Milteforan, a promising veterinary commercial product against feline sporotrichosis.

Sporotrichosis, the cutaneous mycosis most commonly reported in Latin America, is caused by the Sporothrix clinical clade species, including Sporothrix brasiliensis and Sporothrix schenckii sensu stricto. Due to its zoonotic transmission in Brazil, S. brasiliensis represents a significant health threat to humans and domestic animals. Itraconazole, terbinafine, and amphotericin B are the most used antifungals for treating sporotrichosis. However, many strains of S. brasiliensis and S. schenckii have shown resistance to these agents, highlighting the importance of finding new therapeutic options. Here, we demonstrate that milteforan, a commercial veterinary product against dog leishmaniasis, whose active principle is miltefosine, is a possible therapeutic alternative for the treatment of sporotrichosis, as observed by its fungicidal activity in vitro against different strains of S. brasiliensis and S. schenckii. Fluorescent miltefosine localizes to the Sporothrix cell membrane and mitochondria and causes cell death through increased permeabilization. Milteforan decreases S. brasiliensis fungal burden in A549 pulmonary cells and bone marrow-derived macrophages and also has an immunomodulatory effect by decreasing TNF-α, IL-6, and IL-10 production. Our results suggest milteforan as a possible alternative to treat feline sporotrichosis. IMPORTANCE: Sporotrichosis is an endemic disease in Latin America caused by different species of Sporothrix. This fungus can infect domestic animals, mainly cats and eventually dogs, as well as humans. Few drugs are available to treat this disease, such as itraconazole, terbinafine, and amphotericin B, but resistance to these agents has risen in the last few years. Alternative new therapeutic options to treat sporotrichosis are essential. Here, we propose milteforan, a commercial veterinary product against dog leishmaniasis, whose active principle is miltefosine, as a possible therapeutic alternative for treating sporotrichosis. Milteforan decreases S. brasiliensis fungal burden in human and mouse cells and has an immunomodulatory effect by decreasing several cytokine production.

Aspergillus fumigatus conidial surface-associated proteome reveals factors for fungal evasion and host immunity modulation.

Aspergillus fumigatus causes aspergillosis and relies on asexual spores (conidia) for initiating host infection. There is scarce information about A. fumigatus proteins involved in fungal evasion and host immunity modulation. Here we analysed the conidial surface proteome of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, as well as pathogenic Aspergillus lentulus, to identify such proteins. After identifying 62 proteins exclusively detected on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding these proteins. Deletion of 33 of these genes altered susceptibility to macrophage, epithelial cells and cytokine production. Notably, a gene that encodes a putative glycosylasparaginase, modulating levels of the host proinflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins are important for evasion and modulation of the immune response at the onset of fungal infection.

Brilacidin, a host defense peptide mimetic, potentiates ibrexafungerp antifungal activity against the human pathogenic fungus Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus. BRI + IBX can inhibit the growth of A. fumigatus voriconazole- and caspofungin-resistant clinical isolates. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against viruses, bacteria, and fungi. In vitro, combination of BRI + IBX plays a fungicidal role, increases the fungal cell permeability, decreases the fungal survival in the presence of A549 epithelial cells, and appears as a promising antifungal therapeutic alternative against A. fumigatus. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Aspergillus fumigatus causes a series of distinct invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. A. fumigatus causes a spectrum of distinct clinical entities named aspergillosis, which the most severe form is the invasive pulmonary aspergillosis. There are few therapeutic options for treating aspergillosis and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a synergizer o fibrexafungerp (IBX) against A. fumigatus. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. We propose the combination of BRI and IBX as a new antifungal combinatorial treatment against aspergillosis.

Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus.

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins' involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Brilacidin, a novel antifungal agent against Cryptococcus neoformans.

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

Brilacidin, a host defense peptide mimetic, potentiates ibrexafungerp antifungal activity against the human pathogenic fungus Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic, brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus. CAS-resistant strains with mutations in fks1 that encodes the 1,3-ß-D-glucan synthase are not IBX-resistant and BRI+IBX can inhibit their growth. The combination of BRI+IBX plays a fungicidal role, increases the fungal cell permeability and decreases the fungal survival in the presence of A549 epithelial cells.

