (group SM-S) for 24 h at 37 °C [15].

Determining the pH value and counting the viable C. albicans cells after the exposure to SM-S

After incubating the C. albicans cultures in BHI or BHI with SM-S for 24 h, the pH of the cultures was measured. The culture was centrifuged (2000 rpm for 10 min); the supernatant was discarded, and the pellet was suspended in 6 mL of PBS. This procedure was performed twice. The number of viable Candida cells in the suspensions obtained was determined using a hemocytometer, after staining the cells with methylene blue. Five repetitions were performed for each group, and the assay was repeated twice [15]. Posteriorly, the cell densities of the C. albicans suspensions were adjusted to specific values to standardize them for each of the following experiments.

Effects of the pretreatment with SM-S on the virulence factors of C. albicans: in vitro analysis

Extracellular proteolytic activity

To induce the production of secreted aspartic proteinases (Sap), 5 μL of a C. albicans suspension (1 × 107 cells/mL) was inoculated on agar containing 0.2% bovine serum albumin (BSA), 1.17% yeast carbon base, and 0.01% yeast extract (the medium was sterilized via filtration, and 2% autoclaved agar was added). An incubation at 37 °C for 5 days followed. The proteolytic activity (Pz) was determined by measuring the colony and halo diameters and by using the following formula: Pz = colony diameter/(colony diameter + halo diameter). The value obtained reflected the nature of the proteolytic activity: (i) negative proteolytic activity (Pz = 1 cm); (ii) positive proteolytic activity (0.64 cm < Pz < 1 cm); (iii) and strongly positive proteolytic activity (Pz < 0.64 cm). Five repetitions were performed for each group, and this assay was conducted twice [17].

Filamentation capability

In each well of a 24-well plate, 100 μL of a C. albicans suspension (1 × 107 cells/mL) was inoculated in 1 mL of sterile distilled water supplemented with 10% fetal bovine serum. After 24 h of incubation, 50 μL of the inoculum was transferred to glass slides previously demarcated with 10 fields on the back. The slides were then observed using a light microscope (Carl Zeiss, Primo Star, Germany) at a ×400 magnification. A score was assigned according to the number of hyphae present: 0, 1, 2, 3, 4, and 5 for no hyphae, 1–10, 11–20, 21–40, 31–40, and > 40 hyphae, respectively. Five repetitions were performed for each group, and this assay was conducted twice [15].

Adhesion capability

The glass coverslips were placed in a 6-well plate with 120 μL of a C. albicans suspension (1 × 107 cells/mL) and 2880 μL of BHI broth. After incubation for 8 h, the non-adherent cells were removed with sterile distilled water. The coverslips were stained with 1% (v/v) crystal violet for 5 min and analyzed using an optical microscope at a ×400 magnification. The adherent cells were observed and counted in 30 fields of equal size, and the capacity of adhesion of C. albicans was classified as negative (“−,” no adhering cells in the fields), weak (“+,” 1–10 adhering cells/field in at least 15 fields), moderate (“++,” > 10 adhering cells/field in at least 15 fields), or strong (“+++,” ≥ 25 adhering cells/field in at least 15 fields). Moreover, the adhesion characteristics were also analyzed and classified as: (i) diffuse, when the cells adhered to the whole surface but did not form cell aggregates; (ii) localized adhesion, when groups of yeasts adhered to the surface in localized regions; (iii) aggregative adherence, characterized by yeasts arranged as “stacked bricks” or “grape bunches”; and (iv) filamentous adhesion, when filaments or pseudo-hyphae formed along the surface of the coverslip. Five repetitions were performed for each group, and this assay was conducted twice [18].

Capability to form biofilms

A volume of 100 μL of a C. albicans suspension (1 × 107 cells/mL) was added to each well of a 96-well plate and incubated for 90 min under agitation (75 rpm) for the initial adhesion phase. Afterwards, the supernatant was removed, and the wells were washed with PBS twice to discard the non-adherent cells. Then, 200 μL of yeast nitrogen base (YNB; Himedia) with 100 mM glucose was added to the wells. The capability of C. albicans to form biofilms was investigated after 6, 24, and 48 h of incubation. The biofilms were disaggregated from the microplate using an ultrasonic homogenizer at a power of 25% for 30 s (Sonopuls HD2200, Bandelin Electronic, Berlin, Germany). The generated suspension was diluted, plated on SD agar, and incubated for 48 h at 37 °C to determine the number of colony forming units (CFUs). Five repetitions were performed for each group, and this assay was conducted twice [15].

