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Candida albicans is a commensal yeast of the human intestinal microbiota that, under predisposing conditions, can become pathogenic and cause life-threatening systemic infections (candidiasis). Fungal-host interactions during candidiasis are commonly studied using conventional 2D in vitro models, which have provided critical insights into the pathogenicity. However, microphysiological models with a higher biological complexity may be more suitable to mimic in vivo-like infection processes and antifungal drug efficacy. Therefore, a 3D intestine-on-chip model was used to investigate fungal-host interactions during the onset of invasive candidiasis and evaluate antifungal treatment under clinically relevant conditions. By combining microbiological and image-based analyses we quantified infection processes such as invasiveness and fungal translocation across the epithelial barrier. Additionally, we obtained novel insights into fungal microcolony morphology and association with the tissue. Our results demonstrate that C. albicans microcolonies induce injury to the epithelial tissue by disrupting apical cell-cell contacts and causing inflammation. Caspofungin treatment effectively reduced the fungal biomass and induced substantial alterations in microcolony morphology during infection with a wild-type strain. However, caspofungin showed limited effects after infection with an echinocandin-resistant clinical isolate. Collectively, this organ-on-chip model can be leveraged for in-depth characterization of pathogen-host interactions and alterations due to antimicrobial treatment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alexander S. Mosig and Martin Raasch hold equity in Dynamic42 GmbH. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Candida albicans can cause mucosal infections in humans. This includes oropharyngeal candidiasis, which is commonly observed in human immunodeficiency virus infected patients, and vulvovaginal candidiasis (VVC), which is the most frequent manifestation of candidiasis. Epithelial cell invasion by C. albicans hyphae is accompanied by the secretion of candidalysin, a peptide toxin that causes epithelial cell cytotoxicity. During vaginal infections, candidalysin-driven tissue damage triggers epithelial signaling pathways, leading to hyperinflammatory responses and immunopathology, a hallmark of VVC. Therefore, we proposed blocking candidalysin activity using nanobodies to reduce epithelial damage and inflammation as a therapeutic strategy for VVC. Anti-candidalysin nanobodies were confirmed to localize around epithelial-invading C. albicans hyphae, even within the invasion pocket where candidalysin is secreted. The nanobodies reduced candidalysin-induced damage to epithelial cells and downstream proinflammatory responses. Accordingly, the nanobodies also decreased neutrophil activation and recruitment. In silico mathematical modeling enabled the quantification of epithelial damage caused by candidalysin under various nanobody dosing strategies. Thus, nanobody-mediated neutralization of candidalysin offers a novel therapeutic approach to block immunopathogenic events during VVC and alleviate symptoms.IMPORTANCEWorldwide, vaginal infections caused by Candida albicans (VVC) annually affect millions of women, with symptoms significantly impacting quality of life. Current treatments are based on anti-fungals and probiotics that target the fungus. However, in some cases, infections are recurrent, called recurrent VVC, which often fails to respond to treatment. Vaginal mucosal tissue damage caused by the C. albicans peptide toxin candidalysin is a key driver in the induction of hyperinflammatory responses that fail to clear the infection and contribute to immunopathology and disease severity. In this pre-clinical evaluation, we show that nanobody-mediated candidalysin neutralization reduces tissue damage and thereby limits inflammation. Implementation of candidalysin-neutralizing nanobodies may prove an attractive strategy to alleviate symptoms in complicated VVC cases., Competing Interests: E.D. is the CEO of Q.V.Q who produced the anti-candidalysin nanobodies.