Milteforan, a promising veterinary commercial product against feline sporotrichosis.

Sporotrichosis, the cutaneous mycosis most commonly reported in Latin America, is caused by the Sporothrix clinical clade species, including Sporothrix brasiliensis and Sporothrix schenckii sensu stricto. In Brazil, S. brasiliensis represents a vital health threat to humans and domestic animals due to its zoonotic transmission. Itraconazole, terbinafine, and amphotericin B are the most used antifungals for treating sporotrichosis. However, many strains of S. brasiliensis and S. schenckii have shown resistance to these agents, highlighting the importance of finding new therapeutic options. Here, we demonstrate that milteforan, a commercial veterinary product against dog leishmaniasis whose active principle is miltefosine, is a possible therapeutic alternative for the treatment of sporotrichosis, as observed by its fungicidal activity in vitro against different strains of S. brasiliensis and S. schenckii, and by its antifungal activity when used to treat infected epithelial cells and macrophages. Our results suggest milteforan as a possible alternative to treat feline sporotrichosis.

Aspergillus fumigatus mitogen-activated protein kinase MpkA is involved in gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.

Transcriptional profiling of a fungal granuloma reveals a low metabolic activity of Paracoccidioides brasiliensis yeasts and an actively regulated host immune response.

Granulomas are important immunological structures in the host defense against the fungus Paracoccidioides brasiliensis, the main etiologic agent of Paracoccidioidomycosis (PCM), a granulomatous systemic mycosis endemic in Latin America. We have performed transcriptional and proteomic studies of yeasts present in the pulmonary granulomas of PCM aiming to identify relevant genes and proteins that act under stressing conditions. C57BL/6 mice were infected with 1x106 yeasts and after 8- and 12-weeks of infection, granulomatous lesions were obtained for extraction of fungal and murine RNAs and fungal proteins. Dual transcriptional profiling was done comparing lung cells and P. brasiliensis yeasts from granulomas with uninfected lung cells and the original yeast suspension used in the infection, respectively. Mouse transcripts indicated a lung malfunction, with low expression of genes related to muscle contraction and organization. In addition, an increased expression of transcripts related to the activity of neutrophils, eosinophils, macrophages, lymphocytes as well as an elevated expression of IL-1ß, TNF-α, IFN-γ, IL-17 transcripts were observed. The increased expression of transcripts for CTLA-4, PD-1 and arginase-1, provided evidence of immune regulatory mechanisms within the granulomatous lesions. Also, our results indicate iron as a key element for the granuloma to function, where a high number of transcripts related to fungal siderophores for iron uptake was observed, a mechanism of fungal virulence not previously described in granulomas. Furthermore, transcriptomics and proteomics analyzes indicated a low fungal activity within the granuloma, as demonstrated by the decreased expression of genes and proteins related to energy metabolism and cell cycle.

Coxiella co-opts the Glutathione Peroxidase 4 to protect the host cell from oxidative stress-induced cell death.

The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow-derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress-induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.

Antimicrobial Activity the Essential Oil from Croton pluriglandulosus Carn. Leaves against Microorganisms of Clinical Interest.

Multiresistant pathogens pose a serious threat to human health. The genus Candida is one class of human pathogenic yeasts responsible for infections affecting healthy and immunocompromised patients. In this context, plant essential oils emerged as a future natural alternative to control the diseases caused by these pathogens. Based on that, the present study aimed to evaluate the antimicrobial potential of essential oil from C. pluriglandulosus and understand the mechanism of action. Here, it highlighted antimicrobial activity and the mechanisms of action of the essential oil extracted from C. pluriglandulosus Carn.-Torres & Riina (CpEO) leaves on human pathogenic microorganisms in planktonic and biofilm lifestyles. In addition, for the first time, the oil composition was revealed by GC-MS analysis and the toxicity to human red blood cells (HRBC). Twenty-six chemical compounds were identified in CpEO, elemicin, bicyclogermacrene, caryophyllene, brevifolin, and 2,4,6-trimethoxy-styrene. Through hemolytic assay, it was shown that CpEO has no toxicity to human RBCs. At the concentration of 50 µg mL-1, CpEO did not show great antibacterial potential. However, promising data were found for C. krusei and C. parapsilosis inhibiting by 89.3% and 80.7% of planktonic cell growth and 83.5% and 77.9% the biofilm formation, respectively. Furthermore, the mechanisms of action CpEO were elucidated by fluorescence. Scanning electron microscopy revealed damage to the cell membrane and pore formation, ROS overproduction, and induction of apoptosis in candida cells. Our results reinforce the potential of CpEO as an effective alternative molecule of pharmaceutical interest.

