Interaction Between Macrophages and Cryptococcus neoformans: Distinguishing Phagocytosed Versus External Fungi.

The interaction between macrophages and Cryptococcus neoformans is crucial in the pathogenesis of cryptococcosis. These phagocytes are important immune effectors, but also a niche in which facultative intracellular parasites, such as C. neoformans, thrive. Consequently, phagocytosis of cryptococcal cells and its outcomes are very frequently studied. One major issue with several of the tests used for this, however, is that macrophage-C. neoformans interaction does not always result in phagocytosis, as fungi may be attached to the external surface of the phagocyte. The most used methodologies to study phagocytosis of cryptococcal cells have varying degrees of precision in separating fungi that are truly internalized from those that are outside macrophages. Here we describe two assays to measure phagocytosis that can differentiate internal from external C. neoformans cells.

Measuring Laccase Activity and Melanin Production in Cryptococcus neoformans.

Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.

Semi-synthetic glycoconjugate vaccine candidate against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, posing a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semi-synthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semi-synthetic glycoconjugate vaccines contain the identical synthetic decasaccharide (M2 motif) antigen. This motif is present in serotype A strains, which constitute 95% of clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity towards M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). While these findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. It could serve as a component in a multi-valent GXM motif vaccine, enhancing both strength and breadth of immune responses.

Unbalanced networks and disturbed kinetics of serum soluble mediators associated with distinct disease outcomes in severe COVID-19 patients.

The present study applied distinct models of descriptive analysis to explore the integrative networks and the kinetic timeline of serum soluble mediators to select a set of systemic biomarkers applicable for the clinical management of COVID-19 patients. For this purpose, a total of 246 participants (82 COVID-19 and 164 healthy controls - HC) were enrolled in a prospective observational study. Serum soluble mediators were quantified by high-throughput microbeads array on hospital admission (D0) and at consecutive timepoints (D1-6 and D7-20). The results reinforce that the COVID-19 group exhibited a massive storm of serum soluble mediators. While increased levels of CCL3 and G-CSF were associated with the favorable prognosis of non-mechanical ventilation (nMV) or discharge, high levels of CXCL10 and IL-6 were observed in patients progressing to mechanical ventilation (MV) or death. At the time of admission, COVID-19 patients presented a complex and robust serum soluble mediator network, with a higher number of strong correlations involving IFN-γ, IL-1Ra and IL-9 observed in patients progressing to MV or death. Multivariate regression analysis demonstrates the ability of serum soluble mediators to cluster COVID-19 from HC. Ascendant fold change signatures and the kinetic timeline analysis further confirmed that the pairs "CCL3 and G-CSF" and "CXCL10 and IL-6" were associated with favorable or poor prognosis, respectively. A selected set of systemic mediators (IL-6, IFN-γ, IL-1Ra, IL-13, PDGF and IL-7) were identified as putative laboratory markers, applicable as complementary records for the clinical management of patients with severe COVID-19.

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Hinge influences in murine IgG binding to Cryptococcus neoformans capsule.

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.

Cryptococcal Virulence in Humans: Learning From Translational Studies With Clinical Isolates.

Cryptococcosis, an invasive mycosis caused by Cryptococcus spp, kills between 20% and 70% of the patients who develop it. There are no vaccines for prevention, and treatment is based on a limited number of antifungals. Studying fungal virulence and how the host responds to infection could lead to new therapies, improving outcomes for patients. The biggest challenge, however, is that experimental cryptococcosis models do not completely recapitulate human disease, while human experiments are limited due to ethical reasons. To overcome this challenge, one of the approaches used by researchers and clinicians is to: 1) collect cryptococcal clinical isolates and associated patient data; 2) study the set of isolates in the laboratory (virulence and host-pathogen interaction variables, molecular markers); 3) correlate the laboratory and patient data to understand the roles fungal attributes play in the human disease. Here we review studies that have shed light on the cryptococcosis pathophysiology using these approaches, with a special focus on human disease. Isolates that more effectively evade macrophage responses, that secrete more laccase, melanize faster and have larger capsules in the cerebrospinal fluid are associated with poorer patient outcomes. Additionally, molecular studies have also shown that cryptococcal clades vary in virulence, with clinical impact. Limitations of those studies include the use of a small number of isolates or retrospectively collected clinical data. The fact that they resulted in very important information is a reflection of the impact this strategy has in understanding cryptococcosis and calls for international collaboration that could boost our knowledge.

Molecular and Cellular Biomarkers of COVID-19 Prognosis: Protocol for the Prospective Cohort TARGET Study.

