ABSTRACT The majority of eukaryotes encode some intron-containing pre-tRNAs. Splicing of these pre-tRNAs requires a dedicated tRNA splicing machinery. The fungal and trypanosome tRNA ligase, Trl1, and the human RNA ligase, RTCB, catalyze an essential step in tRNA splicing. However, Trl1 and RTCB are nonhomologous and biochemically and structurally distinct from each other. Therefore, Trl1 could serve as a broad-spectrum antifungal and anti-trypanosomal target. While the functions and requirements of the three catalytic Trl1 domains have been extensively characterized in the model yeast Saccharomyces cerevisiae , the roles of Trl1 orthologs in pathogenic fungi remain unexplored. Here, we validate Trl1 as one of the few promising novel drug targets for the development of antifungal therapeutics. Functional analyses of the three Trl1 domains show that only the “sealing” domain is essential for growth and viability in Candida albicans and Aspergillus fumigatus . In contrast, the two “healing” domains are dispensable in these pathogenic fungi, suggesting the presence of redundant healing enzymes, unlike in S. cerevisiae . These findings indicate that only the sealing domain is a good drug target. Our analysis also shows that the Mucor enzyme, which only contains the sealing domain, is essential. Using a Caenorhabditis elegans infection model of C. albicans , we further demonstrated that inhibiting Trl1 expression protects worms during an established infection. In contrast to these fungal pathogens, we show that all three domains of Trl1 are essential in Trypanosoma brucei. Our findings show that the essentiality of the Trl1 sealing is conserved in important human pathogens and provides an impetus for future drug development. SIGNIFICANCE Fungal infections are an important cause of human disease and death and difficult to treat and there is an urgent need to develop additional drugs. Based on studies in yeast, one promising target for antifungal drug development is the tRNA splicing pathway. Human tRNA ligase is fundamentally distinct from the fungal one. To investigate the possibility of developing tRNA ligase-targeting drugs, we investigated the function of the catalytic domains of fungal tRNA ligase in different fungal pathogens. Surprisingly, only the first domain is essential in these pathogens and yeast is not a good model fungus. In contrast, all three domains of Trypanosome tRNA ligase are essential. These findings provide an impetus for future drug development.

Candida auris is an emerging multidrug-resistant fungal pathogen. The genetic factors contributing to the virulence, drug resistance, and stress-tolerant nature of C. auris are mostly unknown. Additional animal models of virulence are needed, especially those amenable to high-throughput analysis. The nematode Caenorhabditis elegans has been validated as an effective tool for studying multiple fungal and bacterial pathogens. We describe here a C. elegans infection model in which exposure to C. auris is lethal to worms with kinetics similar to killing by Candida albicans; in contrast to C. albicans, C. auris does not form hyphae, indicating distinct virulence mechanisms. Furthermore, an engineered mutant auxotrophic for adenine biosynthesis (ade2∆) is avirulent, as it is in many models, indicating that the nematode can discriminate between virulent and avirulent mutants. Moreover, the C. elegans model can recapitulate strain-to-strain differences in virulence seen in mouse models. We have adapted a live/dead staining methodology using SYTOX Orange to enable a high-throughput assay amenable to analysis of multiple strains or genetic mutants. This model has significant advantages, including cost-effectiveness, a short generation time, and excellent amenability to high-throughput assays. C. elegans represents a valuable preliminary screening platform for C. auris virulence studies. IMPORTANCE Candida auris is a growing clinical problem. This fungal pathogen spread rapidly worldwide after its discovery in 2009. It avidly colonizes the skin and abiotic surfaces, and many strains are multidrug resistant, making them easy to spread in hospital settings and very difficult to treat. Though distantly related to Candida albicans, it is clear that C. auris possesses unique virulence mechanisms, making studies directly in this species imperative. However, existing animal models, including mouse and invertebrate species, have limitations in variability and throughput. We describe here a facile infection model using the nematode Caenorhabditis elegans, which has previously been used for other bacterial and fungal pathogens. We show that this model can identify virulence differences between strains and in mutants predicted to be less virulent. Moreover, this model is amenable to high-throughput screening. This will be a valuable tool in uncovering C. auris-specific virulence traits.

