Voices of Eukaryotic Microbes: Chemical Communication Via Quorum Sensing

Abstract

Quorum sensing (QS) is a cell–cell communication mechanism mediated by secreted hormone-like signaling molecules that operates in both Gram-positive and Gram-negative bacteria, driving coordinated alterations in gene expression once a critical cell density is reached. In these prokaryotic systems, bacteria produce, release, detect, and respond to small autoinducers, such as acyl-homoserine lactones in Gram-negative bacteria, oligopeptides in Gram-positive bacteria, and the universal autoinducer-2, to regulate community behaviors including biofilm formation, virulence factor production, and stress adaptation. The concept of QS in eukaryotic microbes emerged decades ago, and later investigations confirmed that unicellular fungi and protozoa similarly measure population density to regulate collective activities. In Saccharomyces cerevisiae, aromatic alcohols (2-phenylethanol, tryptophol, tyrosol) serve as QS signals to control filamentous growth, biofilm assembly, and environmental stress responses. Candida albicans employs farnesol to suppress hyphal development while utilizing tyrosol to accelerate germ tube emergence and biofilm maturation. African trypanosomes, including Trypanosoma brucei and related species, generate oligopeptides via secreted peptidases that accumulate as stumpy induction factors (SIFs), triggering a density-dependent shift from proliferative slender forms to transmission-competent stumpy forms essential for tsetse fly infection. QS-based mechanisms influence virulence factors in fungal and protozoan pathogens, affecting their ability to colonize hosts. Exploring QS in eukaryotic organisms opens new possibilities for antifungal treatments and parasite management. By interfering with QS signaling, researchers can disrupt fungal biofilm formation and regulate protozoan development, paving the way for innovative disease control methods.

Introduction

Quorum sensing (QS) is a cell-cell communication system in which microorganisms produce diffusible signal molecules, known as autoinducers, that accumulate in proportion to population density. Once a threshold concentration is reached, these signals are detected by specific receptors, initiating transcriptional programs that coordinate collective behaviors such as biofilm formation, virulence, and morphological transitions. This canonical circuit signal synthesis, accumulation, receptor detection, and transcriptional response provides the mechanistic foundation for understanding QS across bacteria and eukaryotic microbes [1, 2].

Bacteria utilize the QS system to monitor environmental densities of other bacteria, as well as to modulate population-wide behavior in response to changes in numbers or species within a community. One of the most extensively studied quorum-sensing systems is Vibrio fischeri, a light-producing marine bacterium. V. fischeri lives as a symbiotic association within some eukaryotic hosts. In all these associations, there is a specialized light organ containing a pure strain culture of a specific V. fischeri at extremely high cell density. Within these symbiotic relationships, the eukaryotic host supplies bacteria with a nutrient-rich environment, while the bacteria contribute by producing light for the host [3].

In living biological and interactive communities, microorganisms employ quorum-sensing mechanisms as a means of communication. Bacteria and fungi are capable, depending on cell density, of producing a signaling molecule such as secondary metabolites, controlling gene expression, and coordinating behavior within their natural environment. The presence of secondary metabolites has a central function in competence, colonization of surface and host tissue, biofilm development, and morphogenesis [4, 5].

In recent years, there have been several reports of quorum-sensing-related behavior in eukaryotes, especially fungal species. In fungi, QS mechanisms have been described to regulate processes such as secondary metabolite production, sporulation, enzyme secretion, and morphological transition. Various fungal genera exhibit QS mechanisms, employing a diverse array of signaling molecules with distinct structures, including aromatic alcohols, terpenes, and peptides, and this review concentrates on the best-characterized yeast systems to illustrate core principles. Moreover, fungi can interact not only with bacteria but also with their plant or mammalian hosts [1]. In yeasts, Candida albicans, farnesol is a substance produced during the synthesis of the mevalonate/sterol pathway. It plays a crucial role in preventing the formation of biofilms and also affects the morphological transition. Farnesol inhibits the production of hyphae in a concentration-dependent manner. On the other hand, tyrosol, which is derived from tyrosine, has the opposite effect. It accelerates the formation of germ tubes without compensating for the inhibitory effects of farnesol on germination [6]. In Saccharomyces cerevisiae, pheromones, ammonia, and two aromatic alcohols like phenylethanol and tryptophol, play a role in its communication system. Although other fungi, such as Histoplasma capsulatum, Ceratocystis ulmi, and Neurospora crassa, possess a QS system, the specific signaling molecules they use have not yet been discovered [7].

In the subkingdom Protozoa, trypanosomes use density sensing in their mammalian host to prepare for transmission. The essential component for the initiation of QS is the stumpy induction factor (SIF). This factor is produced by both pleomorphic and monomorphic forms, with the key distinction being their ability to respond to SIF. SIF plays a role in QS by detecting population density within the mammalian host, facilitating preparation for transmission. This molecule promotes the transition from the slender form to the transmissible stumpy form [8].

Besides bacterial and fungal QS systems, QS-like mechanisms have also been described in viruses [9, 10] and even higher-order organisms like ants [11]. ). In some environments, in fact, QS controls the synthesis of the secreted molecules responsible for providing social signals in order to coordinate group behaviors. QS has more recently expanded its scope to signaling between phyla or even interkingdom, as well as being included in the widespread, emerging communication between microorganisms and hosts.

This review describes the QS system in eukaryotic microorganisms, such as fungi and protozoan unicellular parasites. It also explains the molecular mechanisms of QS and its role in pathogenicity. This review focused on QS in Candida albicans, Saccharomyces cerevisiae, and Trypanosoma spp. in detail. For the design of new antibacterial or antifungal agents, inhibiting the secretion of QS molecules could hinder microbial infections. This area seems to be interesting and efficient in the upcoming years.

