Virulence-Related Genes Expression in Planktonic Mixed Cultures of Candida albicans and Non-Albicans Candida Species

Candida albicans, a component of the human microbiota, may cause infections as an opportunistic pathogen, ranging from superficial mycoses to disseminated candidemia, especially in high-risk groups such as immunosuppressed or patients in the intensive care unit [1]. C. albicans is a primary cause of nosocomial infections accounting for 15% of all cases progressing to sepsis [2]. Furthermore, it is the third most frequently isolated microorganism in intravascular catheters and the fourth most common cause of candidemia associated with a mortality rate exceeding 50% [3]. Although C. albicans remains the most frequently identified species, there has been an increase in infections caused by non-albicans Candida (NAC) species in recent years [4, 5]. The incidence of fungal infections due to various Candida spp. has doubled in the last decade [6‒8]. Despite the importance of studying mixed infections related to Candida species, many studies have focused on understanding the fungal-bacterial relationship [9], with limited research on biological interactions involving inter-Candida species.

Candida species exhibit two distinct life forms in nature; they can exist either as free-floating planktonic cells or as organized structures within complex biofilms. Planktonic cells are crucial in the early stages of biofilm formation through surface cell attachment, growing both as single yeast cells and hyphal filaments [10, 11]. In addition, there is an active phase after complete biofilm maturation known as dispersion, where fungal cells detach from the biofilm and revert to their planktonic phenotype [12]. The event of cells detaching can disperse to other sites, initiating a new cycle of biofilm formation. In C. albicans, this event is responsible for candidemia and dissemination leading to the establishment of invasive disease [13]. Plasticity of hyphal formation switching from yeast, pseudohyphal to hyphae is a determinant factor for the disease progression. Virulence factors associated with adhesion, hyphal formation, and subsequent mature biofilm present different expression levels throughout biofilm development [14, 15]. Among a wide range of Candida virulence factors, HWP1, ALS3, and SAP1 gene expression mainly contribute to the early stage of biofilm formation. The HWP1 gene encodes a fungal cell wall mannose protein, necessary for hyphal growth [16]. The transition of yeast to its hyphal form has been associated with greater virulence in candidemia. In vivo studies have shown that overexpression of the HWP1 gene improves C. albicans adherence to venous catheters, thereby increasing the risk of candidemia [17]. The ALS3 gene encodes adhesins [18], involved in catrin-dependent endocytosis through its binding to cellular receptors such as E and N-cadherin in the host. In vitro studies have shown a direct correlation between C. albicans ALS3 gene expression and its ability to damage host epithelial cells [1] but also is involved in co-adhesion of oropharyngeal infections between C. glabrata and C. albicans [19]. Finally, C. albicans produce aspartyl proteinases 1 (SAP1), a proteinase important in the degradation of cell membranes and extracellular matrix, which alters structural components and facilitates penetration into host tissues [20].

Various mechanisms have been proposed to explain the interaction between different Candida species and their impact on virulence during the planktonic stage. These mechanisms include direct contact between hyphae, which serve as anchoring sites for other species, enhancing their adherence [6]. However, no evidence establishes a correlation between the virulence factors during the planktonic stage in co-infections. Interactions between different microbial species, and even more so between those of the same genus are topics of great interest to human health [21]. Research focused on understanding the interactions within Candida species in co-culture during the planktonic stage is crucial to elucidate the mechanism of posterior biofilm formation and encourage the implementation of antifungals with synergistic action, as most current pharmacological substances commonly tend to have a single target [22]. Therefore, this study aimed to evaluate the expression of genes related to adhesion, invasion, and secretion of proteases in C. albicans and NAC bloodstream isolates, cultured as single and mixed cultures during the initial planktonic stage.

A laboratory reference strain C. albicans ATCC MYA-3573 (thereafter C. albicans ATCC) and three clinical strains (C. albicans DCA53, C. tropicalis DCT6, and C. parapsilosis DCP1) isolated from blood sources were used in this study. All isolates were previously identified by the VITEK 2 system (bioMérieux, Inc., Hazelwood, MO, USA), followed by molecular identification through the amplification of the intergenic regions of rDNA using ITS-1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS-4 universal primers (5′-TCC TCC GCT TAT TGA TAT GC-3′) [23]. The taxonomic affiliation was verified by the BLAST alignment algorithm. All Candida strains were grown in HiCrome Candida medium and stored in 40% glycerol at −80°C. Before the experiment, the samples were subcultured on LB Broth Base supplemented with peptone and incubated at 37°C with shaking at 250 rpm for 24 h.

