The research team, led by Olivia K. A. Paulin and Jonathan P. Richardson, constructed an entire panel of mutant C. albicans strains in a single genetic background, using a recyclable CRISPR-Cas9 genome editing system. Rather than comparing knockout strains generated in different laboratories with different histories—a practice that has historically made cross-study comparisons unreliable—the researchers built homozygous single, double, triple and quadruple gene deletions additively, recycling the CRISPR cassette after each round of mutagenesis. The four genes targeted were ALS3, which encodes the hypha-associated adhesin Als3p that binds host receptors such as E-cadherin; ECE1, which encodes candidalysin, the cytolytic peptide toxin critical for host cell damage; HGC1, which encodes a G1-cyclin protein essential for maintaining hyphal morphogenesis; and SAP2, which encodes a secreted aspartyl proteinase that degrades host proteins for nutrient acquisition and immune evasion. This architecture allowed the team to evaluate each factor individually and in every meaningful combination during systemic infection in mice.

The experimental design was straightforward but powerful. Immunocompetent female C57BL/6 mice, six to seven weeks old, were injected intravenously via the tail vein with five hundred thousand C. albicans cells of either the wild-type parental strain or one of the mutant derivatives. One cohort was sacrificed three days later for quantitative organ fungal burden analysis—kidneys and brains were harvested, homogenised, serially diluted and plated onto agar in triplicate to count colony forming units. A parallel cohort was monitored daily for three weeks to measure survival, with animals humanely euthanised upon becoming moribund or losing more than twenty percent of their initial body weight. The results from these two readouts, collected across biological replicates, told strikingly different stories about the same set of fungal mutants.

The kidney findings were, at first glance, underwhelming. On day three, no mutant strain showed a significant difference in kidney fungal burden compared with wild-type, regardless of which combination of virulence genes had been deleted. The kidney is generally regarded as the principal target organ in murine systemic candidiasis, so this result suggested that Als3p, candidalysin, Hgc1p and Sap2p are collectively dispensable for controlling fungal growth in that organ. But the brain told an entirely different story. Wild-type-infected mice carried a median of approximately 5,620 colony forming units per gram of brain tissue. Deletion of ECE1 alone drove the burden up to 55,900 CFU/g, and deletion of HGC1 alone to 42,950 CFU/g. When the two deletions were combined, fungal burdens exploded to 802,000 CFU/g—a more than hundredfold increase over wild-type. Adding further deletions pushed the numbers higher still: the triple mutant lacking ALS3, ECE1 and HGC1 reached 1,230,000 CFU/g, the SAP2/ECE1/HGC1 triple mutant 1,058,000 CFU/g, and the quadruple mutant 1,375,000 CFU/g. Meanwhile, mutants retaining either ECE1 or HGC1 showed brain burdens indistinguishable from wild-type.

This pattern revealed something biologically important: hyphal formation and maintenance, governed by Hgc1p, and candidalysin production, governed by Ece1p, act in concert to restrict fungal growth in the brain—apparently by provoking protective innate immune responses. The authors connect this to established mechanisms of cerebral antifungal immunity. Previous work has shown that CARD9-positive microglia in the brain promote antifungal defence through interleukin-1β and CXCL1-mediated recruitment of neutrophils, and that microglial Toll-like receptor 4 and CD11b coordinate the eradication of cerebral C. albicans infection. Candidalysin, with its capacity to damage host cells and trigger pro-inflammatory signalling, appears to function as a double-edged sword: it injures tissue, but it also sounds the alarm that summons the immune cells needed to contain the fungus. Without both hyphae and toxin, the brain becomes permissive territory for uncontrolled fungal proliferation.

The twenty-one-day survival experiments complicated the picture further, and in doing so exposed the limits of using fungal burden as a proxy for disease severity. Wild-type infection yielded a survival rate of only twenty-five percent, confirming the lethality of the strain. Among single-deletion mutants, loss of HGC1 raised survival to sixty-seven percent and loss of SAP2 to seventy-five percent, while loss of ALS3 or ECE1 barely moved the needle at thirty-three and twenty-three percent respectively—indicating that hyphal growth and Sap2p, not candidalysin, are the strongest individual drivers of mortality in systemic disease. The multi-deletion data, however, introduced a fascinating nuance. The double mutant lacking ECE1 and HGC1 showed seventy-five percent survival, and the triple mutant lacking SAP2, ECE1 and HGC1 reached eighty-two percent, the highest of any strain tested. Yet the SAP2/ECE1 double mutant survived at only fifty percent—worse than the SAP2 single mutant at seventy-five percent—suggesting that the effect of losing candidalysin depends profoundly on the morphological and genetic context of the strain.