Fungal Quorum Sensing

Fungi harness cell-density–regulated metabolites to coordinate transitions between yeast and filamentous forms and to structure biofilms. This density-dependent signaling allows fungal populations to tailor morphology, community architecture, and virulence strategies to changing environmental or host conditions. Aromatic alcohols, such as 2-phenylethanol and tyrosol, drive filamentation, matrix assembly, and adaptation to stress in Saccharomyces cerevisiae. Candida albicans employs the antagonistic pair of farnesol and tyrosol to fine-tune hyphal induction, virulence gene expression, and biofilm maturation. Through the QS system, individual fungal cells can behave like a coordinated multicellular group, responding collectively to environmental signals and cues [5]. Beyond these model systems, QS-mediated regulation has been reported in diverse fungi, including Cryptococcus neoformans, Histoplasma capsulatum, several Aspergillus and Penicillium species, as well as Ceratocystis ulmi, Neurospora crassa, Aureobasidium spp., and Debaryomyces hansenii. Collectively, these findings highlight quorum sensing as a widespread strategy that allows fungi to act as coordinated multicellular communities [12, 13]. Numerous instances of QS have been reported in fungi. In this article, we focus on two representative cases examined in detail: Candida albicans and Saccharomyces cerevisiae. Some known QS systems in fungal microbes were described in Table 1.

Table 1.

QS systems in fungal microbes

Fungal genera	QS molecule	Effect	References
Aspergillus (A. terreus)	butyrolactone I	inhibits cyclin-dependent kinases	[1, 14]
Aspergillus (A. nidulans)	oxylipin	The cleistothecium conidium switch is regulated by oxylipin	[1, 15]
Debaryomyces	phenylethanol, tyrosol	responsible for biofilm formation	[16]
Ceratocystis (Ce. Ulmi)	2- methyl-1-butanol, methylvaleric acid, 4-hydroxyphenylacetic acid	The regulation of germ tube formation	[17]
Histoplasma (H. capsulatum)	α-(1,3) glucan	continuously produced by the fungus inside macrophages, involved in the morphogenesis and subsequent pathogenesis	[18, 19]
Candida species	farnesol	switch from yeast to mycelial form, affecting the formation of chlamydospore	[20–22]
	tyrosol	enhances mycelial growth by initiating the germ tube formation
Candida albicans ATCC 10,231	farnesoic acid	Weak anti-filamentation activity compared to farnesol	[23, 24]
Saccharomyces cerevisiae	phenylethanol, tryptophol, tyrosol	induce pseudohyphal growth when nitrogen is limited	[25, 26]

Candida Albicans

C. albicans is a member of the human body’s normal flora or commensal microbial community, but it is also the leading fungal species responsible for oral and genital infections. This organism can exist in three distinct forms: budding yeast, pseudohyphae, and filamentous hyphae [27]. The shift from the yeast form to the hyphal form plays a key role in its ability to cause infection and is controlled by QS molecules [28].

Candida albicans employs multiple QS molecules to regulate morphology. Farnesol inhibits the yeast-to-hypha transition and slows biofilm maturation, tyrosol accelerates germ tube formation and promotes biofilm development, and farnesoic acid displays only weak anti-filamentation activity compared to farnesol, and it is present in strain ATCC 10,231. Farnesoic acid has only 3.3% of the activity of farnesol in blocking filamentation. Together, these molecules illustrate the nuanced, density-dependent regulation of morphogenesis in C. albicans [24, 29].

In addition to the molecules previously mentioned, two compounds, phenylethyl alcohol and tryptophol, have also been identified in Candida; their role in quorum sensing is yet to be confirmed [20].

Farnesol is an extracellular QS molecule, continuously produced in biofilms, during growth over a temperature range from 23 to 43 °C, and in amounts roughly proportional to the colony-forming units per ml (CFU/ml). Chemically, farnesol is an acyclic sesquiterpene alcohol, endogenously synthesized via the ergosterol pathway, and it is a heat-stable molecule, unaffected by extreme pH (partly responsible for this protective reaction). Its production is not dependent on the type of carbon or nitrogen source, or on the chemical nature of the growth medium [6, 22]. The secretion of farnesol was confirmed under various conditions in eight Candida species, including C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, C. guilhiermondi, C. kefyr, C. krusei, and C. glabrata, but its concentration and the effect on biofilm formation are the highest for C. albicans [30].

Tyrosol belongs to a group of phenolic compounds called phenylethanoids. In a proposed production pathway, tyrosine is converted into tyramine by tyrosine decarboxylase. Subsequent oxidation and reduction of tyramine result in the formation of tyrosol. However, growing evidence indicates that tyrosol is synthesized via tyramine, as tyrosine decarboxylase was identified in Rhodiola sachalinensis. Tyrosol is a powerful antioxidant compound, possibly more due to intracellular accumulation than to the antioxidant activity itself, which is weak compared with other molecules. Antioxidant activity is induced by scavenging ROS and nitrogen species that are related to human disease. On the other side, its antibacterial activity is exerted by binding and inhibiting bacterial ATP synthase [6].

Farnesoic acid is a natural product that is derived endogenously from isoprene compounds in prokaryotes and eukaryotes, including bacteria and fungi. The best-known biological functions of farnesoic acid are as a diffusible signal factor that regulates virulence in Xanthomonas campestris and as a QS molecule that inhibits hyphal formation in C. albicans. Farnesoic acid has been observed in strain ATCC 10,231 and shows weak anti-filamentation potency compared to farnesol [31].

There are still some key gaps in the complete understanding of the C. albicans QS system. The bona fide receptor(s) for farnesol remain undefined, and the molecular crosstalk between QS networks, central metabolism, and epigenetic regulation under host-like conditions is still unexplored. Future efforts integrating high-throughput receptor screens, single-cell transcriptomics, in vivo infection models, and computational QS network modeling will be essential to map the complete signaling cascade and reveal new antifungal intervention points.

Quorum Sensing Regulation in C. Albicans

Following the discovery of farnesol in C. albicans cultures, numerous studies have explored how this molecule influences morphogenesis. RT-PCR analyses revealed that farnesol inhibits MAP kinase signaling pathways by suppressing the expression of key genes like HST7 and CPH1. Hog1p phosphorylation also increased in the presence of farnesol, demonstrating that several morphogenesis-associated genes were downregulated (e.g., CRK1 and PDE2) and some upregulated (e.g., TUP1) by the presence of farnesol [32].

