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.

Single nucleotide polymorphisms and chromosomal copy number variation may impact the Sporothrix brasiliensis antifungal susceptibility and sporotrichosis clinical outcomes.

Feline-transmitted sporotrichosis has garnered attention due to the recent high incidence and the lack of efficient control in the epicenter of the epidemic, Rio de Janeiro, Brazil. Sporothrix brasiliensis is the major pathogen involved in feline-to-human sporotrichosis in Brazil and displays more virulent genotypes than the closely related species S. schenckii. Over the last two decades, several reports of antifungal-resistant strains have emerged. Sequencing and comparison analysis of the outbreak strains allowed us to observe that the azole non-wild-type S. brasiliensis strain CFP 1054 had significant chromosomal variations compared to wild-type strains. One of these variants includes a region of 231 Kb containing 75 duplicated genes, which were overrepresented for lipid and isoprenoid metabolism. We also identified an additional strain (CFP 1055) that was resistant to itraconazole and amphotericin B, which had a single nucleotide polymorphism in the tac1 gene. The patients infected with these two strains showed protracted clinical course and sequelae. Even though our sample size is modest, these results suggest the possibility of identifying specific point mutations and large chromosomal duplications potentially associated with antifungal resistance and clinical outcomes of sporotrichosis.

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.

Association of Convalescent Plasma Treatment With Clinical Status in Patients Hospitalized With COVID-19: A Meta-analysis.

Importance: COVID-19 convalescent plasma (CCP) is a potentially beneficial treatment for COVID-19 that requires rigorous testing. Objective: To compile individual patient data from randomized clinical trials of CCP and to monitor the data until completion or until accumulated evidence enables reliable conclusions regarding the clinical outcomes associated with CCP. Data Sources: From May to August 2020, a systematic search was performed for trials of CCP in the literature, clinical trial registry sites, and medRxiv. Domain experts at local, national, and international organizations were consulted regularly. Study Selection: Eligible trials enrolled hospitalized patients with confirmed COVID-19, not receiving mechanical ventilation, and randomized them to CCP or control. The administered CCP was required to have measurable antibodies assessed locally. Data Extraction and Synthesis: A minimal data set was submitted regularly via a secure portal, analyzed using a prespecified bayesian statistical plan, and reviewed frequently by a collective data and safety monitoring board. Main Outcomes and Measures: Prespecified coprimary end points-the World Health Organization (WHO) 11-point ordinal scale analyzed using a proportional odds model and a binary indicator of WHO score of 7 or higher capturing the most severe outcomes including mechanical ventilation through death and analyzed using a logistic model-were assessed clinically at 14 days after randomization. Results: Eight international trials collectively enrolled 2369 participants (1138 randomized to control and 1231 randomized to CCP). A total of 2341 participants (median [IQR] age, 60 [50-72] years; 845 women [35.7%]) had primary outcome data as of April 2021. The median (IQR) of the ordinal WHO scale was 3 (3-6); the cumulative OR was 0.94 (95% credible interval [CrI], 0.74-1.19; posterior probability of OR <1 of 71%). A total of 352 patients (15%) had WHO score greater than or equal to 7; the OR was 0.94 (95% CrI, 0.69-1.30; posterior probability of OR <1 of 65%). Adjusted for baseline covariates, the ORs for mortality were 0.88 at day 14 (95% CrI, 0.61-1.26; posterior probability of OR <1 of 77%) and 0.85 at day 28 (95% CrI, 0.62-1.18; posterior probability of OR <1 of 84%). Heterogeneity of treatment effect sizes was observed across an array of baseline characteristics. Conclusions and Relevance: This meta-analysis found no association of CCP with better clinical outcomes for the typical patient. These findings suggest that real-time individual patient data pooling and meta-analysis during a pandemic are feasible, offering a model for future research and providing a rich data resource.

Development and Validation of a Treatment Benefit Index to Identify Hospitalized Patients With COVID-19 Who May Benefit From Convalescent Plasma.