BACKGROUND: Since the beginning of the COVID-19 pandemic, the world's attention has been focused on better understanding the relation between the human host and the SARS-CoV-2 virus, as its action has led to hundreds of thousands of deaths. OBJECTIVE: In this context, we decided to study certain consequences of the abundant cytokine release over the innate and adaptive immune systems, inflammation, and hemostasis, comparing mild and severe forms of COVID-19. METHODS: To accomplish these aims, we will analyze demographic characteristics, biochemical tests, immune biomarkers, leukocyte phenotyping, immunoglobulin profile, hormonal release (cortisol and prolactin), gene expression, thromboelastometry, neutralizing antibodies, metabolic profile, and neutrophil function (reactive oxygen species production, neutrophil extracellular trap production, phagocytosis, migration, gene expression, and proteomics). A total of 200 reverse transcription polymerase chain reaction-confirmed patients will be enrolled and divided into two groups: mild/moderate or severe/critical forms of COVID-19. Blood samples will be collected at different times: at inclusion and after 9 and 18 days, with an additional 3-day sample for severe patients. We believe that this information will provide more knowledge for future studies that will provide more robust and useful clinical information that may allow for better decisions at the front lines of health care. RESULTS: The recruitment began in June 2020 and is still in progress. It is expected to continue until February 2021. Data analysis is scheduled to start after all data have been collected. The coagulation study branch is complete and is already in the analysis phase. CONCLUSIONS: This study is original in terms of the different parameters analyzed in the same sample of patients with COVID-19. The project, which is currently in the data collection phase, was approved by the Brazilian Committee of Ethics in Human Research (CAAE 30846920.7.0000.0008). TRIAL REGISTRATION: Brazilian Registry of Clinical Trials RBR-62zdkk; https://ensaiosclinicos.gov.br/rg/RBR-62zdkk. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/24211.

Transcriptional Remodeling Patterns in Murine Dendritic Cells Infected with Paracoccidioides brasiliensis: More Is Not Necessarily Better.

Most people infected with the fungus Paracoccidioides spp. do not get sick, but approximately 5% develop paracoccidioidomycosis. Understanding how host immunity determinants influence disease development could lead to novel preventative or therapeutic strategies; hence, we used two mouse strains that are resistant (A/J) or susceptible (B10.A) to P. brasiliensis to study how dendritic cells (DCs) respond to the infection. RNA sequencing analysis showed that the susceptible strain DCs remodeled their transcriptomes much more intensely than those from the resistant strain, agreeing with a previous model of more intense innate immunity response in the susceptible strain. Contrastingly, these cells also repress genes/processes involved in antigen processing and presentation, such as lysosomal activity and autophagy. After the interaction with P. brasiliensis, both DCs and macrophages from the susceptible mouse reduced the autophagy marker LC3-II recruitment to the fungal phagosome compared to the resistant strain cells, confirming this pathway's repression. These results suggest that impairment in antigen processing and presentation processes might be partially responsible for the inefficient activation of the adaptive immune response in this model.

A novel Sporothrix brasiliensis genomic variant in Midwestern Brazil: evidence for an older and wider sporotrichosis epidemic.

Sporotrichosis is a subcutaneous infection caused by fungi from the genus Sporothrix. It is transmitted by inoculation of infective particles found in plant-contaminated material or diseased animals, characterizing the classic sapronotic and emerging zoonotic transmission, respectively. Since 1998, southeastern Brazil has experienced a zoonotic sporotrichosis epidemic caused by S. brasiliensis, centred in the state of Rio de Janeiro. Our observation of feline sporotrichosis cases in Brasília (Midwestern Brazil), around 900 km away from Rio de Janeiro, led us to question whether the epidemic caused by S. brasiliensis has spread from the epicentre in Rio de Janeiro, emerged independently in the two locations, or if the disease has been present and unrecognized in Midwestern Brazil. A retrospective analysis of 91 human and 4 animal cases from Brasília, ranging from 1993 to 2018, suggests the occurrence of both sapronotic and zoonotic transmission. Molecular typing of the calmodulin locus identified S. schenckii as the agent in two animals and all seven human patients from which we were able to recover clinical isolates. In two other animals, the disease was caused by S. brasiliensis. Whole-genome sequence typing of seven Sporothrix spp. strains from Brasília and Rio de Janeiro suggests that S. brasiliensis isolates from Brasília are genetically distinct from those obtained at the epicentre of the outbreak in Rio de Janeiro, both in phylogenomic and population genomic analyses. The two S. brasiliensis populations seem to have separated between 2.2 and 3.1 million years ago, indicating independent outbreaks or that the zoonotic S. brasiliensis outbreak might have started earlier and be more widespread in South America than previously recognized.

Paracoccidioides HSP90 Can Be Found in the Cell Surface and Is a Target for Antibodies with Therapeutic Potential.

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the host's antifungal immunity. In this work we show the generation of and promising results with an mAb against Heat Shock Protein (HSP)90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM.

Laccase Affects the Rate of Cryptococcus neoformans Nonlytic Exocytosis from Macrophages.

Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans, we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis.IMPORTANCECryptococcus neoformans is a yeast that causes severe disease, primarily in immunosuppressed people. It has many attributes that allow it to survive and cause disease, such as a polysaccharide capsule and the dark pigment melanin produced by the laccase enzyme. Upon infection, the yeast is ingested by cells called macrophages, whose function is to kill them. Instead, these fungal cells can exit from macrophages in a process called nonlytic exocytosis. We know that this process is controlled by both host and fungal factors, only some of which are known. As part of an ongoing study, we observed that C. neoformans isolates that produce melanin faster are more-frequent targets of nonlytic exocytosis. Further experiments showed that this is probably due to higher production of laccase, because fungi lacking this enzyme are nonlytically exocytosed less often. This shows that laccase is an important signal/regulator of nonlytic exocytosis of C. neoformans from macrophages.