Researchers have extensively studied the signaling pathways that convert environmental cues into morphological changes. The Ras-cAMP-PKA pathway, along with the general repressor TUP1, plays a key role in how cells respond to farnesol [33]. Farnesol exerts broad regulatory effects on C. albicans signaling pathways, most prominently through inhibition of the Ras1-cAMP-PKA cascade. Farnesol disrupts the Ras-cAMP-Efg1 signaling cascade, which is essential for hyphal formation. However, this hyphal growth can be restored by supplementing with external dibutyryl-cAMP [34]. Transcriptional profiling of C. albicans in response to farnesol has revealed an increase in the expression of genes specifically regulated by the MAPK signaling pathway [35].

It has been suggested that farnesol can repress hyphal formation triggered by the cAMP-PKA pathway by inhibiting the Ras1-CDC35 signaling pathway [34]. There is a linear Ras1-cAMP-PKA signaling cascade in C. albicans, where Ras1 activates the adenylate cyclase Cdc35 to synthesize cAMP, which in turn relieves inhibition of the Protein Kinase A (PKA) subunits. Efg1 is a key downstream transcription factor phosphorylated and activated by PKA to drive the yeast-to-hypha transition [36].

One mechanism behind this repression appears to involve the upregulation of the global repressor TUP1. This idea is supported by findings showing that farnesol could not block hyphal development in tup1/tup1 and nrg1/nrg1 null mutants [37]. Additionally, farnesol interferes with the activation of Cek1p, a key component of the MAPK cascade involved in filamentation [32].

A key question regarding farnesol-mediated QS control is how its regulatory specificity is ensured in C. albicans. Shchepin et al. compared 40 natural and synthetic analogs of farnesol for their inhibition of C. albicans filamentation and demonstrated that farnesol has the greatest morphogenesis inhibitory activity. Accordingly, they hypothesized that there likely exists a farnesol cognate receptor that determines ligand binding specificity [38]. Figure 1 describes the QS-mediated functions and gene regulation in C. albicans.

Fig. 1.

QS-mediated functions and gene regulation in C. albicans. While tyrosol promotes filamentation and biofilm formation, decreasing the length of the lag phase, farnesol inhibits these processes and increases the lag phase. Likewise, farnesol inhibits MAP kinase cascades by suppressing the expression of HST7 and CPH1 genes. Hog1p phosphorylation also increased in the presence of farnesol, demonstrating that several morphogenesis-associated genes were downregulated (e.g., CRK1 and PDE2) and some upregulated (e.g., TUP1) by the presence of farnesol. Farnesoic acid, secreted by strain ATCC 10,231, has low quorum-sensing activity but induces neutrophil apoptosis by activating Cas-3 and Cas-7, preventing NET release. The figure was created using BioRender.com

Role of QS in C. Albicans Infections

Candida species rank among the most commonly isolated fungi in wound environments, which are highly conducive to biofilm formation, a key driver of persistent and recurrent infections [39]. Oral carriage of fungi, particularly Candida spp., occurs in virtually all individuals at some point in life, with continuous colonization observed in roughly 40% of humans. C. albicans has emerged as one of the most prevalent microorganisms in hospital-acquired infections, where candidemia carries a mortality rate approaching 50%. Opportunistic infections by Candida arise when host immune defenses are compromised, ranging from reduced salivary flow due to ill-fitting dentures (denture stomatitis) to HIV-1 infection or postsurgical immunosuppression. Importantly, the pathogenic success of Candida is closely linked to its ability to undergo morphological transitions and establish biofilms, processes that directly enhance adhesion, persistence, and tissue invasion [40]. This critical role of morphological plasticity is further underscored by findings that mutant strains lacking the ability to form hyphae exhibit significantly reduced virulence in both mucosal and systemic infection models, highlighting the contribution of filamentation and biofilm architecture to pathogenicity [41].

Beyond providing structural integrity, Candida utilizes QS molecules, mainly farnesol and tyrosol, to regulate its virulence repertoire. Farnesol, produced endogenously during early biofilm development, modulates filamentation to limit excessive tissue damage and enhance fungal persistence. In murine models of systemic candidiasis, farnesol promotes endothelial adhesion and translocation by activating host MAPK and NF-κB pathways, thereby fostering a cytokine milieu favorable to fungal survival [6].

In contrast, tyrosol secretion under nutrient-rich and hypoxic conditions accelerates hyphal extension and epithelial invasion. Elevated tyrosol levels in oropharyngeal candidiasis models are associated with increased epithelial barrier disruption and upregulation of proinflammatory cytokines such as IL-1β and TNF-α [42].

Sessile Candida biofilms demonstrate a five- to eightfold increase in resistance to all clinically approved antifungal agents compared to planktonic cells. This multifactorial resistance stems from heightened metabolic activity during early biofilm formation and the emergence of dormant persister cells with exceptional drug tolerance. Additionally, the extracellular polymeric matrix comprising up to 90% of the biofilm’s dry mass acts as a physical barrier that impedes antifungal penetration [43, 44].

Given the dual role of QS in both virulence and drug resistance, therapeutic strategies have shifted from simple exogenous application of QS molecules to precise modulation of endogenous signaling pathways. Farnesol-based small-molecule inhibitors not only disrupt biofilm maturation in vivo but also suppress QS-regulated virulence mechanisms [22]. Similarly, engineered probiotic strains capable of sequestering tyrosol restore the yeast–hypha equilibrium, mitigate tissue damage, and enhance the efficacy of conventional antifungal treatments [45].

Saccharomyces Cerevisiae

S. cerevisiae, baker’s or brewer’s yeast, is a unicellular fungus vital for fermentation in baking, brewing, and winemaking. It ferments sugars to produce carbon dioxide and alcohol, enhancing the texture and flavor of products. As the first eukaryotic genome sequenced, it serves as a key model organism in molecular biology and biotechnology, contributing to research and the production of biofuels and pharmaceuticals. S. cerevisiae yeast is the most common microorganism in industrial ethanol production due to its safety and tolerance to various physiological stresses compared to other yeasts and bacteria. It can create different multicellular forms in response to environmental conditions by using adhesive proteins, adhesins, and flocculins. These forms include clots, flowers, biofilms, and filaments [46, 47].

