Ashraf S. Ibrahim

Mucormycosis is a highly lethal invasive fungal infection caused by fungi of the order Mucorales, with mortality rates exceeding 50% despite current antifungal therapies with polyenes or azoles. Because iron acquisition is essential for Mucorales growth and virulence, we investigated whether transferrin, the primary physiological iron-sequestering protein in plasma, could serve as a host-directed therapeutic strategy against mucormycosis. Transferrin inhibited the growth of clinically relevant Mucorales species, including Rhizopus delemar (R. delemar), Rhizomucor, and Lichtheimia corymbifera (L. corymbifera) in a concentration-dependent manner. Checkerboard assays demonstrated synergistic interactions between transferrin and liposomal amphotericin B (LAMB), with fractional inhibitory concentration index values below 0.5 for all tested species. In endothelial cell infection model, transferrin significantly reduced expression of the fungal invasion ligand spore coat protein homolog 3 (CotH3) and the host receptor glucose-regulated protein 78 (GRP78), key mediators of angioinvasion during mucormycosis. In a neutropenic murine model of pulmonary mucormycosis, transferrin enhanced the therapeutic efficacy of both LAMB and isavuconazole (ISAV), resulting in improved survival compared with antifungal monotherapy. Collectively, these findings provide preclinical evidence that transferrin modulates pathogenic determinants associated with mucormycosis and enhances the efficacy of current antifungal therapies, supporting further investigation of transferrin as a potential host-directed adjunctive treatment.

Candidozyma auris is an emerging, multidrug-resistant (MDR) fungal pathogen that persistently colonizes human skin and disproportionately causes invasive infections in patients with metabolic or immune dysfunction. Despite strong epidemiological links to diabetes and immunosuppression, how these host conditions shape skin colonization, immune defense, and dissemination remain poorly defined. Here, we establish complementary murine model of C. auris skin colonization under immunocompetent, immunosuppressed, and diabetic ketoacidosis (DKA) conditions. DKA mice exhibited significantly increased skin fungal burden, impaired clearance, and frequent systemic dissemination. Notably, DKA permitted dissemination despite preserved granulocyte and neutrophil recruitment to the skin, indicating a functional rather than quantitative defect in innate immunity. Consistent with this, phagocytes from DKA mice displayed impaired antifungal activity characterized by reduced phagocytosis and killing despite elevated reactive oxygen species production. Hyperglycemic and ketone-rich conditions (BHB) remodel the C. auris cell wall, reducing mannan, increasing chitin, and upregulating adhesins, thereby enhancing adhesion and inflammatory activation while impairing neutrophil killing. Together, these findings reveal host metabolic dysfunction as a primary driver of persistent C. auris skin colonization and dissemination, identify qualitative defects in innate antifungal immunity as a key determinant of invasive risk and highlight metabolic condition as a critical target for infection prevention strategies. Short Summary: This study establishes the first physiologically relevant murine model of Candidozyma auris skin colonization under diabetic ketoacidosis and immunosuppression, revealing distinct immune dysfunction and systemic dissemination that can inform targeted antifungal strategies.

Multidrug-resistant fungi, Candida auris, persist in the healthcare environment and colonize human skin and medical devices, causing life-threatening infections with mortality rates approaching ~60% in immunocompromised patients. Because some isolates are resistant to nearly all available antifungal drugs, alternative therapeutic approaches are urgently needed. C. auris cell wall contains β-glucan and highly conserved fungal glycan structures. Moreover, many cell wall proteins are heavily β-glycosylated, and a monoclonal antibody raised against β-glucan has been shown to recognize C. albicans hyphal-regulated cell wall protein (Hyr1p), which has homologous proteins in C. auris. Based on this conversation, we evaluated β-glucan chitosan particles (GCP) as a vaccine candidate and evaluated the humoral and cellular immune responses. GCP induced robust IgG antibody and IFN-γ, IL-4, and IL17 producing T cells that recognized purified C. auris cell wall proteins. Anti-GCP antibodies recognized the cell walls of C. auris isolates from four phylogenetic clades through binding to cell wall glycoproteins. Mice vaccinated with GCP were protected from disseminated C. auris infection (40% vs. 0% survival, p = <0.0001). GCP-vaccinated mice had significantly lower fungal burden than placebo in target organs (kidney, heart, and brain) and fewer fungal abscesses. These findings support GCP vaccination as a promising strategy against MDR C. auris.