The Fifth International Symposium on Fungal Stress (ISFUS) brought together in Brazil many of the leaders in the field of fungal stress responses, from fourteen countries, for four days of outstanding science ranging from basic research to studies with agricultural, medical, industrial, and environmental significance. In addition to the excellent oral and poster presentations, the Symposium organisers ensured that all participants had ample opportunity to engage, socialise, and network to exchange ideas and share research. The conference was enhanced by the world-class venue near Iguazu Falls, probably the greatest natural phenomenon in South America.

Candida is one of the most frequent causes of bloodstream infections, and our first line of defense against these invasive infections is the innate immune system. The early immune response is critical in controlling Candida albicans infection, but C. albicans has several strategies to evade host immune attack. Phagocytosis of C. albicans blocks hyphal growth, limiting host damage and virulence, but how C. albicans limits early recruitment and phagocytosis in vertebrate infection is poorly understood. To study innate immune evasion by intravital imaging, we utilized the transparent larval zebrafish infection model to screen 131 C. albicans mutants for altered virulence and phagocyte response. Infections with each of the seven hypovirulent mutants led to altered phagocyte recruitment and/or phagocytosis, falling into four categories. Of particular interest among these is NMD5, a predicted β-importin and newly identified virulence factor. The nmd5∆/∆ mutant fails to limit phagocytosis, and its virulence defects are eliminated when phagocyte activity is compromised, suggesting that its role in virulence is limited to immune evasion. These quantitative intravital imaging experiments are the first to document altered Candida-phagocyte interactions for several additional mutants and clearly distinguish recruitment from phagocytic uptake, suggesting that Candida modulates both events. This initial large-scale screen of individual C. albicans mutants in a vertebrate, coupled with high-resolution imaging of Candida-phagocyte interactions, provides a more nuanced view of how diverse mutations can lead to more effective phagocytosis, a key immune process that blocks germination and drives anti-fungal immunity. IMPORTANCE:Candida albicans is part of the human microbial community and is a dangerous opportunistic pathogen, able to prevent its elimination by the host immune system. Although Candida avoids immune attack through several strategies, we still understand little about how it regulates when immune phagocytes get recruited to the infection site and when they engulf fungal cells. We tested over 130 selected Candida mutants for their ability to cause lethal infection and found several hypovirulent mutants, which provoked altered innate immune responses, resulting in lower overall inflammation and greater host survival. Of particular interest is NMD5, which acts to limit fungal phagocytosis and is predicted to regulate the activity of stress-associated transcription factors. Our high-content screening was enabled by modeling Candida infection in transparent vertebrate zebrafish larva. Our findings help us understand how Candida survives immune attack during commensal and pathogenic growth, and may eventually inform new strategies for controlling disease.

The ubiquitous fungal pathogen Candida albicans has the potential to either asymptomatically colonize the gastrointestinal (GI) tract or become an invasive pathogen through mechanisms that remain incompletely understood. Here we explored the fungal, host, and environmental factors that influence the ability of C. albicans to colonize the mouse GI tract using a representative clinical strain, CLCA10. After a single gavage challenge (5 × 106 CFU C. albicans), specific pathogen-free (SPF) mice remained colonized with C. albicans strain CLCA10, but not other Candida species, for at least 58 days with the fungus confined largely to the gut luminal contents. Colonized mice exhibited no weight loss or other signs of active infection, and CLCA10 did not disrupt the gut microbiome. Moreover, C. albicans colonization with CLCA10 was not substantially affected by the mouse commercial source or the method used to cultivate the fungus prior to gavage. Although some genetically manipulated C. albicans strains were unable to robustly colonize, strain SC5314 also colonized the mouse gut despite having enhanced pathogenicity. C. albicans CLCA10 gut colonization in part depended on the hypha-associated adhesins Als3 and Hwp1 and the peptide toxin candidalysin and could not be eradicated by potent antifungal therapy. Thus, this study concludes that C. albicans gut colonization in the mouse is critically dependent on fungal hyphal factors, the targeting of which could enhance strategies to reduce C. albicans gut colonization and the intractable threat to human health it represents.IMPORTANCECandida albicans is an important human fungal pathogen and a ubiquitous colonizer of the gastrointestinal (GI) tract. However, it is not understood how C. albicans persists within the GI tract and from which it may disperse to cause disease. Here, we demonstrated that multiple strains of C. albicans, including the widely used SC5314 strain, robustly colonize the mouse GI tract for at least 2 months. This colonization caused no disruption to the host tissue or bacterial microbiome and was resistant to clearance by antifungal drugs. Importantly, colonization was mediated by proteins expressed by C. albicans that are known to be involved in fungal virulence and unrelated to experimental conditions. Overall, this work identifies mechanisms by which C. albicans persists in the GI tract, enhancing our knowledge of host-fungal interactions during commensal colonization and potentially how to reduce such colonization.

