Candida albicans is a human commensal fungus and the etiologic agent of infections in skin, oral and esophageal cavities, gastrointestinal tract, lungs, and bloodstream in immunocompromised individuals (Kadosh and Lopez-Ribot, 2013; Lemberg et al., 2022). Although it is considered an opportunistic fungus, C. albicans is the most common cause of nosocomial fungal infections, with a mortality rate of ∼40% in patients with systemic infections (Gudlaugsson et al., 2003; Perlroth et al., 2007; Erami et al., 2022; Jeong et al., 2022). Due to its severity, nosocomial candidemia contributes to prolonged hospitalization, increased healthcare costs, and increased morbidity and mortality rates (Perlroth et al., 2007; Arendrup, 2010; Kullberg and Arendrup, 2015; Erami et al., 2022; Jeong et al., 2022).

Candida albicans presents several biological characteristics that contribute to its pathogenicity (Calderone and Fonzi, 2001). The expression of adhesins and invasins contributes to an effective adherence and invasion of endothelial and epithelial cells, enabling their dissemination into the bloodstream. Adhesin and invasin also contribute to biofilm formation in implantable medical devices, which are the main source of long-term candidiasis (Schmidt et al., 2012; Desai et al., 2014; Sheppard and Filler, 2014; Kullberg and Arendrup, 2015; McCarty and Pappas, 2016). The secretion of aspartyl proteases and phospholipases promotes fungal tissue invasion and organ damage while simultaneously activating the innate immune response (Felk et al., 2002; Gabrielli et al., 2016), and the ability to alter their morphotypes among unicellular yeast cells—pseudohyphae, and hyphae favor the evasion of the immune system (Netea et al., 2006, 2015; Goodridge and Underhill, 2008; Wheeler et al., 2008; Marakalala et al., 2009; Gow et al., 2011). Finally, the ability to resist oxidative stress by inducing the expression of antioxidants such as catalase (CAT1), glutathione peroxidase (GPX), and superoxide dismutase (SOD) plays critical roles in repairing oxidatively damaged proteins (Enjalbert et al., 2003, 2006, 2007; Fradin et al., 2003, 2005; Rubin-Bejerano et al., 2003; Lorenz et al., 2004).

The mechanisms by which Candida albicans can evade the immune system affecting immunosuppressed individuals have been widely studied. However, there is much to be understood in variations among lineages, specifically different clinical isolates of C. albicans. Comparative studies among strains are important, given that intraspecific diversification is the first step in the evolutionary process, and the diversity among lineages may result in virulence differences and, more importantly, in their resistance to antifungal drugs.

L3881, isolated from a 44-year-old patient’s bloodstream, has achieved its hypovirulent status based on the presence of a mutation in HWP1 gene, which reduces the biofilm production and hyphal formation in the homozygous lineage (Padovan et al., 2009) – referred to as L757 strain. Conversely, a study carried out by Maza et al. (2017) showed, in vitro, that L3881 presented higher resistance to oxidative stress the wild-type SC5314, a characteristic that may be correlated to higher virulence (Maza et al., 2017). Therefore, there is no adequate experimental evidence to consider L3881 hypovirulent.

To better understand the main mechanisms modulated in C. albicans clinical isolates that lead to changes in its virulence ability, we analyzed both L3881 and SC5314 in vitro and in vivo behaviors. Overall, our findings demonstrated that L3881 was less phagocytized by macrophages and presented higher resistance to oxidative stress than wild-type SC5314. These characteristics prevented higher mortality rates due to L3881 infection in animal models of candidiasis. However, L3881 induced an upregulation of pro-inflammatory mediators both in vitro and in vivo. These data indicated that in vitro and in vivo functional characterizations are necessary for virulence determination in different clinical isolates as a response to modulation of the host–pathogen interactions.

Candida albicans is the most studied human commensal fungus and the most common etiologic agent of nosocomial fungal infections. As an opportunistic fungus, the ability of C. albicans to cause disease is due to its capacity to invade, evade the immune system, and infect different organs. The diversity of clinical isolates, in terms of virulence, makes the understanding of the biological/genetic factors are responsible for their virulence more complex. In this sense, using different clinical isolates, several studies have assessed the biological features of C. albicans that may serve as virulence factors (Reviewed by Dadar et al., 2018).

SC5314 has been widely acknowledged as a virulent lineage in mouse models, and since its isolation, it has been used as a wild-type reference in previous studies (Padovan et al., 2009; Lionakis et al., 2011; Maza et al., 2017; Shankar et al., 2020; Lemberg et al., 2022). In turn, the studies that employed L3881 (first described as L757 lineage, Padovan et al., 2009) have not yet reached a conclusion about its virulence status.