In liquid environments, it can be sessile or planktonic and may also form flocs and flors. In solid and semi-solid environments, it may also be observed in the form of biofilms, colonies, filaments, and mats. S. cerevisiae grows in filaments in conditions of a lack of nutrients and searches for and receives food [48, 49].

The type of filamentous growth in S. cerevisiae varies depending on the cell type. In diploid cells, the growth is false hyphal, where the cells grow as long filaments. While in haploid cells, the growth is aggressive, where the cells penetrate the agar. This feature helps the cell to function best in different environmental conditions. Saccharomyces causes cellular responses in different environmental conditions, such as temperature, osmotic pressure, toxins, hunger, and radiation level [50, 51].

In S. cerevisiae, the synthesis of QS molecules is influenced by cell density. Yeast cells in high-density populations produce more QS molecules per cell compared to those in low-density conditions. Upon reaching a critical population threshold or quorum, the expression of the genes ARO9 and ARO10 is significantly upregulated, leading to enhanced biosynthesis of aromatic alcohols. Phenylethanol (Phe-OH), tryptophol (Trp-OH), and tyrosol (Tyr-OH) have been identified as the main QS molecules in S. cerevisiae. Factors such as cell density, amount of nitrogen, and ethanol for growth play a role in the production of these molecules [52]. Phe-OH, Trp-OH, and Tyr-OH are made from the amino acids phenylalanine, tryptophan, and tyrosine, respectively, during the processes of transamination, decarboxylation, and reduction, where nitrogen is low [53]. Chen and Fink declared ARO genes responsible for the production of QS molecules, including 2-phenylethanol, tryptophol, and tyrosol [54].

The studies conducted by Kang et al. showed that ARO80 is the main gene involved in the QS process, such that its deletion can reduce the oxidative tolerance of yeast cells [55]. Aro80p is a transcription factor of the Ehrlich pathway that causes the expression of ARO9 and ARO10 genes. ARO10 and ARO9 gene expression increase with the increase in the number of cells, causing the production of aromatic alcohols [56].

Previous research has shown that different aromatic alcohols have different effects on cell morphology depending on the type of yeast, which was described earlier in Table 1. In S. cerevisiae, Phe-OH and Trp-OH compounds act as effective stimuli in changing the shape and structure of cells, and Tyr-OH has less effect in this context [54]. So far, the mechanism of Phe-OH and Trp-OH has been studied more, and the mechanism of Tyr-OH has rarely been studied. The level of cAMP plays an important role in cell filament growth, regulating the activity of PKA and TPK2, the catalytic subunit of PKA, and increasing filament growth [57]. TPK2 leads to the phosphorylation and activation of FLO8p [58]. The QS molecules Phe-OH and Trp-OH induce pseudohyphal growth when nitrogen is limited. The research conducted by Nath et al. shows that Tyr-OH plays an important role in improving the tolerance of S. cerevisiae to heavy metals [59]. Gonzalez et al. showed the effect of food-related aromatic alcohols on invasive and pseudohyphal growth [60]. The response of S. cerevisiae haploid cells to mating pheromones is one of the most well-known QS mechanisms, where QS molecules stimulate filamentous growth through the MAPK pathway [61, 62].

Biofilm regulation is one of the other processes regulated by QS in which Phe-OH has a positive effect on biofilm formation [63]. Flocculation is a social behavior in microorganisms that appears as the accumulation of cells in a coherent and connected structure. This behavior helps cells to survive better in harsh environmental conditions. Smukala et al. investigated the increase of flocculation by adding the Trp-OH in a strain of S. cerevisiae [64].

Quorum Sensing Regulation in S. Cerevisiae

S. cerevisiae synthesizes QS molecules such as phenylethanol (Phe-OH), tryptophol (Trp-OH), and tyrosol (Tyr-OH) from amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr) through transamination, decarboxylation, and reduction by the Ehrlich pathway under low concentrations of nitrogen [53]. When aromatic amino acids are the sole nitrogen source, the expression of GAP1 is induced, which leads to the activation of the general amino acid permease Gap1p. Furthermore, the amino acid osmotic factor (Ssy1p) works like a sensor for extracellular aromatic amino acids and paves the way for the aromatic amino acids’ transportation into the cells [65, 66]. Subsequently, Aromatic amino acid transaminase, which is expressed by the ARO8 and ARO9 genes, catalyzes the transamination of phenylalanine, tyrosine, and tryptophan into α-keto acids such as phenylpyruvate (PPA), indole-3-pyruvate (IPA), and 4-hydroxyphenylpyruvate (4-HPPA), respectively. After these acids are decarboxylated, phenylacetaldehyde (PAA) [67], indole-3-acetaldehyde (IAAld) [54, 56], and hydroxyphenyl acetaldehyde (4-HPAA) [68] are produced by the products of the ARO10, PDC1, PDC5, and PDC6 genes, which encode decarboxylases [69, 70]. Then, under the action of dehydrogenase expressed by alcohol dehydrogenase genes (ADH1-5) and formaldehyde dehydrogenase gene (SFA1), these aldehydes are reduced to QS molecules such as Phe-OH, Trp-OH, and Tyr-OH [71]. Finally, transporters carry aromatic alcohols out of the cell, where they accumulate to a certain concentration and cause quorum-sensing-related phenotypes such as filamentous growth or flocculation. These Tyr-OH and Phe-OH, through the transcription factor Flo8p and the cAMP-dependent PKA component Tpk2p, influence the up-regulation of FLO11. The product of FLO11, Flo11p, is involved in cell adhesion, unipolar/bipolar growth pattern, pseudomycelium growth, filamentous growth, and cell elongation [48, 64, 72].

Thus far, the regulation procedure of the pathway remains unclear, but advances in some fields, like bioinformatics and different studies on yeast responses to aromatic alcohols, have predicted several potential regulators. One such regulator is the transcription factor Aro80p, which controls the expression of genes in the Ehrlich pathway’s response to aromatic amino acids [54]. Wuster and Babu in 2010 suggested that five transcription factors, such as Cat8p, Mig1p, Sip4p, Rgm1p, and Msn2p, possibly are the key transcriptional regulators in the yeast’s Ehrlich pathway, influencing the expression of genes affected by aromatic alcohols [73]. In more detail, Cat8p, which is a zinc cluster protein, represses the ADH2 gene while Mig1p, with two zinc finger motifs, regulates CAT8. These genes are proposed to participate in quorum sensing and are also crucial for aromatic alcohol-mediated communication. Hence, Wang et al. confirmed that the CAT8 overexpression and MIG1 deletion strains can show a higher rate of Phe-OH production [74, 75]. Figure 2 illustrates the production of QS molecules and the QS-mediated functions in S. cerevisiae.