Candida albicans, an opportunistic fungal pathogen, causes systemic and superficial infections, especially in immunocompromised patients. Treatment of fungal infections is complicated by limited antifungal options and the development of drug resistance. Previous work from our group demonstrated the efficacy of the anti-virulence peptide EntV and shorter variants against C. albicans infection in various animal models, including mouse models of oropharyngeal candidiasis and disseminated infection and a rat venous catheter model. However, the mechanism of action, which abrogates fungal virulence without fungicidal or fungistatic activity, has remained unknown. We used a combination of cell biological, biochemical, genomic, and genetic approaches to identify this mechanism. We demonstrate that EntV-based peptides bind to the fungal cell envelope in a punctate and dynamic manner, co-localizing with extracellular vesicles (EVs), which play a critical role in fungal biofilm formation and virulence. Transcriptomic and genetic analyses further indicate that this activity is linked to the intracellular vesicular trafficking machinery, especially the ESCRT pathway, as mutations in this pathway alter sensitivity to EntV peptides and regulate virulence. We also show that EntV treatment significantly reduces EV secretion in C. albicans, supporting a novel mechanism of antifungal action through inhibition of EV-mediated virulence. These findings further develop EntV as a promising anti-virulence agent with potential for therapeutic development against drug-resistant fungal pathogens.

A recent study in mBio reports the construction and preliminary screening of a library containing mutants of 99 of the 119 predicted protein kinases in Candida albicans (the majority of the remaining 20 are probably essential) (J. Kramara, M.-J. Kim, T. L. Ollinger, L. C. Ristow, et al., mBio e01249-24, 2024, https://doi.org/10.1128/mbio.01249-24). Using a quantitative competition assay in 10 conditions that represent nutritional, osmotic, cell wall, and pH stresses that are considered to model various aspects of the host environment allowed them to phenotypically cluster kinases, which highlight both the integration and specialization of signaling pathways, suggesting novel functions for many kinases. In addition, they tackle two complex and partially overlapping differentiation events, hyphal morphogenesis and biofilm formation. They find that a remarkable 88% of the viable kinase mutants in C. albicans affect hyphal growth, illustrating how integrated morphogenesis is in the overall biology of this organism, and begin to dissect the regulatory relationships that control this key virulence trait.

Eukaryotic organisms are composed of different cell types with defined shapes and functions. Specific cell types are produced by the process of cell differentiation, which is regulated by signal transduction pathways. Signaling pathways regulate cell differentiation by sensing cues and controlling the expression of target genes whose products generate cell types with specific attributes. In studying how cells differentiate, fungi have proved valuable models because of their ease of genetic manipulation and striking cell morphologies. Many fungal species undergo filamentous growth—a specialized growth pattern where cells produce elongated tube-like projections. Filamentous growth promotes expansion into new environments, including invasion into plant and animal hosts by fungal pathogens. The same signaling pathways that regulate filamentous growth in fungi also control cell differentiation throughout eukaryotes and include highly conserved mitogen-activated protein kinase (MAPK) pathways, which is the focus of this review. In many fungal species, mucin-type sensors regulate MAPK pathways to control filamentous growth in response to diverse stimuli. Once activated, MAPK pathways reorganize cell polarity, induce changes in cell adhesion, and promote the secretion of degradative enzymes that mediate access to new environments. However, MAPK pathway regulation is complicated because related pathways can share components with each other yet induce unique responses (i.e. signal specificity). In addition, MAPK pathways function in highly integrated networks with other regulatory pathways (i.e. signal integration). Here, we discuss signal specificity and integration in several yeast models (mainly Saccharomyces cerevisiae and Candida albicans) by focusing on the filamentation MAPK pathway. Because of the strong evolutionary ties between species, a deeper understanding of the regulation of filamentous growth in established models and increasingly diverse fungal species can reveal fundamentally new mechanisms underlying eukaryotic cell differentiation.