Computer simulations of mathematical models open up the possibility of assessing hypotheses generated by experiments on pathogen immune evasion in human whole-blood infection assays. We apply an interdisciplinary systems biology approach in which virtual infection models implemented for the dissection of specific immune mechanisms are combined with experimental studies to validate or falsify the respective hypotheses. Focusing on the assessment of mechanisms that enable pathogens to evade the immune response in the early time course of a whole-blood infection, the least-square error (LSE) as a measure for the quantitative agreement between the theoretical and experimental kinetics is combined with the Akaike information criterion (AIC) as a measure for the model quality depending on its complexity. In particular, we compare mathematical models with three different types of pathogen immune evasion as well as all their combinations: (i) spontaneous immune evasion, (ii) evasion mediated by immune cells, and (iii) pre-existence of an immune-evasive pathogen subpopulation. For example, by testing theoretical predictions in subsequent imaging experiments, we demonstrate that the simple hypothesis of having a subpopulation of pre-existing immune-evasive pathogens can be ruled out. Furthermore, in this study we extend our previous whole-blood infection assays for the two fungal pathogens Candida albicans and C. glabrata by the bacterial pathogen Staphylococcus aureus and calibrated the model predictions to the time-resolved experimental data for each pathogen. Our quantitative assessment generally reveals that models with a lower number of parameters are not only scored with better AIC values, but also exhibit lower values for the LSE. Furthermore, we describe in detail model-specific and pathogen-specific patterns in the kinetics of cell populations that may be measured in future experiments to distinguish and pinpoint the underlying immune mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Background: Candida albicans and Candida glabrata are the 2 most prevalent Candida species causing bloodstream infections. Patterns of innate immune activation triggered by the 2 fungi differ considerably., Methods: To analyze human natural killer (NK) cell activation by both species, we performed ex vivo whole-blood infection assays and confrontation assays with primary human NK cells., Results: C. albicans was a stronger activator for isolated human NK cells than C. glabrata. In contrast, activation of blood NK cells, characterized by an upregulated surface exposure of early activation antigen CD69 and death receptor ligand TRAIL, as well as interferon-γ (IFN-γ) secretion, was more pronounced during C. glabrata infection. NK cell activation in blood is mediated by humoral mediators released by other immune cells and does not depend on direct activation by fungal cells. Cross-talk between Candida-confronted monocyte-derived dendritic cells (moDC) and NK cells resulted in the same NK activation phenotype as NK cells in human blood. Blocking experiments and cytokine substitution identified interleukin-12 as a critical mediator in regulation of primary NK cells by moDC., Conclusions: Activation of human NK cells in response to Candida in human blood mainly occurs indirectly by mediators released from monocytic cells., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Rationale: The liver is a central organ not only for metabolism but also immune function. Life-threatening infections of both bacterial and fungal origin can affect liver function but it is yet unknown whether molecular changes differ depending on the pathogen. We aimed to determine whether the hepatic host response to bacterial and fungal infections differs in terms of hepatic metabolism and liver function. Methods: We compared murine models of infection, including bacterial peritoneal contamination and infection (PCI), intraperitoneal and systemic C. albicans infection, at 6 and 24 h post-infection, to sham controls. The molecular hepatic host response was investigated by the detection of regulatory modules based on large-scale protein-protein interaction networks and expression data. Topological analysis of these regulatory modules was used to reveal infection-specific biological processes and molecular mechanisms. Intravital microscopy and immunofluorescence microscopy were used to further analyze specific aspects of pathophysiology such as cholestasis. Results: Down-regulation of lipid catabolism and bile acid synthesis was observed after 6 h in all infection groups. Alterations in lipid catabolism were characterized by accumulation of long chain acylcarnitines and defective beta-oxidation, which affected metabolism by 6 h. While PCI led to an accumulation of unconjugated bile acids (BA), C. albicans infection caused accumulation of conjugated BA independent of the route of infection. Hepatic dye clearance and transporter expression revealed reduced hepatic uptake in fungal infections vs. defects in secretion following polybacterial infection. Conclusion: Molecular phenotypes of lipid accumulation and cholestasis allow differentiation between pathogens as well as routes of infection at early stages in mice. Targeted metabolomics could be a useful tool for the profiling of infected/septic patients and the type of pathogen, with subsequent customization and targeting of therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans , a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans , candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. IMPORTANCE Candida albicans , usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer., (Copyright © 2018 Allert et al.)

The condition of neutropenia, i.e., a reduced absolute neutrophil count in blood, constitutes a major risk factor for severe infections in the affected patients. Candida albicans and Candida glabrata are opportunistic pathogens and the most prevalent fungal species in the human microbiota. In immunocompromised patients, they can become pathogenic and cause infections with high mortality rates. In this study, we use a previously established approach that combines experiments and computational models to investigate the innate immune response during blood stream infections with the two fungal pathogens C. albicans and C. glabrata . First, we determine immune-reaction rates and migration parameters under healthy conditions. Based on these findings, we simulate virtual patients and investigate the impact of neutropenic conditions on the infection outcome with the respective pathogen. Furthermore, we perform in silico treatments of these virtual patients by simulating a medical treatment that enhances neutrophil activity in terms of phagocytosis and migration. We quantify the infection outcome by comparing the response to the two fungal pathogens relative to non-neutropenic individuals. The analysis reveals that these fungal infections in neutropenic patients can be successfully cleared by cytokine treatment of the remaining neutrophils; and that this treatment is more effective for C. glabrata than for C. albicans .

Candida albicans and Candida glabrata account for the majority of candidiasis cases worldwide. Although both species are in the same genus, they differ in key virulence attributes. Within this work, live cell imaging was used to examine the dynamics of neutrophil activation after confrontation with either C. albicans or C. glabrata. Analyses revealed higher phagocytosis rates of C. albicans than C. glabrata that resulted in stronger PMN (polymorphonuclear cells) activation by C. albicans. Furthermore, we observed differences in the secretion of chemokines, indicating chemotactic differences in PMN signalling towards recruitment of further immune cells upon confrontation with Candida spp. Supernatants from co-incubations of neutrophils with C. glabrata primarily attracted monocytes and increased the phagocytosis of C. glabrata by monocytes. In contrast, PMN activation by C. albicans resulted in recruitment of more neutrophils. Two complex infection models confirmed distinct targeting of immune cell populations by the two Candida spp.: In a human whole blood infection model, C. glabrata was more effectively taken up by monocytes than C. albicans and histopathological analyses of murine model infections confirmed primarily monocytic infiltrates in C. glabrata kidney infection in contrast to PMN-dominated infiltrates in C. albicans infection. Taken together, our data demonstrate that the human opportunistic fungi C. albicans and C. glabrata are differentially recognized by neutrophils and one outcome of this differential recognition is the preferential uptake of C. glabrata by monocytes., (© 2015 John Wiley & Sons Ltd.)