Fig. 2.

QS molecules and the QS-mediated functions in S. cerevisiae. The modulation of the Ehrlich pathway in Saccharomyces cerevisiae focuses on the biosynthesis of higher alcohols through transamination, decarboxylation, and dehydrogenation steps. Key genes such as ARO8, ARO9, ARO10, PDC1, PDC5, PCD6, and ADH1–5 are involved in these processes, with evidence of up-regulation under conditions with specific cell density and Nitrogen amount, which are fundamental in converting phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr) to aromatic alcohols like phenylethanol (Phe-OH), tryptophol (Trp-OH), and tyrosol (Tyr-OH). Regulatory elements, such as the cAMP/PKA pathway and the FL011 gene, influence this response, linking metabolic output to environmental signals, including unipolar/bipolar growth patterns, pseudomycelium growth, cell elongation, and filamentous growth. Furthermore, Tyr-OH, Trp-OH, and Phe-OH have effects on metal tolerance, flocculation, and biofilm formation, respectively. The figure was created using BioRender.com

Beyond these transcription-level regulatory mechanisms, a recent multi-omics study revealed how QS signals reshape broader cellular physiology in S. cerevisiae. Britton et al. demonstrated through proteomic analysis that nitrogen limitation has a strong impact on ribosomal proteins and related pathways, highlighting extensive cellular reprogramming under nutrient stress. Interestingly, no proteomic shift was observed in response to 2-phenylethanol (2-PE) across the tested strains, raising questions about its precise role as a QS signal in S. cerevisiae. This finding suggests that 2-PE may act in a more nuanced manner, subtly influencing targeted biochemical processes rather than driving broad proteomic changes. In contrast, lipidomic and metabolomic profiling revealed clear and substantial alterations, pointing to 2-PE’s primary influence on membrane architecture and metabolic fluxes. Although these changes appear less dramatic than proteomic responses, they likely play pivotal roles in yeast adaptation during high-density, resource-limited fermentations, supporting both survival and metabolic efficiency [76].

Different Factors Related to QS Molecule Production in S. Cerevisiae

S. cerevisiae’s production of QS molecules (QSMs) is affected by various environmental factors, including density of cells [26, 56], nitrogen concentration [54], ethanol concentration [56], and aerobic/anaerobic growth conditions [77]. Among the factors, Cell density plays a crucial role in regulating QSM production when higher densities result in greater production per yeast cell [50]. Owing to the higher concentration of QSMs, the expression of ARO9 and ARO10 genes is regulated, which leads to increased production of aromatic alcohols [56]. Trp-OH also activates the transcription factor Aro80p, which in turn induces the transaminase and decarboxylase genes’ expression and leads to the formation of a loop [26, 73].

The production of Phe-OH, Trp-OH, and Tyr-OH begins when cell mass reaches between 2 × 10⁷ and 3 × 10⁷ cells/mL, 3 × 10⁷ and 4 × 10⁷ cells/mL, and 2 × 10⁷ and 4 × 10⁷ cells/mL, respectively, indicating these concentrations as the quorum for synthesis under fermentation conditions. Nitrogen content in the medium also affects QSM production, with high ammonium concentrations inhibiting the expression of enzymes necessary for aromatic alcohol synthesis. Aromatic alcohol production peaks when the concentration of ammonium is ≤ 50 mM and significantly decreases at concentrations > 500 mM. Additionally, ethanol produced during S. cerevisiae fermentation negatively impacts the overall rate and process of aromatic alcohol synthesis and inhibits cell growth as well [56].

Last but not least, the aerobic and anaerobic conditions also influence aromatic alcohol production, with alcohol dehydrogenase genes upregulated under anaerobic conditions, leading to enhanced production. S. cerevisiae alters phenylalanine to a ratio of 1:9 phenylacetate and Phe-OH in the presence of oxygen, while anaerobic conditions result in a nearly complete proportion of Phe-OH [78, 79].

QS in S. cerevisiae acts beyond its role as a model system and has an important role in industrial-scale fermentation. Aromatic alcohols such as phenyl ethanol and tryptophol have a significant effect on shaping aroma and flavor profiles in fermented beverages. QS-driven control of biofilm development and flocculation also affects the fermentation process. Flocculent yeast strains precipitate more efficiently and simplify downstream processing. In addition, QS signaling contributes to ethanol tolerance and enables yeast populations to endure high alcohol profiles and maintain their efficiency. Thus, targeted manipulation of QS pathways through strain engineering or precise control of environmental conditions represents a promising approach to enhance bioethanol production and improve the overall quality of fermented products [80, 81].

Basic biological processes responsible for these kinds of effects, as well as the reasons behind the observed differences in responses of different strains to these compounds, have not been fully understood. Nevertheless, these results highlight QS as a novel approach for enhancing ethanol yield. A deeper understanding of QS in S. cerevisiae can enable precise control over cell growth and carbon flux during the fermentation process and ultimately lead to improved production of valuable metabolites.

Parasitic Protozoa Quorum Sensing

The process of QS, previously reported in a limited number of protozoa such as Tetrahymena [82] and Paramecium [83, 84], demonstrates that these unicellular organisms are capable of regulating population density and coordinating collective behaviors through chemical and physical signaling. In this article, however, the focus is directed more specifically toward Trypanosoma, where emerging evidence indicates that QS mechanisms not only govern the parasite’s life cycle but also play critical roles in differentiation and adaptation within the host. These findings provide new perspectives on protozoan biology and suggest potential therapeutic avenues against trypanosome-related diseases.