Candida auris is an emerging fungal pathogen with several concerning qualities. First recognized in 2009, it has arisen in multiple geographically distinct genomic clades nearly simultaneously. C. auris strains are typically multidrug resistant and colonize the skin much better than most other pathogenic fungi; it also persists on abiotic surfaces, enabling outbreaks due to transmission in health care facilities. All these suggest a biology substantially different from the 'model' fungal pathogen, Candida albicans and support intensive investigation of C. auris biology directly. To uncover novel virulence mechanisms in this species requires the development of appropriate animal infection models. Various studies using mice, the definitive model, are inconsistent due to differences in mouse and fungal strains, immunosuppressive regimes, doses, and outcome metrics. At the same time, developing models of skin colonization present a route to new insights into an aspect of fungal pathogenesis that has not been well studied in other species. We also discuss the growing use of nonmammalian model systems, including both vertebrates and invertebrates, such as zebrafish, C. elegans, Drosophila, and Galleria mellonella, that have been productively employed in virulence studies with other fungal species. This review will discuss progress in developing appropriate animal models, outline current challenges, and highlight opportunities in demystifying this curious species.

Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcμR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.

ABSTRACT Fungal resistance to commonly used medicines is a growing public health threat, and there is a dire need to develop new classes of antifungals. We previously described a peptide produced by Enterococcus faecalis , EntV, that restricts Candida albicans to a benign form rather than having direct fungicidal activity. Moreover, we showed that one 12-amino acid (aa) alpha helix of this peptide retained full activity, with partial activity down to the 10aa alpha helix. Using these peptides as a starting point, the current investigation sought to identify the critical features necessary for antifungal activity and to screen for new variants with enhanced activity using both biofilm and C. elegans infection assays. First, the short peptides were screened for residues with critical activity by generating alanine substitutions. Based on this information, we used synthetic molecular evolution (SME) to rationally vary the specific residues of the 10aa variant in combination to generate a library that was screened to identify variants with more potent antifungal activity than the parent template. Five gain-of-function peptides were identified. Additionally, chemical modifications to the peptides to increase stability, including substitutions of D-amino acids and hydrocarbon stapling, were investigated. The most promising peptides were additionally tested in mouse models of oropharyngeal and systemic candidiasis where their efficacy in preventing infection was demonstrated. The expectation is that these discoveries will contribute to the development of new therapeutics in the fight against antimicrobial resistant fungi. IMPORTANCE Since the early 1980s, the incidence of disseminated life-threatening fungal infections has been on the rise. Worldwide, Candida and Cryptococcus species are among the most common agents causing these infections. Simultaneously, with this rise of clinical incidence, there has also been an increased prevalence of antifungal resistance, making treatment of these infections very difficult. For example, there are now strains of Candida auris that are resistant to all three classes of currently used antifungal drugs. In this study, we report on a strategy that allows for the development of novel antifungal agents by using synthetic molecular evolution. These discoveries demonstrate that the enhancement of antifungal activity from naturally occurring peptides is possible and can result in clinically relevant agents that have efficacy in multiple in vivo models as well as the potential for broad-spectrum activity.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Adelman, Max; Minze, Laurie; Tran, Truc; Detranaltes, Andrea; Rydell, Kirsten; Schettino, Marissa; Malikzad, Husna; Amaya, Abigail; Virk, Muhammad; Atterstrom, Rachel; Jones, Mary; Lorenz, Michael; Corry, David; Arias, Cesar Author Information