Significant differences in growth rates between C. albicans and NAC species were observed in our study, as described by Bordallo-Cardina et al. [24]. Therefore, initial suspensions of each Candida spp. were quantified and adjusted to 10⁷ viable cells/mL using a hemocytometer (online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000540991). Initially, 1,200 μL of LB Broth Base supplemented with peptone was pipetted into 24-well microtiter plates. Next, 70 μL of each Candida species were added in the single culture and mixed cultures, with a 1:1 relation in dual-species suspensions. The combinations used for the mixed cultures were C. albicans ATCC + C. parapsilosis DCP1, C. albicans ATCC + C. tropicalis DCT6, C. albicans DCA53 + C. parapsilosis DCP1, C. albicans DCA53 + C. tropicalis DCT6, and C. tropicalis DCT6 + C. parapsilosis DCP1. The plates were incubated at 37°C with stirring at 150 rpm for 20 h according to the established growth curve. The experiments were performed by three biological replicates. Subsequently, 400 μL of culture was used for RNA extraction. Finally, the samples were placed in a cryotube, exposed to liquid nitrogen, and stored at −80°C. Additionally, 400 μL of each Candida species cultivated as a single and mixed culture were used for SEM analysis.

Sample preparation was performed using the t-butanol freeze-drying method [25]. Briefly, all samples were fixed with 3% glutaraldehyde and washed thrice with phosphate buffer. Osmium tetroxide was added for 1 h at room temperature and subsequently washed with distilled water 3 times. Afterward, the samples were dehydrated with a series of dilutions of tert-butanol at 70, 80, 90, and 100%, each for 1 h. The samples were freeze-dried, covered with a thin layer of gold, and examined using a scanning electron microscope (JSM-IT500 InTouchScope^™ Scanning Electron Microscope).

The sequences of the genes analyzed in the present study (HWP1, ALS3, and SAP1) were downloaded from NCBI and aligned in A Plasmid Editor (ApE) program [26]. Primer specificity was evaluated against BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). To determine their specificity, all primers were tested using quantitative real-time polymerase chain reaction (qPCR) with melting curve analysis (online suppl. Figs. S1–S3). ACT1 and EF1 were used as the reference housekeeping genes. The primer sequences are listed in Table 1.

A 600-bp region of the HWP1 gene was amplified from a clinical strain of C. albicans using primers F1 HWP1 and R3 HWP1. The fragment was then cloned into the pGEMT-Easy vector (Promega) following the manufacturer’s instructions. The ligation reaction was incubated for 16 h at 4°C followed by transformation of the recombinant plasmid into E. coli DH5α (endA1, gyrA, hsdR17, relA, recA1, supE44, thi1, Δ(lacaya-argF) U169, φ80(lacZΔM15), F-). The constructs were verified by colony PCR and electrophoresis on a 1% agarose. The plasmid was extracted using the PureLink Quick Plasmid Miniprep Kit (Invitrogen), following the manufacturer’s protocol. The plasmid concentration was determined using the Qubit dsDNA BR Assay kit (Invitrogen) and then converted into copy number concentration using the formula described by [27].

The plasmid DNA sample was serially diluted (from 1 × 10⁹, 1 × 10⁸, 1 × 10⁷, 1 × 10⁶, 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², 1 × 10¹, 1 × 10⁰ copies/μL) with ultrapure water and used to prepare the standard curve. Bio-Rad CFX software provided by the CFX96 thermocycler (Bio-Rad) was used to calculate the standard curve.

Total sample RNA was extracted using the Total RNA Kit I (Omega BioTek, Inc., Norcross, GA, USA) following the manufacturer's instructions, with slight modifications involving running the samples through the ruptor at 5V for 5 min in step two. RNA quality and concentration were evaluated using a spectrophotometer (Synergy HTX, – BioTek). The extracted total RNA was transcribed into complementary DNA using the Superscript Double-Stranded complementary DNA Synthesis Kit (Invitrogen), according to the manufacturer’s protocols. Then, the qPCR assays were conducted using the FastGene^® IC Green 2x qPCR Universal Mix (Nippon Genetics Europe Gmbh) according to the manufacturer’s instructions with the following cycling parameters: 50°C for 3 min, followed by denaturation at 95°C for 3 min (one cycle) and 95°C for 10 s, and annealing and elongation at 66°C for 30 s. All samples were subjected to melting curve analysis. Absolute quantification of each reaction was performed using a previously described standard curve.