Effects of pretreatment with SM-S on the pathogenicity of C. albicans in a G. mellonella model: in vivo analysis

Determination of the survival curve of G. mellonella after infection with C. albicans

Viable cells of C. albicans were quantified after pretreatment in BHI (control group) and BHI with SM-S using trypan blue staining. Then, each G. mellonella larva was infected by 10 μL of a C. albicans inoculum (at 1 × 107 or 1 × 108 cells/mL) to evaluate the pathogenicity of different concentrations, as well as to select the most appropriate concentration for the subsequent tests. After inoculation, the larvae were incubated in Petri dishes at 37 °C. The number of dead G. mellonella larvae was counted during seven consecutive days. Touch-insensitive larvae were considered dead. For this study, 16 larvae (weighing 200–250 mg) were used per group, and each experiment was repeated three times [18].

Recovery of C. albicans from the hemolymph of G. mellonella

After 4 and 12 h of infection with C. albicans (1 × 108 cells/mL), the larvae were euthanized through a cut in the ventral part in the cephalocaudal direction using a scalpel. Then, their hemolymph was extracted by gently squeezing the larvae body after creating an incision in the head-caudal direction using a scalpel blade. Posteriorly, a pool of hemolymph from five larvae was serially diluted and added to SD agar supplemented with chloramphenicol (0.1 mg/mL of medium). After 48 h of incubation, the C. albicans concentration (in CFU/mL) was determined for each group [19].

Counting the hemocytes present in the hemolymph of G. mellonella

For each experimental group, five larvae were infected by C. albicans (1 × 108 cells/mL) per group were used, and each experiment was repeated three times. After 4 and 12 h of incubation, the hemolymph was extracted from the larvae and 20 μL of this biological material was diluted in 180 μL of sterile anticoagulant buffer composed of 150 nM sodium chloride, 5 nM potassium chloride, 10 nM tris-HCl (pH 6.9), 10 nM EDTA, and 30 nM citrate sodium in an ice-cold microtube. Then, the hemocytes were counted using a hemocytometer [20].

Statistical analysis

The SM-S and BHI groups were compared using a Student’s t-test or a Mann–Whitney test. The survival curves of G. mellonella larvae were drawn using the Kaplan-Meier method, and the level of significance was calculated using the Log-rank test (Mantel-Cox). For all tests, the GraphPad Prism software was used at a significance level of 5% (P ≤ 0.05).

Results

Exposure to SM-S reduced the pH and viability of Candida albicans cells

After incubating C. albicans in BHI or BHI containing the S. mutans supernatant (SM-S) for 24 h, the pH and number of viable cells of the cultures were evaluated. The pH values obtained were 6.3 and 6.0 for the C. albicans culture in BHI and BHI with SM-S, respectively (Fig. 1a). A cell density of 8.7 × 108 and 5.2 × 108 viable cells/mL was obtained for the BHI and SM-S groups, respectively, showing that the exposure of C. albicans cells to SM-S significantly reduced their cell viability (Fig. 1b).

Fig. 1.

Fig. 1

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a Comparison of the pH levels of the analyzed products (BHI and SM-S). b Comparison of the concentrations of viable C. albicans cells after the exposure to BHI or SM-S for 24 h. Student’s t-test, P ≤ 0.05

Exposure to SM-S affected hyphae formation and adhesion of Candida albicans

The proteolytic activity, filamentation, adherence, and biofilm formation abilities of the C. albicans cells that survived the exposure to SM-S were analyzed. The Candida cells of both the BHI and SM-S pretreated groups showed a strongly positive proteolytic activity, with no statistically significant difference between the groups (Fig. 2a). Conversely, compared with that of the group exposed to BHI broth alone, a significant inhibition of hyphae production was observed in the group exposed to SM-S (Figs. 2b and 3A–B). Regarding the adherence capability, C. albicans SM-S pretreated cells showed a strong adherence to the glass coverslips surface, similar to that of the cells of the BHI group. However, different adhesion characteristics were observed; for example, an aggregative adherence pattern in the BHI group (Fig. 3C–D), whereas a diffuse adherence pattern in the SM-S pretreated group. Regarding biofilm formation, compared with that of the BHI control group, the ability of C. albicans pretreated with SM-S to form biofilms was impaired in the initial adhesion period (1 h 30 min), as well as in the microbial community formation period (6 h and 24 h) and maturation phase (48 h). Statistically significant differences between the BHI and SM-S pretreated groups were observed for the periods of 1 h 30 min, 6, and 24 h periods (Fig. 4).