Platelets are first responders in thrombosis and hemostasis, but also central players in inflammation. Compared to platelets recruited to thrombi, immune-responsive platelets use distinct effector functions including Arp2/3-dependent migration along adhesive substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cellular level is incompletely understood. Here, we use time-resolved morphodynamic profiling of individual platelets to show that migration, in contrast to clot retraction, requires anisotropic myosin IIa-activity at the platelet rear which is preceded by polarized actin polymerization at the front to initiate and maintain migration. Polarization of migrating platelets is coordinated by integrin GPIIb-dependent outside-in signaling via Gα13 to trigger tyrosine kinase c-Src/14-3-3ζ-dependent lamellipodium formation and functions independent of soluble agonists or chemotactic signals. Inhibitors of this signaling cascade, including the clinically employed ABL/c-Src inhibitor dasatinib, interfere predominantly with the migratory capacity of platelets, without major impairment of classical platelet functions. In murine inflammation models, this translates to reduced migration of platelets visualized by 4D intravital microscopy, resulting in increased inflammation-associated hemorrhage in acute lung injury. Finally, platelets isolated from dasatinib-treated leukemia patients prone to clinically relevant hemorrhage exhibit prominent migration defects, while other platelet functions are only partially affected. In summary, we define a distinct signaling pathway essential for migration, and provide novel mechanistic insights explaining dasatinib-related platelet dysfunction and bleeding.

ABSTRACT Co-infection with Streptococcus mutans and Candida albicans is associated with dental caries, and their co-cultivation results in enhanced biofilm matrix production that contributes to increased virulence and caries risk. Moreover, the catalase-negative S. mutans demonstrates increased oxidative stress tolerance when co-cultivated in biofilms with C. albicans, a catalase-producing yeast. Here, we sought to obtain mechanistic insights into the increased H2O2 tolerance of S. mutans when co-cultivated with clinical isolates of Candida glabrata, Candida tropicalis, and C. albicans. Additionally, the C. albicans SC5314 laboratory strain, its catalase mutant (SC5314Δcat1), and S. mutans UA159 and its glucosyltransferase B/C mutant (UA159ΔgtfB/C) were grown as single- and dual-species biofilms. Time-kill assays revealed that upon acute H2O2 challenge, the survival rates of S. mutans in dual-species biofilms with the clinical isolates and C. albicans SC5314 were greater than when paired with SC5314Δcat1 or as a single-species biofilm. Importantly, this protection was independent of glucan production through S. mutans GtfB/C. Transwell assays and treatment with H2O2-pre-stimulated C. albicans SC5314 supernatant revealed that this protection is contact-dependent. Biofilm stability assays with sublethal H2O2 or peroxigenic Streptococcus A12 challenge resulted in biomass reduction of single-species S. mutans UA159 and dual-species with SC5314Δcat1 biofilms compared to UA159 biofilms co-cultured with C. albicans SC5314. S. mutans oxidative stress genes were upregulated in single-species biofilms when exposed to H2O2, but not when S. mutans was co-cultivated with C. albicans SC5314. Here, we uncovered a novel, contact-dependent, synergistic interaction in which the catalase of C. albicans protects S. mutans against H2O2. IMPORTANCE It is well established that co-infection with the gram-positive caries-associated bacterium Streptococcus mutans and the yeast pathobiont Candida albicans results in aggressive forms of caries in humans and animal models. Together, these microorganisms form robust biofilms through enhanced production of extracellular polysaccharide matrix. Further, co-habitation in a biofilm community appears to enhance these microbes’ tolerance to environmental stressors. Here, we show that catalase produced by C. albicans protects S. mutans from H2O2 stress in a biofilm matrix-independent manner. Our findings uncovered a novel synergistic trait between these two microorganisms that could be further exploited for dental caries prevention and control.

Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production.