A study carried out by Padovan and colleagues characterized L3881 as hypovirulent based on hyphal wall protein 1 (HWP1) gene. This gene is responsible for encoding a cell surface protein expressed only in hyphae, whose main function is to bind to epithelial cells. L3881 was characterized as a homozygous HWP1-2 lineage that showed reduced HWP1 expression, lower biofilm formation levels, and defective hyphae development (Padovan et al., 2009). In another study carried out by Maza, Bonfim-Melo and colleagues, in addition to producing shorter hyphae, L3881 showed lower internalization into HeLa cells and damage than the SC5314 lineage, but conversely was more resistant to oxidative stress (Maza et al., 2017). Despite genetic factors, the ability to cause disease is strongly related to the host–pathogen relationship outcomes, as discussed in Bahia et al. (2018). Therefore, aiming at overcoming these previous discrepancies, we have performed a detailed analysis of the main mechanisms that enable C. albicans SC5314 and L3881 lineages to cause disease.

To investigate host–pathogen interactions, we analyzed both in vitro and in vivo behavior of these lineages. Tissue-residing macrophages are the first innate immune cells that are key effector in antifungal defense, and previous studies have demonstrated the relevance of macrophages in anti-C. albicans host defense. In a study by Qian et al. (1994), macrophage-depleted mice showed accelerated fungal proliferation in tissues and increased mortality (Qian et al., 1994). Here, we showed that the L3881 lineage was better able to face the macrophage antifungal defense in vitro given that it was less phagocytized and displayed higher proliferation rate after been phagocytized than the reference lineage SC5314.

After phagocytosis, respiratory burst with ROS production is an important antimicrobial defense mechanism. The ROS generated within the phagosome creates a toxic environment that induces oxidative stress in C. albicans, leading to irreversible damage. Previous studies have demonstrated that C. albicans is resistant to ROS and has a well-known ability to induce an antioxidative response to phagocytosis by secreting a range of antioxidants, such as catalase (CAT) and glutathione (GSH) (Enjalbert et al., 2003, 2006, 2007; Fradin et al., 2003, 2005; Rubin-Bejerano et al., 2003; Lorenz et al., 2004; Brown et al., 2014).

Our data demonstrated that in addition to better dealing with macrophage phagocytosis, L3881 was more resistant to oxidative stress, in agreement with Maza, Bonfim-Melo and colleagues (Maza et al., 2017). In this study, we demonstrated, for the first time, some mechanisms responsible for this resistant phenotype: L3881 showed higher CAT and GSH enzymatic activity creating a more efficient detoxification of host-derived ROS than SC5314.

Subsequently, we evaluated the cytokine profile secreted by macrophages upon fungi invasion. L3881 induced lower secretion of TNF-α whose pro-inflammatory effects may have been modulated by the progressive increase in IL-4. Rocha et al. (2017) demonstrated that TNF-α can prevent in vitro biofilm development of C. albicans yeast forms (Rocha et al., 2017).

Taken together, we hypothesized that L3881 takes advantage of macrophage activity in vitro due to its persistence against phagocytosis, higher resistance to oxidative stress, and an altered stimulation of these cells, generating distinct profiles of the secreted cytokines analyzed, suggesting that L3881 has a virulent potential. To understand deeper about L3881 ability to cause disease, we analyzed it in an in vivo model of candidemia. The pathogenesis of C. albicans infection involves a complex interaction between fungal and host features. The properties that are central to C. albicans pathogenesis are as follows: first, the ability to morphologically switch between yeast and hyphal forms and, second, biofilm formation, both allowing this pathogen to invade, spread, and infect tissues (Jabra-Rizk et al., 2004; Ramage et al., 2005; Calderone, 2012; Chauvel et al., 2012). Although L3881 presented HWP1 natural deletion and shorter hyphae, our data demonstrated that both lineages were able to spread through the bloodstream and invade the liver, lungs, and kidney of infected mice, confirming their virulent status. Emphasis is placed on the fact that L3881 presented higher CFU counts in the blood, liver, and lungs, while the kidney was the only organ in which SC5314 presented higher CFU counts. The SC5314 infection profile in the kidneys was consistent with previously published data (MacCallum et al., 2009; Lionakis et al., 2011; Lüttich et al., 2013). Lüttich and colleagues showed that the SC5314 lineage can persist in the kidney over time, even in animals showing no clinical symptoms (Lüttich et al., 2013). Similarly, other studies have shown that the kidney is a common target of disseminated Candida infection in mice (Louria, 1985; Papadimitriou and Ashman, 1986).