Trypanosoma

African trypanosomes are single-celled protozoan parasites that live both in mammalian hosts and in the Tsetse fly vector during their life cycle. These organisms are evolutionarily distinct from animals, fungi, and plants, belonging to a very particular order, Kinetoplastida, within the phylum Euglenozoa. A defining feature of this group is their unique mitochondrial DNA, known as kinetoplastid DNA (kDNA). Within the Kinetoplastida, the Trypanosomatids have evolved to lead an obligatory parasitic lifestyle. Notably, species within the Trypanosoma and Leishmania genera spend their lives between a mammalian host and an insect vector [85, 86].

Inside the mammalian host, African trypanosomes initially exist as slender, replicative forms that establish and sustain the infection. As parasite levels in the bloodstream rise and reach a critical threshold, these slender forms begin to differentiate, first into intermediate forms, and eventually into non-dividing short stumpy forms, which are arrested in the G0/G1 phase of the cell cycle [87]. This density-dependent transformation is a type of QS that helps prolong the host’s survival and prepares the parasite for successful uptake and transmission to the Tsetse fly [8]. Research into this QS mechanism has revealed that both pleomorphic strains (capable of differentiating into stumpy form) and monomorphic strains (which have lost this ability through repeated passage in animals or culture) produce a soluble, low-molecular-weight, heat-stable compound known as the stumpy induction factor (SIF) [88].

Stumpy Inducing Factor (SIF) refers to parasite-derived oligopeptides generated by Zn-dependent peptidases, which act as density-dependent signals promoting differentiation into the non-proliferative stumpy form. Although initially described as a soluble, low-molecular-weight heat-stable compound, subsequent studies suggest that SIF may represent a mixture of trypanosome-derived peptides rather than a single defined molecule. Host contributions have been proposed but remain less substantiated [89]. For clarity, this review adopts the consensus view that SIF is a parasite-derived signaling mixture.

The life cycle of Trypanosoma. brucei involves several key stages: (1) After a Tsetse fly feeds on an infected mammal, the stumpy forms of T. brucei enter the fly’s midgut and differentiate into procyclic forms. (2) The procyclic cells migrate through the midgut and eventually reach the proventriculus. (3) In the proventriculus, the parasites differentiate into epimastigote forms, which then move to the salivary glands where they develop into metacyclic forms, the infective stage for mammals. (4) During a subsequent bite, the Tsetse fly injects metacyclic trypanosomes into a new mammalian host, where they differentiate into slender bloodstream forms. (5) In the mammalian bloodstream, slender forms multiply and increase parasitemia. Once the parasite density reaches a critical level, a QS signal triggers their differentiation, via an intermediate form, into stumpy cells that are preadapted for uptake by the Tsetse fly [90, 91]. Figure 3 describes the life cycle of Trypanosoma in humans and the tsetse fly.

Fig. 3.

The life cycle of Trypanosoma in humans and the tsetse fly. In the mammalian host, Trypanosoma exists as long, slender proliferative forms in the blood, and density-dependent QS peptide signals trigger differentiation into short-stumpy non-dividing forms upon reaching a certain threshold. These are pre-adapted for transmission to the tsetse fly vector, where they differentiate into procyclic proliferative forms in the midgut, then into epimastigote forms. The parasites migrate to the salivary glands, transforming into metacyclic, non-dividing forms. These are infectious and ready to be transmitted back to a mammal, restarting the cycle. The figure was created using BioRender.com

The slender-to-stumpy differentiation could be a density-dependent phenomenon that involves the inhibition by contact. The exogenous factor or inducer could be either a host product, a trypanosome product, or a product of the host-parasite interaction [92].

More recently, an experimentally validated model for density-dependent stumpy induction has been developed, sharing similarities with the QS response of Gram-positive bacteria, which utilize secreted peptides as self-inducers. Typically, the peptides are released through a specific ATP-binding cassette (ABC) transporter. A member of the GPR89 protein family that spans the membrane has been demonstrated to be involved in stumpy formation [89].

Genomic studies of trypanosomes led to the discovery of a gene encoding a protein from the orphan GPCR-like family, known as TbGPR89 [92]. Structural predictions using the iTASSER software indicated that TbGPR89 shares similarities with proton-oligopeptide transporters (POTs), which are found in both bacteria and humans [8]. Supporting this idea, researchers found that introducing a bacterial oligopeptide transporter into slender-form parasites triggered early development into stumpy forms. Similarly, exposing trypanosomes to complex oligopeptide mixtures or synthetic dipeptides and tripeptides also induced premature stumpy formation [89].

To investigate the role of oligopeptides in stumpy form differentiation, researchers exposed Trypanosoma brucei parasites to varying concentrations of broths, which served as a source of oligopeptides. This treatment led to reduced cell proliferation and the expression of stumpy-specific markers. Additionally, mixtures of chemically synthesized dipeptides and tripeptides were tested, with tripeptides proving more effective at inducing stumpy formation, especially those containing N-terminal amino acids such as Asn, Gln, His, Phe, Asp, and Trp [8]. These findings support the conclusion that oligopeptide signaling works through the parasite’s natural SIF (stumpy induction factor) pathway. Parasites that are unresponsive to SIF also fail to respond to oligopeptides or to the ectopic expression of TbGPR89, which normally induces differentiation [93, 94].

Molecular Mechanism of QS Regulation of Trypanosoma

The slender to stumpy differentiation is a density-dependent response similar to QS in other microbial systems and is crucial for the parasite life cycle, including infection chronicity and disease. The first regulators linked to stumpy formation in Trypanosoma brucei were two protein kinases, a MAP kinase and another kinase, whose deletion promoted stumpy differentiation. This suggests they act as negative regulators of the process. Similarly, silencing TbTOR4, an unusual component of the target of rapamycin (TOR) complex in T. brucei, induced laboratory-adapted cell lines unresponsive to the physiological QS signal to become stumpy in vivo. This finding points to the TOR signaling pathway as a key inhibitor of stumpy development. Similar to the nutrient-sensing mechanisms mediated by TOR in many eukaryotes in response to ATP/AMP balance, a cellular energy level sensor, AMPK, promotes stumpy formation [93]. Among the AMPK isoforms in T. brucei, AMPKα1 has been identified as a key driver of stumpy formation, activated through phosphorylation [95]. However, there may be redundancy between AMPKα1 and AMPKα2, as both must be depleted using RNA interference (RNAi) to block AMP-induced growth inhibition. Interestingly, AMPKα2, but not AMPKα1, was identified in a genetic screen as an activator of stumpy formation, suggesting both isoforms contribute but in distinct ways [96].