The authors propose a hypothesis to explain this context-dependence: candidalysin may play a dual role in systemic virulence. In strains that retain hyphal growth, such as the SAP2 mutant background, candidalysin may contribute to protective host responses including activation of the NLRP3 inflammasome and the induction of adaptive Th17 CD4 T-cell responses, which promote fungal clearance. Deleting ECE1 in such a background could reduce inflammation and thereby impair clearance, explaining the counterintuitively poor survival of the SAP2/ECE1 double mutant. Conversely, in a yeast-locked background such as the HGC1 mutant, where hyphal damage is already absent, candidalysin’s contribution tilts back toward pure virulence, and its deletion becomes protective. The authors are appropriately cautious, noting that group sizes limit statistical power and that dedicated studies interrogating host responses to this mutant panel will be required to confirm the hypothesis. Still, the idea that a fungal toxin could simultaneously be a virulence factor and an immune adjuvant whose net effect flips depending on fungal morphology is a provocative reframing of toxin biology.

Equally surprising was the behaviour of Als3p. Long regarded as a textbook virulence factor for its role in epithelial and endothelial adhesion and invasion, Als3p showed minimal influence on early brain fungal burden, consistent with prior reports that the adhesin is dispensable for virulence in disseminated candidiasis. But across all multi-deletion mutants, deletion of ALS3 consistently reduced survival, hinting at a previously underappreciated protective role. One possibility raised by the authors is that hyphal Als proteins act as ligands for complement receptor 3 on host immune cells, promoting receptor engagement and protective immune responses, or that Als3p contributes to NLRP3 inflammasome activation. If confirmed, this would add Als3p to the growing list of fungal virulence attributes that stimulate the very immune machinery they also subvert. Sap2p, meanwhile, showed only a modest reduction in brain burdens despite its known involvement in brain entry, a discrepancy the authors attribute to differences in experimental conditions and genetic backgrounds across studies—a caution about how strongly methodology shapes conclusions in this field.

The deeper lesson of the study is the uncoupling of fungal burden from disease outcome. High brain fungal loads correlated with deletion of HGC1 and ECE1, yet the same genes that failed to restrict brain growth also contributed, paradoxically, to long-term mortality: hyphal formation and Sap2p secretion were the key drivers of death in the survival experiments, while candidalysin played a lesser individual role. Fungal burden, the authors emphasise, does not necessarily reflect pathogenicity, because host-mediated tissue damage and organ dysfunction—rather than the sheer number of fungi—can determine disease severity. Virulence attributes simultaneously promote protective immune responses that restrict fungal growth and host damage that contributes to death, making any single snapshot of infection misleading. This framing challenges a widespread convention in preclinical infectious disease research, where organ colony counts are routinely treated as the definitive readout of virulence.

Taken together, the study identifies the critical combinatorial core of C. albicans systemic pathogenicity: hyphal growth accompanied by simultaneous candidalysin and Sap2p secretion. Deletion of all three factors produced the most attenuated strain, with survival climbing to eighty-two percent, while every partial combination left the fungus significantly dangerous. Beyond its mechanistic conclusions, the work demonstrates the value of building additive mutant panels in a single genetic background—a strategy that eliminates the confounding of strain background effects and permits genuinely systematic dissection of virulence networks. As fungal infections rise globally and antifungal drug development remains slow, understanding which virulence traits actually matter, and in what combinations, may guide the selection of drug targets and vaccine antigens that disarm the fungus without dismantling the immune signals that help clear it. The paradox that candidalysin can be both poison and alarm bell may prove central to that effort.

Oropharyngeal candidiasis remains a significant health issue, especially among immunocompromised individuals, affecting mucosal tissues in the oral cavity and impairing quality of life. Despite the clinical importance of macrophages as first responders, the precise mechanisms enabling these cells to sense and counteract Candida infections at the molecular level have remained obscure. This study fills a critical knowledge gap by demonstrating that TREM2, a receptor known for sensing tissue damage in various pathological contexts, directly recognizes the fungal toxin candidalysin and orchestrates an effective antifungal response.