Current diagnostic approaches for mucormycosis are often limited by low sensitivity and prolonged turnaround times, which result in delayed treatment and poor clinical outcomes. We developed a novel diagnostic method utilizing a colorimetric loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of mucormycosis. The assay incorporates specifically designed primers capable of detecting as low as 0.001 picograms (pg) of spiked genomic DNA from Mucorales fungi. This LAMP assay demonstrated a high sensitivity of 98% and a 100% specificity of detecting fungal ribosomal DNA (rDNA) in bronchoalveolar lavage (BAL) samples collected from mice infected with Mucorales fungi ( n = 48) or from an uninfected control group ( n = 15). To align the assay with clinical antifungal therapy, a subset of infected mice was treated with either liposomal amphotericin B (LAMB) or a combination of LAMB and a humanized monoclonal antibody (VX-01) targeting the Mucorales-specific surface protein CotH3. Consistent with the treatment efficacy, the LAMP assay detected significantly lower fungal burdens in BAL samples from mice receiving the combination therapy compared to those treated with LAMB alone or placebo. Further validation was conducted using BAL samples from patients diagnosed with mucormycosis ( n = 24) or aspergillosis ( n = 17). The assay demonstrated a sensitivity of 79% and a specificity of 94%. These findings highlight the diagnostic potential of this LAMP-based assay as a point-of-care. Its high sensitivity, specificity, and rapid turnaround time position this assay as a promising tool for early and accurate detection of mucormycosis, with the potential to improve patient management and clinical outcomes. IMPORTANCE: Mucormycosis is a rapidly progressive and fatal fungal infection. Timely diagnosis is critical for effective treatment, yet current diagnostic tools are slow, insensitive, or require complex laboratory procedures. In this study, we developed and validated a colorimetric loop-mediated isothermal amplification (LAMP) assay that enables rapid and reliable detection of Mucorales DNA directly from bronchoalveolar lavage (BAL) specimens. The assay demonstrated high sensitivity and specificity in both experimental mouse models and clinical samples, producing results within 1 h without the need for sophisticated equipment. This simple, robust, and cost-effective molecular diagnostic tool holds great potential for early detection of mucormycosis, facilitating prompt antifungal therapy and improving patient survival.

Mucormycosis is an emerging, life-threatening human infection caused by Mucorales fungi^1,2,3. Metabolic disorders uniquely predispose an ever-expanding group of patients to mucormycosis through poorly understood mechanisms^1,2,4,5, suggesting that uncharacterized host metabolic effectors may confer protective immunity against this infection. Here we uncover a master regulatory role of albumin in host defence against Mucorales through the modulation of fungal pathogenicity. Our initial studies identified severe hypoalb uminaemia as a prominent metabolic abnormality and an independent biomarker of poor mucormycosis outcome across three distinct cohorts of patients with mucormycosis. Notably, purified albumin selectively inhibits Mucorales growth among a range of pathogens, and albumin-deficient mice display susceptibility specifically to mucormycosis. The antifungal activity of albumin is mediated by the release of bound free fatty acids (FFAs). Albumin prevents FFA oxidation, which otherwise abolishes their antifungal properties, and sera from patients with mucormycosis display high levels of oxidized FFAs. Physiologically, albumin-bound FFAs suppress the expression of key virulence factors by inhibiting protein synthesis, the reby rendering Mucorales avirulent in vivo. Overall, we identify a host defence mechanism that directs the pathogen to suppress its pathogenicity program in response to unfavourable metabolic cues regulated by albumin. These findings have major implications for the pathogenesis and management of mucormycosis.

Invasive pulmonary aspergillosis (IPA), mainly caused by A. fumigatus, remains a life-threatening fungal infection, with rising reports of infections caused by resistant species such as Aspergillus lentulus and Aspergillus calidoustus . EL219 (formerly known as SF001) is a novel, next-generation polyene with enhanced ergosterol selectivity and reduced nephrotoxicity. We evaluated the efficacy of EL219 compared to liposomal amphotericin B (LAMB) in vitro using the Clinical Laboratory and Standards Institute M38 methodology and in immunosuppressed murine models of IPA caused by A. fumigatus , A. lentulus , and A. calidoustus . Immunosuppressed ICR mice were infected via inhalation ( A. fumigatus ) or via intratracheal instillation ( A. lentulus and A. calidoustus ), then treated intravenously once daily with placebo, EL219 (0.3, 1.5, 7.5, and 30 mg/kg), or LAMB (5 mg/kg) starting 16 h post-infection. Treatment lasted 4 days ( A. fumigatus and A. lentulus ) or 7 days ( A. calidoustus ). Survival through Day 21 and lung fungal burden at Day 4 were assessed. EL219 showed enhanced in vitro activity against A. lentulus and A. calidoustus compared to LAMB. EL219 significantly improved survival in a dose-dependent manner, with 30 mg/kg outperforming LAMB. EL219 also resulted in >5-log fungal burden reductions in A. fumigatus -infected lungs. EL219 demonstrated broad-spectrum efficacy, improved survival, and reduced lung fungal burden, supporting its potential as a novel therapy for IPA.