Vacuoles and peroxisomes are involved in Aspergillus fumigatus gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi.

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections.

Behind the Curtain: In Silico and In Vitro Experiments Brought to Light New Insights into the Anticryptococcal Action of Synthetic Peptides.

Cryptococcus neoformans is the pathogen responsible for cryptococcal pneumonia and meningitis, mainly affecting patients with suppressed immune systems. We have previously revealed the mechanism of anticryptococcal action of synthetic antimicrobial peptides (SAMPs). In this study, computational and experimental analyses provide new insights into the mechanisms of action of SAMPs. Computational analysis revealed that peptides interacted with the PHO36 membrane receptor of C. neoformans. Additionally, ROS (reactive oxygen species) overproduction, the enzymes of ROS metabolism, interference in the ergosterol biosynthesis pathway, and decoupling of cytochrome c mitochondrial membrane were evaluated. Three of four peptides were able to interact with the PHO36 receptor, altering its function and leading to ROS overproduction. SAMPs-treated C. neoformans cells showed a decrease in scavenger enzyme activity, supporting ROS accumulation. In the presence of ascorbic acid, an antioxidant agent, SAMPs did not induce ROS accumulation in C. neoformans cells. Interestingly, two SAMPs maintained inhibitory activity and membrane pore formation in C. neoformans cells by a ROS-independent mechanism. Yet, the ergosterol biosynthesis and lactate dehydrogenase activity were affected by SAMPs. In addition, we noticed decoupling of Cyt c from the mitochondria, which led to apoptosis events in the cryptococcal cells. The results presented herein suggest multiple mechanisms imposed by SAMPs against C. neoformans interfering in the development of resistance, thus revealing the potential of SAMPs in treating infections caused by C. neoformans.

Antifungal Potential of Synthetic Peptides against Cryptococcus neoformans: Mechanism of Action Studies Reveal Synthetic Peptides Induce Membrane-Pore Formation, DNA Degradation, and Apoptosis.

Cryptococcus neoformans is a human-pathogenic yeast responsible for pneumonia and meningitis, mainly in patients immunocompromised. Infections caused by C. neoformans are a global health concern. Synthetic antimicrobial peptides (SAMPs) have emerged as alternative molecules to cope with fungal infections, including C. neoformans. Here, eight SAMPs were tested regarding their antifungal potential against C. neoformans and had their mechanisms of action elucidated by fluorescence and scanning electron microscopies. Five SAMPs showed an inhibitory effect (MIC50) on C. neoformans growth at low concentrations. Fluorescence microscope (FM) revealed that SAMPs induced 6-kDa pores in the C. neoformans membrane. Inhibitory assays in the presence of ergosterol revealed that some peptides lost their activity, suggesting interaction with it. Furthermore, FM analysis revealed that SAMPs induced caspase 3/7-mediated apoptosis and DNA degradation in C. neoformans cells. Scanning Electron Microscopy (SEM) analysis revealed that peptides induced many morphological alterations such as cell membrane, wall damage, and loss of internal content on C. neoformans cells. Our results strongly suggest synthetic peptides are potential alternative molecules to control C. neoformans growth and treat the cryptococcal infection.

Aspergillus fumigatus FhdA Transcription Factor Is Important for Mitochondrial Activity and Codon Usage Regulation during the Caspofungin Paradoxical Effect.

Aspergillus fumigatus is the main etiological agent of aspergillosis. The antifungal drug caspofungin (CSP) can be used against A. fumigatus, and CSP tolerance is observed. We have previously shown that the transcription factor FhdA is important for mitochondrial activity. Here, we show that FhdA regulates genes transcribed by RNA polymerase II and III. FhdA influences the expression of tRNAs that are important for mitochondrial function upon CSP. Our results show a completely novel mechanism that is impacted by CSP.

