Use of 2D minilungs from human embryonic stem cells to study the interaction of Cryptococcus neoformans with the respiratory tract.

Organoids can meet the needs between the use of cell culture and in vivo work, bringing together aspects of multicellular tissues, providing a more similar in vitro system for the study of various components, including host-interactions with pathogens and drug response. Organoids are structures that resemble organs in vivo, originating from pluripotent stem cells (PSCs) or adult stem cells (ASCs). There is great interest in deepening the understanding of the use of this technology to produce information about fungal infections and their treatments. This work aims the use 2D human lung organoid derived from human embryonic stem cells (hESCs), to investigate Cryptococcus neoformans-host interactions. C. neoformans is an opportunistic fungus acquired by inhalation that causes systemic mycosis mainly in immunocompromised individuals. Our work highlights the suitability of human minilungs for the study of C. neoformans infection (adhesion, invasion and replication), the interaction with the surfactant and induction of the host's alveolar pro-inflammatory response.

Minilungs from Human Embryonic Stem Cells to Study the Interaction of Streptococcus pneumoniae with the Respiratory Tract.

The new generation of organoids derived from human pluripotent stem cells holds a promising strategy for modeling host-bacteria interaction studies. Organoids recapitulate the composition, diversity of cell types, and, to some extent, the functional features of the native organ. We generated lung bud organoids derived from human embryonic stem cells to study the interaction of Streptococcus pneumoniae (pneumococcus) with the alveolar epithelium. Invasive pneumococcal disease is an important health problem that may occur as a result of the spread of pneumococcus from the lower respiratory tract to sterile sites. We show here an efficient experimental approach to model the main events of the pneumococcal infection that occur in the human lung, exploring bacterial adherence to the epithelium and internalization and triggering an innate response that includes the interaction with surfactant and the expression of representative cytokines and chemokines. Thus, this model, based on human minilungs, can be used to study pneumococcal virulence factors and the pathogenesis of different serotypes, and it will allow therapeutic interventions in a reliable human context. IMPORTANCE Streptococcus pneumoniae is responsible for high morbidity and mortalities rates worldwide, affecting mainly children and adults older than 65 years. Pneumococcus is also the most common etiologic agent of bacterial pneumonia and nonepidemic meningitis, and it is a frequent cause of bacterial sepsis. Although the introduction of pneumococcal vaccines has decreased the burden of pneumococcal disease, the rise of antibiotic-resistant strains and nonvaccine types by serotype replacement is worrisome. To study the biology of pneumococcus and to establish a reliable human model for pneumococcal pathogenesis, we generated human minilungs from embryonic stem cells. The results show that these organoids can be used to model some events occurring during the interaction of pneumococcus with the lung, such as adherence, internalization, and the initial alveolar innate response. This model also represents a great alternative for studying virulence factors involved in pneumonia, drug screening, and other therapeutic interventions.

Immunoproteomic and Immunopeptidomic Analyses of Histoplasma capsulatum Reveal Promiscuous and Conserved Epitopes Among Fungi With Vaccine Potential.

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species.

Cell Wall Integrity Pathway Involved in Morphogenesis, Virulence and Antifungal Susceptibility in Cryptococcus neoformans.

Due to its location, the fungal cell wall is the compartment that allows the interaction with the environment and/or the host, playing an important role during infection as well as in different biological functions such as cell morphology, cell permeability and protection against stress. All these processes involve the activation of signaling pathways within the cell. The cell wall integrity (CWI) pathway is the main route responsible for maintaining the functionality and proper structure of the cell wall. This pathway is highly conserved in the fungal kingdom and has been extensively characterized in Saccharomyces cerevisiae. However, there are still many unknown aspects of this pathway in the pathogenic fungi, such as Cryptococcus neoformans. This yeast is of particular interest because it is found in the environment, but can also behave as pathogen in multiple organisms, including vertebrates and invertebrates, so it has to adapt to multiple factors to survive in multiple niches. In this review, we summarize the components of the CWI pathway in C. neoformans as well as its involvement in different aspects such as virulence factors, morphological changes, and its role as target for antifungal therapies among others.

Immunotherapy against Systemic Fungal Infections Based on Monoclonal Antibodies.