Fig. 2.

Fig. 2

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a Analysis of the extracellular proteolytic activity in the absence and presence of SM-S for 5 days. Student’s t-test, P ≤ 0.05. b Analysis of the filamentation capability of cells evaluated based on a score system: 0 (no hyphae), 1 (1–10 hyphae), 2 (11–20 hyphae), 3 (21–40), 4 (31–40), and 5 (> 40 hyphae). Mann–Whitney test, P ≤ 0.05

Fig. 3.

Fig. 3

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Optical microscopy images of C. albicans cells. A Analysis of the filamentation ability (using fetal bovine serum) of the cells in the BHI control group and B the SM-S group. C Analysis of the capacity of cells of the BHI control group to adhere to the glass coverslips. D Analysis of the capacity of the cells of the SM-S group to adhere to the glass coverslips

Fig. 4.

Fig. 4

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Means and standard deviation of the CFUs present in the C. albicans biofilms. Comparison between the BHI and SM-S groups at the different stages of the biofilm formation process (1 h 30 min, 6 h, 24 h, and 48 h). Student’s t-test, P ≤ 0.05

Inoculation with pre-treated cells increased immune response of Galleria mellonella against Candida albicans

In the in vivo assays, the infected G. mellonella larvae died faster as the inoculum concentration increased. The non-infected larvae alone remained alive during the 7 days of the analysis. A total of 56% and 75% of the larvae infected by 107 cells/mL of C. albicans from the BHI control and SM-S pretreated groups survived, respectively. However, the infection with C. albicans SM-S pretreated cells did not significantly affect the survival rate of G. mellonella larvae when compared to BHI group (P = 0.2959) (Fig. 5a). When G. mellonella larvae were infected by an inoculum of 108 cells/mL of C. albicans, 100% of the larvae of both groups died 72 h post-infection (P = 0.7788) (Fig. 5b).

Fig. 5.

Fig. 5

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Survival rate of the non-infected G. mellonella larvae (no inoculation or inoculation only with PBS) and the larvae infected by C. albicans previously cultivated in BHI (BHI group) or BHI with SM-S (SM-S group). a Larvae infected by an inoculum of 107 CFU/mL of a C. albicans suspension. b Larvae infected by an inoculum of 108 CFU/mL of a C. albicans suspension. Log-rank test (Mantel-Cox), P ≤ 0.05

Although no statistically significant differences were observed, the fungal burden recovered from the hemolymph of G. mellonella larvae infected by C. albicans SM-S pretreated cells was slightly lower than those recovered from the larvae infected by C. albicans not exposed to S. mutans metabolites (BHI group) for both periods of observation (4 and 12 h post-infection) (Fig. 6a). Moreover, the number of hemocytes in the hemolymph of larvae was higher in the SM-S pretreated group than in the BHI control group, with a statistically significant difference between the groups for the period of 12 h post-infection (Fig. 6b).

Fig. 6.

Fig. 6

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Analysis of the hemolymph of G. mellonella larvae 4 and 12 h post-infection with C. albicans previously cultivated in BHI (BHI group) or BHI with SM-S (SM-S group). a Mean and standard deviation of the number of CFUs of C. albicans recovered from the hemolymph of the larvae. b Mean and standard deviation of the concentration of hemocytes in the hemolymph of the larvae. c Mean and standard deviation of the concentration of hemocytes in the hemolymph of the larvae inoculated with PBS, and heat-killed cells of C. albicans grown in BHI, BHI with SM-S and YPD. Student’s t-test, P ≤ 0.05

Discussion

S. mutans can release metabolic products, such as mutacins, which can inhibit the growth of C. albicans and other microorganisms in the same environment [21]. S. mutans and C. albicans coexist in the oral cavity and establish ecological interactions that can result either in a synergistic action that favors the development of diseases or in an antagonistic association, which is disadvantageous to one of the species [22]. The antagonistic interactions have been widely explored to identify natural compounds that can be used as antifungal agents for treating candidiasis. Previous studies have reported that the supernatant of an S. mutans culture can kill Candida cells and decrease their growth in both the planktonic and biofilm stages. However, in these studies, the S. mutans supernatant caused a partial reduction in the number of Candida cells, and a total fungal elimination could not be achieved [15, 16]. Here, the effects of the pretreatment with the culture supernatant filtrate of S. mutans (SM-S) on the surviving Candida cells were evaluated. For this, C. albicans was cultured in contact with SM-S for 24 h, and the virulence mechanisms of the surviving cells, along with their ability to cause systemic infection in G. mellonella, were investigated.