Two-way ANOVA was performed between the quantification data of single and mixed culture, followed by Tukey’s multiple-comparison test. When data for only one Candida spp. in a mixed biofilm were available, comparisons were made using the Mann-Whitney U test. Statistical significance was set at p < 0.05. Statistical analysis was performed using Prism software 7.0, and figures were constructed in R version 4.2.2 for Mac using the packages ggplot2 [28], ggbreak [29], and ggsignif [30].

SEM analysis showed that C. albicans proliferated on planktonic suspension consisting of both spherical yeast cells and hyphae (Fig. 1a), while single cultures of C. tropicalis and C. parapsilosis were found to form exclusively spherical or elongated yeast cells (Fig. 1b, c). Interestingly, mixed cultures formed by C. albicans DCA53 + C. parapsilosis (Fig. 1d) and C. albicans DCA53 + C. tropicalis (Fig. 1e) exhibited abundant hyphal morphology compared to single cultures of each species.

Gene expression of both C. albicans and NAC species was examined in single and mixed cultures. It was suspected that HWP1, ALS3, and SAP1 genes might undergo changes in the expression levels and potentially be a determinant factor in triggering filamentation within mixed cultures. Our findings confirmed that the expression levels indeed vary between single and mixed cultures. C. albicans isolates, growing with NAC species originating from the bloodstream, showed distinct expression levels compared with C. albicans ATCC. We also highlighted that in a single culture, C. parapsilosis had the highest expression of all the studied genes compared to the other Candida species (Figs. 2-4).

Comparison between single and mixed cultures about HWP1 gene expression showed that DCP1, ATCC, and DCT6 expressed pronouncedly in a mixture culture depending on the combination. Both C. albicans DCA53 and C. parapsilosis upregulated HWP1 gene expression in mixed cultures with 1.94- and 1.53-fold increase, respectively. Similar results were found for C. albicans DCA53 and C. tropicalis interaction, where the HWP1 gene was also upregulated in both species with 4.56- and 2.57-fold increase, respectively. These results suggest a synergistic interaction between C. albicans DCA53 and NAC species in mixed cultures. In contrast, C. parapsilosis versus C. tropicalis showed an antagonistic interaction, with a HWP1 gene downregulation for C. parapsilosis (0.74-fold decrease) and a significant upregulation for C. tropicalis (15.29-fold increase).

There was a high variation during analysis of gene expression specifically on species growing within a mixed culture. C. parapsilosis showed high levels of HWP1 gene expression in combination of C. albicans ATCC and C. albicans DCA53. In addition, C. albicans ATCC showed a significantly higher gene expression level of HWP1 in interaction with C. tropicalis compared to C. albicans DCA53. Whereas C. tropicalis growing in mixed culture with C. parapsilosis presents high levels of HWP1 gene expression (Fig. 2) (online suppl. Table 2).

Concerning ALS3 gene expression, C. albicans ATCC and DCA53 had no gene expression during a single culture, however, when exposed to NAC species, the ALS3 gene showed a slight expression (Fig. 3). C. tropicalis mixed culture with C. albicans wild-type and clinical isolate showed a synergistic interaction when compared to its single culture, with a 43.69-fold increase in interaction with C. albicans wild-type and a 8.60-fold increase with C. albicans clinical isolate. In contrast, ALS3 gene expression of C. parapsilosis showed overexpression growing in single culture conditions. However, ALS3 gene expression of C. parapsilosis showed downregulation in mixed cultures with C. albicans ATCC (0.58-fold decrease), as well as with C. albicans DCA53 (0.56-fold decrease) compared to its single culture, demonstrating an antagonistic interaction between this species, regardless of the strain source. Finally, ALS3 gene expression of C. parapsilosis growing with C. tropicalis also exhibits an antagonistic interaction, with a 0.47-fold decrease and 22.74-fold-increase, respectively (Fig. 3) (online suppl. Table 2).

There was a significant overexpression regarding the SAP1 gene, particularly on C. parapsilosis compared to the rest of the species in single cultures. However, C. parapsilosis was significantly downregulated in mixed cultures with C. albicans DCA53 (0.006-fold decrease) and C. albicans ATCC (0.045-fold decrease), suggesting an antagonistic interaction between this species (Fig. 4) (online suppl. Table 2). Comparison between single and mixed cultures exhibits that SAP1 gene expression in C. albicans clinical isolated DCA53 was constant during co-incubation with NAC species, whereas C. albicans ATCC showed overexpression in mixed cultures with C. tropicalis.Figure 4 does not present SAP1 gene expression in C. tropicalis due to the absence of specific primers.