Mechanisms of action of antimicrobial peptides ToAP2 and NDBP-5.7 against Candida albicans planktonic and biofilm cells.

Candida albicans is a major cause of human infections, ranging from relatively simple to treat skin and mucosal diseases to systemic life-threatening invasive candidiasis. Fungal infections treatment faces three major challenges: the limited number of therapeutic options, the toxicity of the available drugs, and the rise of antifungal resistance. In this study, we demonstrate the antifungal activity and mechanism of action of peptides ToAP2 and NDBP-5.7 against planktonic cells and biofilms of C. albicans. Both peptides were active against C. albicans cells; however, ToAP2 was more active and produced more pronounced effects on fungal cells. Both peptides affected C. albicans membrane permeability and produced changes in fungal cell morphology, such as deformations in the cell wall and disruption of ultracellular organization. Both peptides showed synergism with amphotericin B, while ToAP2 also presents a synergic effect with fluconazole. Besides, ToAP2 (6.25 µM.) was able to inhibit filamentation after 24 h of treatment and was active against both the early phase and mature biofilms of C. albicans. Finally, ToAP2 was protective in a Galleria mellonella model of infection. Altogether these results point to the therapeutic potential of ToAP2 and other antimicrobial peptides in the development of new therapies for C. albicans infections.

Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans.

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

Paracoccidioides HSP90 can be found in the cell surface and Is a target for antibodies with therapeutic potential

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the host’s antifungal immunity. In this work we show the generation of and promising results with an mAb against Heat Shock Protein (HSP)90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM

A hidden battle in the dirt: Soil amoebae interactions with Paracoccidioides spp.

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae-all potential predators of fungi-grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits.

Integrin ß1 Promotes the Interaction of Murine IgG3 with Effector Cells.

Abs exert several of their effector functions by binding to cell surface receptors. For murine IgG3 (mIgG3), the identity of its receptors (and the very existence of a receptor) is still under debate, as not all mIgG3 functions can be explained by interaction with FcγRI. This implies the existence of an alternate receptor, whose identity we sought to pinpoint. We found that blockage of integrin ß1 selectively hampered binding of mIgG3 to macrophages and mIgG3-mediated phagocytosis. Manganese, an integrin activator, increased mIgG3 binding to macrophages. Blockage of FcγRI or Itgb1 inhibited binding of different mIgG3 Abs to variable extents. Our results are consistent with the notion that Itgb1 functions as part of an IgG receptor complex. Given the more ancient origin of integrins in comparison with FcγR, this observation could have far-ranging implications for our understanding of the evolution of Ab-mediated immunity as well as in immunity to microorganisms, pathogenesis of autoimmune diseases, and Ab engineering.

Antifungal drugs: New insights in research & development.

The need for better antifungal therapy is commonly accepted in view of the high mortality rates associated with systemic infections, the low number of available antifungal classes, their associated toxicity and the increasing number of infections caused by strains with natural or acquired resistance. The urgency to expand the range of therapeutic options for the treatment of fungal infections has led researchers in recent decades to seek alternative antifungal targets when compared to the conventional ones currently used. Although new potential targets are reported, translating the discoveries from bench to bedside is a long process and most of these drugs fail to reach the patients. In this review, we discuss the development of antifungal drugs focusing on the approach of drug repurposing and the search for novel drugs for classical targets, the most recently described gene targets for drug development, the possibilities of immunotherapy using antibodies, cytokines, therapeutic vaccines and antimicrobial peptides.

Imaging Cryptococcus spp. Capsule by Differential Interference Contrast Microscopy Using Percoll®.

The most important virulence factor in the Cryptococcus genus is the polysaccharide capsule. This genus includes several species that cause life-threatening invasive disease. An increase in capsule thickness is important during fungal infection. The capsule is usually imaged using India ink, and crucial insights on the dynamics of its growth have been obtained using capsule-binding proteins such as specific antibodies or complement. We have developed an alternative method that allows both static and time-lapse imaging of the capsule using Percoll®, a suspension of nanometric spheres that do not penetrate the capsule. Given that these particles have a higher refractive index than the capsule, the latter can be imaged by differential interference contrast (DIC) microscopy. Static observation of the capsule with DIC and Percoll® results in capsule thickness measurements that match those made with India ink. Using capsule-inducing media, a glass-bottom incubation chamber and a live-imaging system equipped for DIC microscopy, this method allows time-lapse imaging of capsule growth. In contrast with India ink staining, DIC imaging of Percoll® exclusion halos result in crisp images. The greatest advantage of this method, though, is that unlike India ink, the Percoll® particles are non-toxic and unlike opsonins they do not bind the capsule, resulting in observations of capsule growth that are free from interference of bound proteins on capsule physiology.