Initially, the pH values and the number of viable cells were determined after culturing C. albicans in BHI or BHI containing SM-S for 24 h. The pH values were analyzed since the pH changes can influence the growth and morphological transition of C. albicans [23]. Both Candida cultures in BHI and BHI with SM-S had similar pH values (approximately 6). In addition, the number of CFUs obtained from the culture of C. albicans in BHI with SM-S was lower than that obtained from the culture in BHI alone, corroborating the effects of SM-S against C. albicans as previously demonstrated [15]. Furthermore, our results confirm that the inhibition of C. albicans cells by SM-S was not attributed to the pH environmental conditions. Then, the proteolytic activity, filamentation, adherence, and biofilm forming abilities of the surviving C. albicans cells were analyzed in vitro using different assays.

The proteolytic activity was evaluated based on the production of proteinases in BSA. The presence of proteinase, which belongs to the aspartic proteinase class, is a primary indicator of Candida virulence [24], and these proteinases are responsible for degrading numerous proteinaceous targets within the host organism, providing nutrients for fungal propagation and allowing Candida to invade the mucosal tissues [25]. Based on the methodology used in this study, the C. albicans SM-S pretreated cells exhibited a strong proteolytic activity, similar to Candida cells cultured in BHI alone (control group), indicating that the S. mutans supernatant was unable to alter the capacity of C. albicans to produce proteinases. Reportedly, this is the first study that investigated the effects of the S. mutans supernatant on the proteolytic activity of C. albicans. Fernandes et al. [26] studied the proteolytic capacity of C. albicans treated with farnesol, an acyclic alcohol considered an important quorum sensing molecule in microbial interactions. Although farnesol inhibits the growth of C. albicans, it does not affect its proteolytic activity. Both groups (treated with farnesol and non-treated) showed moderate proteinase and phospholipase and strong hemolysin activities. Although we observed that the supernatant containing S. mutans extracellular vesicles did not affect the proteolytic activity of C. albicans, metabolomic and proteomic analysis of C. albicans biofilms co-cultivated with extracellular vesicles isolated from S. mutans increased the expression of C. albicans proteins and metabolites related to carbohydrate metabolism [27].

Promisingly, the filamentation capability of C. albicans was impaired by the pretreatment with the S. mutans culture supernatant. Candida albicans SM-S pretreated cells showed a significantly lower hyphal formation ability than the non-treated cells. A previous study reported that S. mutans can release some metabolites, such as mutanobactin A, which acts as a signaling molecule that regulates the morphogenesis of C. albicans [12]. In addition, Santos et al. [16] verified that the treatment of C. albicans cells with the extract of the S. mutans supernatant led to a decrease in the filamentation ability of these cells by downregulating the genes related to hyphae formation, such as CPH1, EFG1, HWP1, and UME6. Furthermore, environments with lower pH, as observed for SM-S, favor the maintenance of blastoconidia rather than hyphae [28], which may explain the reduced filamentation when compared to the BHI group .

The adhesion stage is essential for the survival of C. albicans in a host and is the first stage of the onset of biofilm formation and infection [29]. Here, C. albicans cultured in BHI with SM-S or BHI alone exhibited a strong ability to adhere to the surface of the glass coverslips. However, the adherence of fungal cells to glass presented different spatial organizations according to the groups studied. The C. albicans cells of the BHI control group presented an aggregative adhesion pattern, whereas SM-S pretreated cells showed a diffuse adhesion pattern. Therefore, these results suggest that the S. mutans metabolites interfere with the adhesion pattern and cellular organization of C. albicans.

Another important virulence factor of C. albicans is its ability to form biofilms, which provides protection against commonly used antifungals and host defense mechanisms [7, 30]. The stages of C. albicans biofilm formation include the initial phase (0–11 h), intermediate phase (12–30 h), and maturation phase (31–72 h) [31]. In this study, we analyzed the effects of the pretreatment with on C. albicans biofilms at different stages. C. albicans SM-S pretreated showed an impaired ability to form biofilms in all the developmental stages, mainly in the initial phase. These results are likely due to the capacity of SM-S to reduce the filamentation ability and alter the adherence pattern of Candida cells. Stepanov et al. [32] investigated the ability of 2,4-diacetylphloroglucinol (2,4-DAPG), a secondary metabolite produced by Pseudomonas spp., to prevent biofilm formation by C. albicans. These authors verified that the exposure of C. albicans to 2,4-DAPG led to a decreased capacity of this fungus to form biofilms, which was directly associated with a reduction in the cell surface hydrophobicity and the inhibition of the yeast-to-hyphae transition.