The outcome of C. albicans infection is also related to host immune response, and we assessed this by quantifying immune cells and inflammatory mediators (Ashman, 2008; Netea et al., 2008). In this study, for the first time, we showed the profile of macrophages and neutrophils recruited and of inflammatory mediators secreted in the L3881-infected mice.

Upon increased fungal load or breach of defensive tissue barriers, macrophages and neutrophils are recruited to infection sites where they kill C. albicans by a combination of mechanisms including phagocytosis, degranulation to release toxic mediators, and ROS production.

Our data demonstrated that neutrophils are the most recruited immune defense cells, regardless of the tissue. Despite the large number of phagocytic cells recruited, L3881 was still able to infect all tissues much more robustly than SC5314. This could be explained by the higher capacity of L3881 to face phagocytosis and its antioxidant system. Moreover, previous studies have shown that the SC5314 lineage is very susceptible to phagocytosis by neutrophils (Shankar et al., 2020). However, there was a higher fungal load in the kidneys, despite the increase in neutrophil recruitment.

The cytokine levels showed variations among lineages and organs. In this sense, distinct C. albicans lineages that lead to diverse immune responses may impact the disease outcome toward severe clinical forms.

Overall, the SC5314-infected mice displayed a milder and less complex immune response than the L3881-infected mice. In the latter, it is evident that there is a loss of control of the inflammatory immune response in the liver, with higher levels of pro- and anti-inflammatory mediators, and accumulation of neutrophils that are unable to efficiently eliminate fungi from the liver. Off note, regardless of the lineage and organ, all infected mice presented increased CXCL1 levels, which may explain higher recruitment of neutrophils, an effect potentiated by the high levels of TNF-α – the most predominant pro-inflammatory cytokine.

TNF-α is a macrophage-derived cytokine which can enhance the antimicrobial activity of neutrophils (Djeu et al., 1986; Cybulsky et al., 1988). Previous studies have already shown the protective effect of TNF-α during candidiasis. Riipi and Carlson (1990) observed that TNF-α plays a crucial role in vivo in response to disseminated Candida. The treatment of mice with monoclonal antibodies against TNF-α enhanced the mortality to experimental disseminated candidiasis (Steinshamn and Waage, 1992; Louie et al., 1994). Likewise, treatment with pharmacological inhibitors of TNF-α production led to enhanced mortality and increased outgrowth of C. albicans during disseminated candidiasis in mice (Netea et al., 1995). Knock-out mice for TNF-α and β were highly susceptible to disseminated candidiasis (Marino et al., 1997).

However, TNF-α works like a double-edged sword. The excessive release of TNF-α during inflammation can become damaging, thus leading to cell infiltration, hypotension, hepatotoxicity, and structural damage to tissues (Streetz et al., 2000; Kalliolias and Ivashkiv, 2016). The high tissue invasiveness and colonization capacity of L3881 together with the increased amount of TNF-α induced by this lineage may lead to premature death. Therefore, to confirm this result, we evaluated the mortality rate caused by each of the lineages in mice. At the end of 15 days, only 10% of the L3881-infected mice survived compared to 70% of the SC5314-infected mice.

Our results have shown that the ability to better deal with the immune system via resistance to oxidative stress, to invade different tissues, and to interfere with the immune response confirmed the classification of L3881 as a virulent lineage, despite its short hyphae. We raised the discussion that the determination of C. albicans virulence should not only be based only on genotypic characterization but also on the host–pathogen interaction outcomes. Furthermore, the virulence of a lineage is also related to the infection model used. Recently, Dunker et al. (2021) have demonstrated that a filament-deficient lineage showed different virulence profiles depending on the infection model used. In this study, the same lineage generated an attenuated infection in an intraperitoneal infection model, whereas in a systemic infection model, it was as virulent as a wild type (Dunker et al., 2021). This study demonstrated how the determination of the virulence status of a given lineage depends on several factors and cannot be determined considering only one feature.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Institutional Ethics Committee (Comitê de Ética em Experimentação Animal, CETEA/UFMG, Protocol number 06/2011).

TM, DB, and FS: designing research study. MT and FS: funding acquisition. PS, IG, and TM: conducting experiments and acquiring data. PS, IG, TM, AP, TP, and FS: analyzing data. PS, IG, DB, and FS: writing and reviewing the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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