Secondary Metabolites Produced during Aspergillus fumigatus and Pseudomonas aeruginosa Biofilm Formation.

In cystic fibrosis (CF), mucus plaques are formed in the patient's lungs, creating a hypoxic condition and a propitious environment for colonization and persistence of many microorganisms. There is clinical evidence showing that Aspergillus fumigatus can cocolonize CF patients with Pseudomonas aeruginosa, which has been associated with lung function decline. P. aeruginosa produces several compounds with inhibitory and antibiofilm effects against A. fumigatus in vitro; however, little is known about the fungal compounds produced in counterattack. Here, we annotated fungal and bacterial secondary metabolites (SM) produced in mixed biofilms under normoxia and hypoxia conditions. We detected nine SM produced by P. aeruginosa. Phenazines and different analogs of pyoverdin were the main compounds produced by P. aeruginosa, and their secretion levels were increased by the fungal presence. The roles of the two operons responsible for phenazine production (phzA1 and phzA2) were also investigated, and mutants lacking one of those operons were able to produce partial sets of phenazines. We detected a total of 20 SM secreted by A. fumigatus either in monoculture or in coculture with P. aeruginosa. All these compounds were secreted during biofilm formation in either normoxia or hypoxia. However, only eight compounds (demethoxyfumitremorgin C, fumitremorgin, ferrichrome, ferricrocin, triacetylfusigen, gliotoxin, gliotoxin E, and pyripyropene A) were detected during biofilm formation by the coculture of A. fumigatus and P. aeruginosa under normoxia and hypoxia conditions. Overall, we showed how diverse SM secretion is during A. fumigatus and P. aeruginosa mixed culture and how this can affect biofilm formation in normoxia and hypoxia. IMPORTANCE The interaction between Pseudomonas aeruginosa and Aspergillus fumigatus has been well characterized in vitro. In this scenario, the bacterium exerts a strong inhibitory effect against the fungus. However, little is known about the metabolites produced by the fungus to counterattack the bacteria. Our work aimed to annotate secondary metabolites (SM) secreted during coculture between P. aeruginosa and A. fumigatus during biofilm formation in both normoxia and hypoxia. The bacterium produces several different types of phenazines and pyoverdins in response to presence of the fungus. In contrast, we were able to annotate 29 metabolites produced during A. fumigatus biofilm formation, but only 8 compounds were detected during biofilm formation by the coculture of A. fumigatus and P. aeruginosa upon either normoxia or hypoxia. In conclusion, we detected many SM secreted during A. fumigatus and P. aeruginosa biofilm formation. This analysis provides several opportunities to understand the interactions between these two species.

Examination of Genome-Wide Ortholog Variation in Clinical and Environmental Isolates of the Fungal Pathogen Aspergillus fumigatus.

Aspergillus fumigatus is both an environmental saprobe and an opportunistic human fungal pathogen. Knowledge of genomic variation across A. fumigatus isolates is essential for understanding the evolution of pathogenicity, virulence, and resistance to antifungal drugs. Here, we investigated 206 A. fumigatus isolates (133 clinical and 73 environmental isolates), aiming to identify genes with variable presence across isolates and test whether this variation was related to the clinical or environmental origin of isolates. The PanOrtho genome of A. fumigatus consists of 13,085 ortholog groups, of which 7,773 (59.4%) are shared by all isolates (core groups) and 5,312 (40.6%) vary in their gene presence across isolates (accessory groups plus singletons). Despite differences in the distribution of orthologs across all isolates, no significant differences were observed among clinical versus environmental isolates when phylogeny was accounted for. Orthologs that differ in their distribution across isolates tend to occur at low frequency and/or be restricted to specific isolates; thus, the degree of genomic conservation between orthologs of A. fumigatus is high. These results suggest that differences in the distribution of orthologs within A. fumigatus cannot be associated with the clinical or environmental origin of isolates. IMPORTANCE Aspergillus fumigatus is a cosmopolitan species of fungus responsible for thousands of cases of invasive disease annually. Clinical and environmental isolates of A. fumigatus exhibit extensive phenotypic differences, including differences related to virulence and antifungal drug resistance. A comprehensive survey of the genomic diversity present in A. fumigatus and its relationship to the clinical or environmental origin of isolates can contribute to the prediction of the mechanisms of evolution and infection of the species. Our results suggest that there is no significant variation in ortholog distribution between clinical and environmental isolates when accounting for evolutionary history. The work supports the hypothesis that environmental and clinical isolates of A. fumigatus do not differ in their gene contents.