Importance: Identifying which patients with COVID-19 are likely to benefit from COVID-19 convalescent plasma (CCP) treatment may have a large public health impact. Objective: To develop an index for predicting the expected relative treatment benefit from CCP compared with treatment without CCP for patients hospitalized for COVID-19 using patients' baseline characteristics. Design, Setting, and Participants: This prognostic study used data from the COMPILE study, ie, a meta-analysis of pooled individual patient data from 8 randomized clinical trials (RCTs) evaluating CCP vs control in adults hospitalized for COVID-19 who were not receiving mechanical ventilation at randomization. A combination of baseline characteristics, termed the treatment benefit index (TBI), was developed based on 2287 patients in COMPILE using a proportional odds model, with baseline characteristics selected via cross-validation. The TBI was externally validated on 4 external data sets: the Expanded Access Program (1896 participants), a study conducted under Emergency Use Authorization (210 participants), and 2 RCTs (with 80 and 309 participants). Exposure: Receipt of CCP. Main Outcomes and Measures: World Health Organization (WHO) 11-point ordinal COVID-19 clinical status scale and 2 derivatives of it (ie, WHO score of 7-10, indicating mechanical ventilation to death, and WHO score of 10, indicating death) at day 14 and day 28 after randomization. Day 14 WHO 11-point ordinal scale was used as the primary outcome to develop the TBI. Results: A total of 2287 patients were included in the derivation cohort, with a mean (SD) age of 60.3 (15.2) years and 815 (35.6%) women. The TBI provided a continuous gradation of benefit, and, for clinical utility, it was operationalized into groups of expected large clinical benefit (B1; 629 participants in the derivation cohort [27.5%]), moderate benefit (B2; 953 [41.7%]), and potential harm or no benefit (B3; 705 [30.8%]). Patients with preexisting conditions (diabetes, cardiovascular and pulmonary diseases), with blood type A or AB, and at an early COVID-19 stage (low baseline WHO scores) were expected to benefit most, while those without preexisting conditions and at more advanced stages of COVID-19 could potentially be harmed. In the derivation cohort, odds ratios for worse outcome, where smaller odds ratios indicate larger benefit from CCP, were 0.69 (95% credible interval [CrI], 0.48-1.06) for B1, 0.82 (95% CrI, 0.61-1.11) for B2, and 1.58 (95% CrI, 1.14-2.17) for B3. Testing on 4 external datasets supported the validation of the derived TBIs. Conclusions and Relevance: The findings of this study suggest that the CCP TBI is a simple tool that can quantify the relative benefit from CCP treatment for an individual patient hospitalized with COVID-19 that can be used to guide treatment recommendations. The TBI precision medicine approach could be especially helpful in a pandemic.

Thromboelastometry demonstrates endogenous coagulation activation in nonsevere and severe COVID-19 patients and has applicability as a decision algorithm for intervention.

In patients with severe forms of COVID-19, thromboelastometry has been reported to display a hypercoagulant pattern. However, an algorithm to differentiate severe COVID-19 patients from nonsevere patients and healthy controls based on thromboelastometry parameters has not been developed. Forty-one patients over 18 years of age with positive qRT-PCR for SARS-CoV-2 were classified according to the severity of the disease: nonsevere (NS, n = 20) or severe (S, n = 21). A healthy control (HC, n = 9) group was also examined. Blood samples from all participants were tested by extrinsic (EXTEM), intrinsic (INTEM), non-activated (NATEM) and functional assessment of fibrinogen (FIBTEM) assays of thromboelastometry. The thrombodynamic potential index (TPI) was also calculated. Severe COVID-19 patients exhibited a thromboelastometry profile with clear hypercoagulability, which was significantly different from the NS and HC groups. Nonsevere COVID-19 cases showed a trend to thrombotic pole. The NATEM test suggested that nonsevere and severe COVID-19 patients presented endogenous coagulation activation (reduced clotting time and clot formation time). TPI data were significantly different between the NS and S groups. The maximum clot firmness profile obtained by FIBTEM showed moderate/elevated accuracy to differentiate severe patients from NS and HC. A decision tree algorithm based on the FIBTEM-MCF profile was proposed to differentiate S from HC and NS. Thromboelastometric parameters are a useful tool to differentiate the coagulation profile of nonsevere and severe COVID-19 patients for therapeutic intervention purposes.

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.

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.

Thioredoxin Reductase 1 Is a Highly Immunogenic Cell Surface Antigen in Paracoccidioides spp., Candida albicans, and Cryptococcus neoformans.

The increasing number of immunocompromised people has made invasive fungal infections more common. The antifungal armamentarium, in contrast, is limited to a few classes of drugs, with frequent toxicity and low efficacy pointing to the need for new agents. Antibodies are great candidates for novel antifungals, as their specificity can result in lower toxicity. Additionally, the immunomodulatory activity of antibodies could treat the underlying cause of many invasive mycoses, immune disfunction. In a previous comparative genomics study, we identified several potential targets for novel antifungals. Here we validate one of these targets, thioredoxin reductase (TRR1), to produce antibodies that could be useful therapeutic tools. Recombinant TRR1 proteins were produced by heterologous expression in Escherichia coli of genes encoding the proteins from Candida albicans, Cryptococcus neoformans, and Paracoccidioides lutzii. These proteins were then used to immunize mice, followed by detection of serum antibodies against them by ELISA and western blot. A first set of experiments in which individual mice were immunized repeatedly with TRR1 from a single species showed that all three were highly immunogenic, inducing mostly IgG1 antibodies, and that antibodies produced against one species cross-reacted with the others. In a second experiment, individual mice were immunized three times, each with the protein from a different species. The high titers of antibodies confirmed the presence of antigenic epitopes that were conserved in fungi but absent in humans. Immunofluorescence with sera from these immunized mice detected the protein in the cytoplasm and on the cell surface of fungi from all three species. These results validate TRR1 as a good target for potentially broad-spectrum antifungal antibodies.