Abstract Background Candida spp. colonization is a risk factor for candidemia in intensive care unit (ICU) patients. Recent data suggest that the epidemiology of Candida infections is shifting away from C. albicans, but prospective data on the epidemiology of Candida spp. gut colonization are limited. Methods We conducted a prospective cohort study of adult patients admitted to an ICU at a tertiary care hospital. Patients had stool samples collected twice weekly for up to four weeks or until ICU discharge. A convenience sample of stool samples was plated on CHROMagar Candida Plus for 48 hours. Colonies of different morphologies were isolated on Sabouraud dextrose agar and identified with MALDI-ToF. Results A total of 102 patients were included in the cohort. We performed screening for stool Candida colonization in 12 patients: 8 (67%) men, median age 65 (IQR 54.5-68.5). Three (25%) were in shock and eight (67%) required mechanical ventilation on ICU admission. In-hospital mortality was 25%. Seven patients (58%) were colonized with Candida at ≥1 time point (Figure 1). Of 30 total time points tested (median per patient=2, range 1-5), 15 (50%) were positive for Candida spp. Most time points (9/15, 60%) were positive for C. glabrata; 4/15 (27%) were positive for both C. glabrata and C. albicans. A minority (1/15; 7%) were positive for C. albicans and C. parapsilosis alone, respectively. None had C. auris colonization. Most colonization was persistent: Of 6 colonized patients with ≥1 time point tested, 5 (83%) were colonized at multiple timepoints. Eleven patients had data collected on anti-fungal administration during the study period, and five of these received an antifungal (Figure 1). Of these five, three (60%) had no Candida colonization. One lost C. albicans colonization but had persistent C. glabrata colonization despite azole therapy, and another developed C. parapsilosis colonization despite azole and amphotericin administration. Figure 1. Results of serial stool samples tested for Candida colonization per individual patient. Conclusion Most ICU patients in this study had Candida spp. gut colonization, commonly with intrinsically azole-resistant C. glabrata. This study is limited by small sample size, and further data are needed to determine the clinical impact of antifungal-resistant Candida in ICU patients at high risk of candidemia. Disclosures All Authors: No reported disclosures

ABSTRACT The opportunistic fungal pathogen Candida albicans has evolved a variety of mechanisms for surviving inside and escaping macrophages, including the initiation of filamentous growth. Although several distinct models have been proposed to explain this process at the molecular level, the signals driving hyphal morphogenesis in this context have yet to be clarified. Here, we evaluate the following three molecular signals as potential hyphal inducers within macrophage phagosomes: CO 2 , intracellular pH, and extracellular pH. Additionally, we revisit previous work suggesting that the intracellular pH of C. albicans fluctuates in tandem with morphological changes in vitro . Using time-lapse microscopy, we observed that C. albicans mutants lacking components of the CO 2 -sensing pathway were able to undergo hyphal morphogenesis within macrophages. Similarly, a rim101Δ strain was competent in hyphal induction, suggesting that neutral/alkaline pH sensing is not necessary for the initiation of morphogenesis within phagosomes either. Contrary to previous findings, single-cell pH-tracking experiments revealed that the cytosolic pH of C. albicans remains tightly regulated both within macrophage phagosomes and under a variety of in vitro conditions throughout the process of morphogenesis. This finding suggests that intracellular pH is not a signal contributing to morphological changes.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

The pathogenic yeast Candida auris represents a global threat of the utmost clinical relevance. This emerging fungal species is remarkable in its resistance to commonly used antifungal agents and its persistence in the nosocomial settings. The innate immune system is one the first lines of defense preventing the dissemination of pathogens in the host. C. auris is susceptible to circulating phagocytes, and understanding the molecular details of these interactions may suggest routes to improved therapies. In this work, we examined the interactions of this yeast with macrophages. We found that macrophages avidly phagocytose C. auris; however, intracellular replication is not inhibited, indicating that C. auris resists the killing mechanisms imposed by the phagocyte. Unlike Candida albicans, phagocytosis of C. auris does not induce macrophage lysis. The transcriptional response of C. auris to macrophage phagocytosis is very similar to other members of the CUG clade (C. albicans, C. tropicalis, C. parapsilosis, C. lusitaniae), i.e., downregulation of transcription/translation and upregulation of alternative carbon metabolism pathways, transporters, and induction of oxidative stress response and proteolysis. Gene family expansions are common in this yeast, and we found that many of these genes are induced in response to macrophage co-incubation. Among these, amino acid and oligopeptide transporters, as well as lipases and proteases, are upregulated. Thus, C. auris shares key transcriptional signatures shared with other fungal pathogens and capitalizes on the expansion of gene families coding for potential virulence attributes that allow its survival, persistence, and evasion of the innate immune system.