Employing cutting-edge single-cell transcriptomic sequencing, the research team meticulously mapped the immune landscape within the tongues of a murine OPC model. This approach revealed a pronounced infiltration of TREM2-expressing macrophages derived from monocytes localized in the infected lingual epithelium. Genetic ablation models, including global and myeloid lineage-specific knockouts of TREM2 as well as targeted depletion of TREM2-expressing cells via diphtheria toxin receptor approaches, definitively established the indispensable role of these macrophages in conferring resistance to OPC.

A pivotal and longstanding question in fungal immunology—how the host detects candidalysin—was expertly addressed through comprehensive biophysical and computational strategies. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) assays unequivocally confirmed the binding of TREM2 to candidalysin. Further structural insights were gleaned through advanced AlphaFold molecular docking combined with site-directed mutagenesis, identifying key amino acid residues on TREM2 (D131, R136, P169) and candidalysin (G65, N73, N91-K92) that mediate this specific interaction. This discovery reveals how the host immune system cleverly co-opts a pathogen-derived toxin as an alarm signal, enhancing immunosurveillance and rapid response.

The signaling cascade triggered by TREM2 engagement with candidalysin is intricate yet effective. Upon toxin recognition, TREM2 recruits the adaptor protein DAP12 to initiate downstream signaling. This activation proceeds through Syk kinase phosphorylation, followed by phosphorylation of the transcription factor NF-κB p65 and the MAP kinase p38. Consequent to these events, macrophages secrete tumor necrosis factor-alpha (TNF-α), a cytokine that acts both autocrinely and paracrinely within the mucosal milieu.

Functionally, TNF-α promotes macrophage activities such as phagocytosis, microbial killing, and reactive oxygen species (ROS) generation, amplifying fungal clearance. Simultaneously, TNF-α enhances neutrophil extracellular trap (NET) formation and bactericidal functions, crucial defenses against secondary bacterial infections that often complicate fungal diseases. Moreover, TNF-α exerts a modulatory effect on IL-17 signaling pathways in TCRαβ+ T cells, underscoring its role in fine-tuning adaptive immune responses during fungal invasion.

This comprehensive elucidation of the TREM2–candidalysin–TNF-α axis underscores its central importance in early innate immunity against OPC, as it bridges the innate and adaptive arms of host defense. The findings dramatically advance our understanding of host-pathogen interactions and highlight the sophistication of immune sentinel cells in detecting molecular signatures of fungal virulence.

Translationally, the study offers exciting new avenues for therapeutic intervention. Targeting TREM2 with agonists or devising candidalysin mimetics could serve as novel strategies to boost local innate immunity, particularly valuable for patients with compromised immune systems, such as those undergoing CAR-T cell therapies or chemotherapy. Such approaches might potentiate the mucosal barrier’s resilience against opportunistic fungal infections.

Furthermore, the delineated epitope on candidalysin recognized by TREM2 represents a promising candidate for vaccine development, potentially inducing robust immune memory and long-lasting protection against Candida infections. Monitoring TREM2 and candidalysin expression levels in oral mucosa could also be harnessed diagnostically to predict patient susceptibility and guide personalized treatment strategies.

Professor Yunsheng Liang and colleagues’ work not only breaks new ground in fungal immunology but also exemplifies the power of integrative methods encompassing molecular biology, structural bioinformatics, and immunology. Their insights profoundly deepen our grasp of the innate immune system’s capacity to leverage pathogen-derived molecules for host protection and open pathways toward innovative antifungal therapeutics and diagnostics.

As fungal infections increasingly pose a threat globally amidst rising numbers of immunocompromised individuals, such cutting-edge research delivers hope for more effective management and prevention of these debilitating diseases. The TREM2 recognition mechanism of candidalysin establishes an elegant paradigm of immune vigilance, turning a microbial weapon into a trigger for host defense.

This landmark study thereby reframes our conceptual understanding of fungal pathogenesis and immunity, urging further exploration into TREM2’s roles across diverse infectious and inflammatory contexts and driving forward the frontier of translational immunology.

Subject of Research: Animals

Article Title: TREM2-mediated recognition of candidalysin by macrophages confers early protective innate immunity in oropharyngeal candidiasis

News Publication Date: 18-May-2026

References: DOI: 10.1007/s44466-026-00041-5

Image Credits: Professor Yunsheng Liang from Central South University, Professor Yingping Xu from Southern Medical University, Professor Xiaowen Wang from Peking University First Hospital

Keywords: Immunology, Infectious diseases, Fungal infections, Innate immunity, Signal transduction, Molecular biology, Host pathogen interactions, Biotechnology