The Candida genus represents a significant medical problem due to its great ability to adhere to mucus membranes and different environmental surfaces such as medical devices, leading to both superficial and systemic diseases [31]. An important aspect of its virulence lies in its ability to form biofilms [16], a process initiated by the transition from planktonic growth and accompanied by the remodeling of phenotypic behavior, such characteristic is supported by numerous changes in gene expression [32]. In recent years, there has been a growing interest in Candida interactions in mixed cultures due to the increase in colonization by multiple Candida species and infections caused by NAC species [33]. These interactions can range from synergistic and antagonistic to neutral [3]. While numerous studies have examined Candida growth in mixed cultures, most of them focus exclusively on the impact on C. albicans and fail to consider the outcomes for other species [34, 35]. Investigating interactions among microorganisms is essential for understanding their pathogenicity and identifying new therapeutic targets [36, 37]. Here, we assess the changes in gene expression of virulence factors HWP1, ALS3, and SAP1 genes associated with the planktonic stage of C. albicans and NAC species growing in mixed cultures.

Our results indicated that under single culture conditions, these genes show variability in gene expression across all species, with some genes even having extremely variable expression, such as ALS3 and SAP1 genes in C. parapsilosis (200-fold and 550-fold, respectively) in comparison to C. albicans ATCC. We also noticed that within specific mixed cultures, gene expression is homogeneous. These findings indicate that genes with different expression levels may contribute as a species-specific regulator of virulence when multiple species coexist. Notably, a bloodstream isolates of C. parapsilosis showed the highest expression of all evaluated genes in single cultures compared to other Candida species. These results can be explained by its ability to adhere to synthetic materials, colonize medical devices, and potentially facilitate their invasion into the bloodstream [38, 39].

The overexpression of HWP1 and ASL3 genes was observed in mixed cultures between C. albicans DCA53 with NAC species. These findings are similar to prior analyses by [40], where C. albicans provided nutritional substratum to NAC species, thereby enhancing future biofilm formation. Pathirana et al. [35] also found that mixed cultures consisting of C. albicans and C. tropicalis mutually benefit from adhering to each other, resulting in enhanced species growth [31]. In contrast, a study conducted on patients with oral candidiasis found that the presence of C. tropicalis downregulated C. albicans HWP1 and ASL3 genes [34]. The overexpression of the HWP1 gene in mixed cultures, compared to single cultures, correlates with SEM analysis, which revealed greater filamentation. However, it is important to emphasize that it was not possible to differentiate which Candida species each hypha belonged to. Our findings suggest that in mixed culture conditions of Candida species, there is a potentially increased capacity for hyphal growth and adhesion during the planktonic stage.

Overall, we observed that the laboratory strain of C. albicans ATCC exhibits a higher copy number of the HWP1 gene than the clinical strain C. albicans DCA53 when co-cultured with NAC species. Additionally, our analysis of HWP1 gene expression indicates that C. albicans ATCC displays synergistic interactions with C. tropicalis, but antagonistic interactions with C. parapsilosis, in contrast to C. albicans DCA53, which shows synergistic interactions with all tested species. These patterns of HWP1 gene expression lend support to our hypothesis that there are distinct virulent gene expression profiles between laboratory reference strains and clinical bloodstream isolates of Candida.

The expression of the SAP1 gene plays a crucial role in the pathogenesis of Candida spp [41]. In our study, C. albicans SAP1 gene expression was downregulated in mixed culture with NAC species, while C. albicans from clinical isolate DCA53 showed a slight decrease in SAP1 gene expression in mixed culture. We also observed a notable deregulation of SAP1 gene expression of C. parapsilosis when co-cultured with DCA53. These results are related with the findings of [42], who demonstrate that in dual cultures of C. albicans and C. parapsilosis, the enzymatic activity of virulence factors decrease compared to the greater production of proteinases and hemolysins of C. parapsilosis growing in single culture. Further, they found that adherence of C. albicans was favored by C. parapsilosis. These studies demonstrate that co-culture of C. albicans and C. parapsilosis modulate gene expression of SAP1, as well as enzymatic production and adhesion, all of which are critical for pathogenesis by promoting a complete adaptation to a shared microenvironment. But in contrast, Gonia et al. [37] found that C. parapsilosis reduces the pathogenicity of C. albicans through the inhibition of membrane-degrading enzymes in intestinal epithelial cells [36]. Based on these results, the authors propose that some noninvasive species of NAC could be used as probiotics-like strains. Given these discrepancies, more studies are necessary to determine the specific virulence factors of C. albicans and C. parapsilosis in mixed cultures.