After the in vitro studies, the invertebrate model G. mellonella was employed to investigate whether the infection ability of C. albicans could change due to the treatment with SM-S. Galleria mellonella larvae are useful in vivo models to study fungal virulence and are strongly correlated with mammalian models [16, 18, 33, 34]. First, Santos et al. [16] demonstrated that SM-S was not toxic to G. mellonella larvae at concentrations from 1 to 15 mg/mL, suggesting good safety at these concentrations. In this study, the infection of G. mellonella larvae with C. albicans pretreated with SM-S did not significantly affect the survival rate when compared to the group inoculated with not-treated C. albicans. Although no statistically significant different, there was a slightly increase in survival rate, instigating us to perform additional studies using higher concentrations and by purifying the S. mutans supernatant. Recently, Tao et al. [22] investigated the effects of mutanocyclin (a tetramic acid produced from the secondary metabolism of S. mutans) as an alternative treatment agent for treating G. mellonella infected by C. albicans. As a result, the authors observed an increase in the larval survival by 20% and 60% after injections of 12.8 and 25.6 μg of mutanocyclin, respectively.

Another important factor to be analyzed in G. mellonella when studying fungal virulence is its innate immune response, which can be evaluated by quantifying the hemocytes that act as phagocytic cells, for example [35]. In the present study, the circulating hemocyte density was higher in the G. mellonella larvae infected by C. albicans pretreated with SM-S than in the larvae infected by non-treated C. albicans. Furthermore, we observed that increased hemocyte recruitment occurs only with viable C. albicans cells, since inoculation with heat-killed cells did not this parameter. Perini et al. [35] also demonstrated that infections of G. mellonella larvae by different morphotypes of C. tropicalis altered the survival rate and hemocyte density of G. mellonella. The hemocyte density was lower in the larvae infected by a more virulent variant strain than in the larvae infected by the parental strain. These results confirm the relationship between the ability of highly pathogenic Candida strains to kill G. mellonella larvae and to reduce their circulating hemocyte density. Therefore, in our study, the increased hemocyte counts observed in G. mellonella infected by C. albicans pretreated with SM-S can indicate that the S. mutans metabolites made the fungal strain less virulent.

Taken together, the results of this study demonstrate that SM-S can modify the virulence factors of C. albicans, including their filamentation ability, adherence pattern, biofilm formation capacity, and the ability to infect G. mellonella larvae. Therapeutic strategies that target the specific virulence factors of C. albicans are a more attractive approach than antifungal agents, which only target fungal growth and can induce antifungal resistance development. Other advantages of treating Candida infections using anti-virulence strategies include the preservation of the host normal microbiome and the reduced toxicity, being effective for both the prophylactic and therapeutic management of candidiasis [25].

Conclusions

We conclude that C. albicans SM-S pretreated cells changed their ability related to adherence, filamentation, and biofilm formation. Furthermore, the pretreatment with SM-S attenuated the in vivo pathogenicity of C. albicans in the experimental G. mellonella model. Therefore, SM-S can be explored for developing new therapeutic strategies for candidiasis that target the virulence factors of C. albicans.

Author contribution

Conceptualization: J.D.S., M.T.G., J.C.J.; methodology: J.D.S., M.T.G.; investigation: M.T.G., J.D.S., J.C.J.; data analysis: J.D.S., M.T.G., J.R.O., J.C.J, G.V.M; writing—original draft preparation, M.T.G, P.H.F.C, J.R.O.; writing—review and editing: L.D.O and J.C.J; supervision: J.C.J.; project administration: M.T.G, J.C.J.; funding acquisition: J.C.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by CNPq - National Council of Scientific and Technological Development (grant number 306330/2018-0) and CAPES - Coordination for the Improvement of Higher Education Personnel (Scholarships).

Data availability

Not applicable.

Declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

All authors contributed and agreed to the publication.

Conflict of interest

The authors declare no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Maíra Terra Garcia and Jéssica Diane dos Santos contributed equally to this work.

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