The developmental shift to the stumpy form is triggered by an elusive signaling molecule known as stumpy induction factor (SIF). Although laboratory-adapted, monomorphic strains of trypanosomes respond poorly to SIF, they can still adopt stumpy-like features when treated with cell-permeable analogs of cAMP and AMP. A recent study has used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate genetic screens, monomorphic T. brucei parasites were treated with two signaling analogs, 8-(4-chlorophenylthio)-cAMP (pCPTcAMP) and 8-pCPT-2’-O-Me-5’-AMP, to identify mutants that failed to respond and remained proliferative. These screens aimed to uncover genes involved in the signaling pathways that trigger stumpy differentiation. Two screens were performed and identified 43 genes that were potentially targeted in either screen. Twelve genes were common to both screens; 5 were 8-pCPT-2’-O-5’-AMP-specific, and 26 were pCPTcAMP-specific. After analyzing the reads for genome alignment and the presence of appropriate RNAi library primer flanks, the search was refined to narrow down to 27–30 distinct gene targets [96].

These included genes encoding molecules associated with purine salvage, likely selected based on their capacity to rebalance AMP levels within the parasites rather than a function in SIF signal transduction. In contrast, several other genes were more directly associated with signal transduction pathways. These included AMPKα2, several protein kinases, protein phosphatases, and predicted RNA-binding proteins. Many of these molecules had already been implicated in separate studies for their roles in regulating mRNA or protein levels, further supporting their involvement in controlling stumpy formation [97].

The predicted RNA-binding protein RBP7 has been shown to play an important role in the ability of trypanosomes to respond to cell-permeable cAMP and to undergo SIF-induced differentiation from pleomorphic to stumpy forms in vivo. Studies using null mutants revealed that loss of the RBP7AB gene reduced the parasites’ capacity to differentiate, although it did not entirely eliminate their ability to form stumpy cells or remain competent for differentiation [98]. The predicted RNA-binding proteins are relatively small, consisting of just 116 amino acids, and contain a single RNA recognition motif (RRM) domain. This is notably similar to RBP6, another RNA-binding protein − 239 amino acids in length- that serves as a major regulator of developmental transitions within the Tsetse fly [99].

After confirming the impact of RBP7AB deletion on stumpy formation, Cayla and McDonald further explored the interaction between RBP7 and YAK, a gene encoding a predicted DYRK-family protein kinase. Their findings showed that RNAi knockdown of YAK also led to reduced stumpy formation. When YAK null mutants were generated, the parasites remained virulent in vivo and showed reduced accumulation in the 1K1N (1 kinetoplast, 1 nucleus arrested cells) configuration. Additionally, the mutants failed to express PAD1, a cell surface marker specific to stumpy cells, which was confirmed using flow cytometry [98].

Detailed mutational and functional studies of YAK’s catalytic activity revealed that this kinase exists in a unique, preactivated state, differing significantly from typical eukaryotic kinases. Unlike the highly conserved DFG motif found in most kinases, trypanosomes’ YAK features a DFS sequence, along with an unusual HxY motif in its activation loop. These sequence variations are uncommon in other eukaryotes but appear more frequently in Trypanosomatids, suggesting that these parasites use a distinct regulatory model for kinase activation. Phosphoproteomic analysis of predicted downstream targets of YAK provided further insights. YAK (TbDYRK) appears to suppress proteins that maintain the slender form, while activating proteins involved in promoting stumpy formation, pointing to a pivotal position in development [93, 98].

In addition to RBP7A/B and YAK, genome-wide RNAi screens targeting resistance to the cell-permeable cAMP analog pCPTcAMP also identified protein phosphatase 1 (PP1) as a potential regulator of stumpy formation. This role was validated in vivo by simultaneously silencing three PP1 genes, PP1-2, PP1-5, and PP1-6, which resulted in a complete loss of stumpy cell formation [96]. Further analysis revealed that PP1-6 could promote premature stumpy differentiation in both wild-type parasites and in parental cells and RBP7AB null mutants, but not in YAK null mutants. In the absence of YAK, PP1-6 activation produced abnormal cells rather than functional stumpy forms. These findings suggest that while PP1-6 acts independently of RBP7AB, its ability to drive differentiation is dependent on YAK, placing PP1-6 downstream of or functionally linked to YAK in the regulatory pathway [98].

In addition to positive regulators, stumpy formation inhibitors have also been discovered. TbTOR4 stands out among them as a slender retainer [45]. If TbTOR4 is organized similarly to other eukaryotes, its effect should be near the top of a signal transduction cascade. Also, MAP kinase kinase kinases (MEKK) and MAP kinase kinases (MEK) are anticipated to be near the top of a traditional signaling cascade [100, 101].

MEKK1 is not required for the effective expression of the stumpy marker PAD1, whereas TbTOR4 depletion causes cell arrest regardless of MEKK1’s existence. Since arrest occurs before PAD1 expression, this shows that both TbTOR4 and MEKK1 are engaged in cell-cycle arrest, but that MEKK1 is necessary for effective development to stumpy forms. TbTOR4 and MEKK1 are both proteins in the development pathway that result in the production of stumpy cells [98].

Trypanosome Coinfection and Interspecies Signaling

The QS mechanism has been recently identified as a between-phyla or inter-kingdom signaling factor and could form the extensive communication between microorganisms and their hosts. For instance, bacterial products can influence mammalian cell signaling, while host hormones can interact and cross-signal with bacterial QS systems, affecting bacterial gene expression. This highlights a complex interplay between host stress responses, microbial QS signals, and disease progression [8]. QS pathways controlling stumpy formation in T. brucei are conserved across African trypanosomes, including T. congolense and T. vivax. Even though these species don’t produce morphologically distinct stumpy forms, they still respond to density-dependent signals that regulate transmission competence. T. brucei develops a clear stumpy form (non-dividing, transmission-competent), whereas T. congolense and T. vivax don’t show this morphology but still undergo cell cycle arrest and metabolic reprogramming, meaning the molecular program is conserved even if the phenotype differs. In mixed infections, QS signals from one species can manipulate the transmission potential of another, reshaping parasite dynamics in the host and vector. Intriguingly, QS signals are shared between trypanosome species, enabling cross-species communication during coinfections. When a host already infected with T. congolense is superinfected with T. brucei, the T. brucei population undergoes premature differentiation into stumpy forms. This response depends on the integrity of the QS signaling pathway, emphasizing the evolutionary conservation and ecological importance of this communication system [102].