Synergistic Antifungal Activity of Synthetic Peptides and Antifungal Drugs against Candida albicans and C. parapsilosis Biofilms.

C. albicans and C. parapsilosis are biofilm-forming yeasts responsible for bloodstream infections that can cause death. Synthetic antimicrobial peptides (SAMPs) are considered to be new weapons to combat these infections, alone or combined with drugs. Here, two SAMPs, called Mo-CBP3-PepI and Mo-CBP3-PepIII, were tested alone or combined with nystatin (NYS) and itraconazole (ITR) against C. albicans and C. parapsilosis biofilms. Furthermore, the mechanism of antibiofilm activity was evaluated by fluorescence and scanning electron microscopies. When combined with SAMPs, the results revealed a 2- to 4-fold improvement of NYS and ITR antibiofilm activity. Microscopic analyses showed cell membrane and wall damage and ROS overproduction, which caused leakage of internal content and cell death. Taken together, these results suggest the potential of Mo-CBP3-PepI and Mo-CBP3-PepIII as new drugs and adjuvants to increase the activity of conventional drugs for the treatment of clinical infections caused by C. albicans and C. parapsilosis.

The Caspofungin Paradoxical Effect is a Tolerant "Eagle Effect" in the Filamentous Fungal Pathogen Aspergillus fumigatus.

Cell responses against antifungals other than resistance have rarely been studied in filamentous fungi, while terms such as tolerance and persistence are well-described for bacteria and increasingly examined in yeast-like organisms. Aspergillus fumigatus is a filamentous fungal pathogen that causes a disease named aspergillosis, for which caspofungin (CAS), a fungistatic drug, is used as a second-line therapy. Some A. fumigatus clinical isolates can survive and grow in CAS concentrations above the minimum effective concentration (MEC), a phenomenon known as "caspofungin paradoxical effect" (CPE). Here, we evaluated the CPE in 67 A. fumigatus clinical isolates by calculating recovery rate (RR) values, where isolates with an RR of ≥0.1 were considered CPE+ while isolates with an RR of <0.1 were classified as CPE-. Conidia produced by three CPE+ clinical isolates, CEA17 (RR = 0.42), Af293 (0.59), and CM7555 (0.38), all showed the ability to grow in high levels of CAS, while all conidia produced by the CPE- isolate IFM61407 (RR = 0.00) showed no evidence of paradoxical growth. Given the importance of the calcium/calcineurin/transcription factor-CrzA pathway in CPE regulation, we also demonstrated that all ΔcrzACEA17 (CPE+) conidia exhibited CPE while 100% of ΔcrzAAf293 (CPE-) did not exhibit CPE. Because all spores derived from an individual strain were phenotypically indistinct with respect to CPE, it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal-tolerant phenotype. Because the RR parameter showed that the strength of the CPE was not uniform between strains, we propose that the mechanisms which govern this phenomenon are multifactorial. IMPORTANCE The "Eagle effect," initially described for bacterial species, which reflects the capacity of some strains to growth above the minimum inhibitory concentration (MIC) of specific antimicrobial agents, has been known for more than 70 years. However, its underlying mechanism of action in fungi is not fully understood and its connection with other phenomena such as tolerance or persistence is not clear yet. Here, based on the characterization of the "caspofungin paradoxical effect" in several Aspergillus fumigatus clinical isolates, we demonstrate that all conidia from A. fumigatus CPE+ strains are able to grow in high levels of the drug while all conidia produced by CPE- strains show no evidence of paradoxical growth. This work fills a gap in the understanding of this multifactorial phenomenon by proposing that CPE in A. fumigatus should be considered a tolerant but not persistent phenotype.