Co-infection with both C. parapsilosis and C. tropicalis is expected due to clinical settings, given their common association with bloodstream infections and prevalence of isolation in Latin American countries [43‒45]. To date, there have been no reported cases of co-infections involving these specific species. However, understanding their interactions is crucial for anticipating their clinical implications. This study is the first to identify antagonism in the interaction between C. parapsilosis and C. tropicalis during the planktonic stage, characterized by downregulation of gene expression of HWP1 and ALS3 in C. parapsilopsis, while the same genes were upregulated in C. tropicalis. These results suggest that the ability of C. parapsilopsis hyphal and adhesion during mixed cultures are negatively affected during the planktonic stage in the presence of C. tropicalis. We additionally determined that NAC species exhibited slower growth rates, as evident from the reduced number of fungal cells in single cultures. This aligns with the findings of Bordallo-Cardona et al. [24], who found that in isolates from patients with candidemia, C. parapsilosis shows a longer lag phase compared to other Candida species, suggesting a greater requirement for adaptation and initial growth under experimental conditions. Despite their disparities in growth rates, our study reveals that C. parapsilosis planktonic cells in both single and mixed cultures exhibited increased expression levels of HWP1 and ALS3 genes in comparison to clinical and wild-type isolates of C. albicans. This provided evidence that there is no correlation between high growth rates and increased expression levels of virulence genes.

Our results showed that the gene expression associated with virulence factors differs under competitive conditions between Candida species in the planktonic stage. A synergistic interaction of C. albicans and NAC species isolated from clinical samples growing in mixed culture is exhibited. In contrast, C. parapsilosis versus C. tropicalis interaction showed an antagonistic interaction during mixed culture, suggesting a decreased virulence profile of C. parapsilosis during initial co-infection with C. tropicalis. The understanding of how virulence factors could potentially be regulated in mixed cultures carries significant implications, not only for comprehending Candida interactions within mixed cultures, but also for elucidating pathogenesis in mixed cultures during host interactions [24, 34, 46]. Further studies focusing on evaluating clinical relevance of interactions of mixed Candida species are needed.

This study had some limitations as it did not consider which of the yeasts form filaments in mixed cultures. Additionally, a more detailed evaluation of the changes in morphological, biochemical, and virulence characteristics within the mixed cultures is necessary. Furthermore, it is important to evaluate the change in gene expression levels at different stages of yeast growth to understand the precise point at which the growth of one species is affected or favored when grown in a co-culture environment. In addition, it is important to compare the expression of virulence-related genes between planktonic and sessile cells of clinical Candida species. Finally, studies are required to evaluate the real impact and underlying mechanisms of this antagonistic interaction in the biofilm, as well as conduct in vivo studies to assess the interactions involved in the development of candidemia caused by multiple species.

We express our acknowledgment to Yessenia Acosta for clinical sample collection, Karen Muñoz and Katheryn Sacheri, for their valuable contributions to the upkeep of the Candida strain collection.

This study was approved by the Ethics Committee of Universidad Espíritu Santo (CEISH-UEES) project N0 2022-001A. Anonymity and the protection of personal data were preserved. The clinical isolates described come from a hospital biobank and each isolate is unidentified, ensuring that it cannot be traced back to individual patients. This project was evaluated by the CEISH (Committee on Ethical Issues in Human Studies), which determined that written informed consent was not required for this research, project N0 2022-001A.

The authors have no conflicts of interest to declare.

We acknowledge the support from Centro de Investigaciones, Universidad Espíritu Santo. The funder had no role in the design, data collection, data analysis, and reporting of this study.

M.S.-A. and J.C.T.: perform experiments and draft manuscript, L.D.-C.: data microscopy acquisition, M.C.-V.: gene expression data analysis, G.M.-L.: manuscript reviewer, J.C.F.-C.: acquired finances and manuscript reviewer, D.A.-M.: conceived the study, supervised the research, and revised the manuscript.

The supplementary data were deposited at fairdomhub (https://fairdomhub.org/projects/410). Further inquiries can be directed to the corresponding author.