The sharing of QS signals among trypanosome species can drive premature differentiation into non-dividing stumpy forms, effectively limiting the parasite’s ability to transmit and reducing their overall fitness during infections involving competing genotypes. This evolutionary pressure can lead to the selection of parasites with reduced sensitivity to QS signals, helping to re-establish a balance between transmission potential and virulence. However, if these co-infection-adapted parasites find themselves in a monoinfection scenario, such as when competing parasites are eliminated by drug treatment or are naturally nonviable due to factors like serum trypanolytic activity, the consequences can shift dramatically. In the absence of competition, their reduced responsiveness to QS signals may result in heightened virulence and more severe disease outcomes for the host [93].

The coexistence of multiple trypanosome species within the same host may help explain the distinct biological strategies each species employs. The prevalence of trypanosome species varies geographically, with T. vivax and T. congolense being more widespread, while T. brucei tends to be relatively rare. As a result, T. brucei is more likely to face interspecies competition, which may have driven selection for extreme developmental flexibility, producing both antigenically diverse slender forms for immune evasion and transmission-optimized stumpy forms for vector uptake. This adaptive flexibility likely helps T. brucei persist in environments dominated by more prevalent species, giving it a competitive edge. Additionally, the ability of T. brucei to generate mosaic variant surface glycoproteins (VSGs), a dense and uniform surface coat made from a single VSG type, serves as a powerful immune shield, allowing the parasite to evade host defenses by continuously varying its surface identity. Frequent genetic recombination in T. brucei has played a key role in its evolutionary success, enabling the development of unique adaptations such as SRA and TgsGP- both believed to have evolved from VSG genes. These adaptations allow T. brucei to infect hosts that possess serum trypanolytic factors, helping it escape competition with other trypanosome species that cannot survive in such environments. Additionally, the distinct tissue tropisms exhibited by different trypanosome species may represent further adaptations for co-infection. For example, T. brucei tends to specialize in tissues beyond the bloodstream, whereas T. congolense is largely restricted to the blood vessels. This spatial separation- local avoidance- may reduce direct competition and allow each species to remain primarily exposed to its own QS signals, thus avoiding cross-species interference. Thereby, each species can optimize its developmental responses and transmission strategies in mixed infections [93, 103].

Roles of QS in Trypanosome Infections

As outlined in Trypanosoma section, SIF is considered a parasite-derived signaling mixture. Subsequent mechanistic studies have demonstrated that African trypanosomes alternate between a mammalian host and the tsetse fly vector, employing a density-dependent QS-like mechanism to orchestrate differentiation from proliferative ‘slender’ forms to transmission-competent ‘stumpy’ forms. In this process, parasite-derived oligopeptides generated by Zn-dependent peptidases accumulate in the bloodstream and, upon reaching a critical threshold, engage the G protein–coupled receptor TbGPR89 on slender cells. This signaling cascade triggers cell cycle arrest and morphological transformation, enabling survival in the tsetse midgut and subsequent transition to procyclic stages [89].

Cutting-edge single-cell transcriptomic and proteomic analyses have revealed that SIF binding induces a rapid rise in cyclic AMP levels, activation of MAPK cascades, and recruitment of the histone acetyltransferase TbHAT2 to differentiation-associated loci, resulting in targeted histone acetylation changes [5]. Heat-shock proteins and other epigenetic regulators modulate this QS signal, buffering the slender-to-stumpy switch against host-induced stressors such as fever and immune pressure [104].

Most drugs used to treat trypanosomiasis, such as diamidines, suramin, and melaminophenyl arsenicals, have been around for a long time and tend to be broadly toxic to all cells. These drug classes were not developed for a specific intracellular target, meaning resistance is unlikely to arise from mutations that alter a particular intracellular protein. Instead, resistance is more commonly linked to mechanisms of cellular uptake or distribution of drugs within cells [105].

For instance, TbGPR89 is expressed on bloodstream trypanosomes in their “slender form,” which responds to the SIF signal. When ectopically expressed, this receptor triggers the transition to the stumpy form through an SIF-dependent mechanism. If a stable oligopeptide signal could be used to induce premature stumpy formation, it might serve as a broad anti-virulence strategy through quorum-sensing interference. Since TbGPR89 is crucial for both slender form survival and stumpy formation, it creates a dual safeguard against drug resistance. Any mutant parasites that evade TbGPR89 would also lose their ability to be transmitted, making resistance unlikely to spread [89]. Emerging approaches, such as vector-targeted interventions and metabolic interference strategies, could interrupt parasite signaling and mitigate transmission potential.

Conclusions

QS is a signaling mechanism that enables microbes to detect population density and regulate collective behavior. However, QS is not exclusive to bacteria; eukaryotic microorganisms, including yeasts, also employ QS-based systems to regulate essential cell processes, such as biofilm formation, morphological transitions, colony development, and hyphal growth. Similarly, protozoan parasites rely on QS for density sensing within their hosts to regulate their transformation, which is crucial for successful transmission.

Ongoing research continues to investigate the complex connections between infectious agents and diseases, aiming to uncover the adaptive mechanisms that enable microbes to reemerge in the environment. The rapid advancement of modern science has significantly improved disease diagnosis and treatment, effectively overcoming various technical challenges. QS mechanisms play a key role in microbial virulence, affecting host colonization by fungal and protozoan pathogens. Investigating QS in eukaryotic microorganisms presents promising avenues for antifungal therapies and parasite control.

Author Contributions

M.H.M. M.A. and H.R.Sh. reviewed the literature and prepared the draft of the manuscript. M.S.D. prepared the figures and reviewed the initial draft. Z.F. supervised the team, finalized the initial draft, and revised it. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Competing Interests

The authors declare no competing interests.