Candida species, including Candida albicans and Candida auris, represent a growing public health concern due to their increasing prevalence and resistance to antifungal agents. C. albicans is known for causing both superficial and invasive infections, while C. auris is a newly emerged, multidrug-resistant pathogen responsible for severe hospital outbreaks with a high mortality rate of ~ 60% in bloodstream infections. Vaccine candidates targeting C. albicans hyphal cell wall proteins Als3p and Hyr1p have shown protective efficacy in mice. NDV-3A, an alum-formulated Als3p-based vaccine, protected against recurrent vulvovaginal candidiasis in women. We earlier showed that both Als3p and Hyr1p have orthologs in C. auris, and that the NDV-3A vaccine, alongside an anti-Hyr1p monoclonal antibody, protected mice from multidrug resistant C. auris candidemia. Here, we optimized VXV-01, an Als3p and Hyr1p dual antigen vaccine formulated with the clinical-stage adjuvant CAF01, demonstrating robust immunity and CD4 T cell-dependent protection against lethal C. albicans and C. auris. The VXV-01 vaccine did not antagonize antifungal drug therapy and showed higher overall mouse survival than mice receiving the vaccine or antifungal drug alone, albeit this difference did not reach statistical significance. This study highlights the potential of VXV-01 in providing durable protective immunity against hematogenously disseminated C. albicans and C. auris and mucosal C. albicans infections.

Summary: Mucormycosis is an emerging, life-threatening infection caused by Mucorales fungi with incompletely understood immunopathogenesis. We found that unlike other fungal pathogens, Mucorales trigger coordinated neutrophil clustering (swarming) upon swelling of fungal spores. Neutrophil swarms efficiently eliminate germinating Mucorales via ROS production, upon sensing of β-glucan by complement receptor 3 (CR3). Impaired neutrophil clustering in mucormycosis-predisposing conditions, allows for mucoricin toxin production by germinating Mucorales. Released mucoricin selectively binds to β-glucan on the surface of Mucorales, creating a molecular trap that induces neutrophil apoptosis and swarming disruption, leading to invasive fungal disease. Suppression of mucoricin, either genetically or physiologically by albumin-bound free fatty acids, stabilizes neutrophil clusters leading to abrogation of fungal pathogenicity. Additionally, prophylactic GM-CSF administration prevents mucoricin-induced neutrophil death, and protects mice from mucormycosis. Our work identifies neutrophil swarming as the primary target of mucoricin and provides compelling evidence for therapeutic harnessing of this pathway to improve mucormycosis outcome.

Oropharyngeal candidiasis (OPC) caused by the fungus Candida albicans triggers a robust inflammatory response that rapidly eliminates the bulk of the fungal load in the oral mucosa. While pro-inflammatory responses to OPC have been extensively studied, little is known about the counterbalance of immunity in the infection milieu that mitigates inflammatory damage. We employed 10× Visium Spatial Transcriptomics, a next-generation technology that preserves the spatial integrity of infected tongue tissues, enabling high-resolution mapping of the host microenvironment during Candida infection in a murine OPC model. This approach provided a view of cellular interactions and immune dynamics, revealing intricate crosstalk between distinct immune cell populations. Our findings highlight a previously underappreciated role of the type-2 immune response and M2 macrophages in maintaining tissue homeostasis, and the predicted role of platelet degranulation in facilitating disease resolution. Finally, we identified a novel family of small proline-rich antimicrobial proteins with potent antifungal activity. These molecules emerge as promising therapeutic candidates, offering a new avenue for combating OPC. IMPORTANCE: Oropharyngeal candidiasis (OPC), a fungal infection caused by Candida albicans , affects individuals with weakened immune systems. Our study used spatial transcriptomics, a cutting-edge technology that preserves tissue architecture while mapping immune interactions at high resolution. This approach allowed us to uncover previously unrecognized cellular crosstalk and regulatory pathways that shape the host response to OPC. We discovered that platelets, beyond their role in clotting, play a key role in antifungal defense. Additionally, M2 macrophages are important for resistance to OPC. Most notably, we identified a new family of antimicrobial proteins with strong antifungal properties, presenting promising therapeutic potential. By uncovering these overlooked immune mechanisms, our research findings may lead to better treatments for OPC, particularly in immunocompromised individuals.