: The increasing incidence in systemic fungal infections in humans has increased focus for the development of fungal vaccines and use of monoclonal antibodies. Invasive mycoses are generally difficult to treat, as most occur in vulnerable individuals, with compromised innate and adaptive immune responses. Mortality rates in the setting of our current antifungal drugs remain excessively high. Moreover, systemic mycoses require prolonged durations of antifungal treatment and side effects frequently occur, particularly drug-induced liver and/or kidney injury. The use of monoclonal antibodies with or without concomitant administration of antifungal drugs emerges as a potentially efficient treatment modality to improve outcomes and reduce chemotherapy toxicities. In this review, we focus on the use of monoclonal antibodies with experimental evidence on the reduction of fungal burden and prolongation of survival in in vivo disease models. Presently, there are no licensed monoclonal antibodies for use in the treatment of systemic mycoses, although the potential of such a vaccine is very high as indicated by the substantial promising results from several experimental models.

Identification of Off-Patent Drugs That Show Synergism with Amphotericin B or That Present Antifungal Action against Cryptococcus neoformans and Candida spp.

Amphotericin B (AmB) is the antifungal with the strongest fungicidal activity, but its use has several limitations, mainly associated with its toxicity. Although some lipidic and liposomal formulations that present reduced toxicity are available, their price limits their application in developing countries. Flucytosine (5FC) has shown synergistic effect with AmB for treatment of some fungal infections, such as cryptococcosis, but again, its price is a limitation for its use in many regions. In the present work, we aimed to identify new drugs that have a minor effect on Cryptococcus neoformans, reducing its growth in the presence of subinhibitory concentrations of AmB. In the initial screening, we found fourteen drugs that had this pattern. Later, checkerboard assays of selected compounds, such as erythromycin, riluzole, nortriptyline, chenodiol, nisoldipine, promazine, chlorcyclizine, cloperastine, and glimepiride, were performed and all of them confirmed for their synergistic effect (fractional inhibitory concentration index [FICI] < 0.5). Additionally, toxicity of these drugs in combination with AmB was tested in mammalian cells and in zebrafish embryos. Harmless compounds, such as the antibiotic erythromycin, were found to have synergic activity with AmB, not only against C. neoformans but also against some Candida spp., in particular against Candida albicans In parallel, we identified drugs that had antifungal activity against C. neoformans and found 43 drugs that completely inhibited the growth of this fungus, such as ciclopirox and auranofin. Our results expand our knowledge about antifungal compounds and open new perspectives in the treatment of invasive mycosis based on repurposing off-patent drugs.

Identification of Off-Patent Compounds That Present Antifungal Activity Against the Emerging Fungal Pathogen Candida auris.

Candida auris is an emerging fungal pathogen of great concern among the scientific community because it is causing an increasing number of hospital outbreaks of difficult management worldwide. In addition, isolates from this species frequently present reduced susceptibility to azole and echinocandin drugs. For this reason, it is necessary to develop new antifungal strategies to better control the disease caused by this yeast. In this work, we screened drugs from the Prestwick chemical library, which contains 1,280 off-patent compounds that are already approved by the Food and Drug Administration, with the aim of identifying molecules with antifungal activity against C. auris. In an initial screening, we looked for drugs that inhibited the growth of three different C. auris strains and found 27 of them which it did so. Ten active compounds were selected to test the susceptibility profile by using the EUCAST protocol. Antifungal activity was confirmed for seven drugs with MICs ranging from 0.5 to 64 mg/L. Some of these drugs were also tested in combination with voriconazole and anidulafungin at sub-inhibitory concentrations. Our results suggest synergistic interactions between suloctidil and voriconazole with fractional inhibitory concentration index (FICI) values of 0.11 to 0.5 and between ebselen and anidulafungin (FICI, 0.12 to 0.44). Our findings indicate that drug repurposing could be a viable alternative to managing infections by C. auris.

Cryptococcus neoformans can form titan-like cells in vitro in response to multiple signals.

Cryptococcus neoformans is an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain them in vitro. In this work, we describe in vitro conditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2 enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and it was independent of the cryptococcal serotype/species. Transition to titan-like cell was impaired by pharmacological inhibition of PKC signaling pathway. Analysis of the gene expression profile during the transition to titan-like cells showed overexpression of enzymes involved in carbohydrate metabolism, as well as proteins from the coatomer complex, and related to iron metabolism. Indeed, we observed that iron limitation also induced the formation of titan cells. Our gene expression analysis also revealed other elements involved in titan cell formation, such as calnexin, whose absence resulted in appearance of abnormal large cells even in regular rich media. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation.

Cryptococcus neoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleria mellonella.

Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans.