Aldo Henrique Tavares

Upregulation of glyoxylate cycle genes upon Paracoccidioides brasiliensis internalization by murine macrophages and in vitro nutritional stress condition

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a facultative intracellular human pathogen that can persist within macrophage phagolysosomes, indicating that the fungus has evolved defense mechanisms in order to survive under nutritionally poor environments. The analysis of P. brasiliensis transcriptome revealed several virulence factor orthologs of other microorganisms, including the glyoxylate cycle genes. This cycle allows the utilization of two-carbon (C2) compounds as carbon source in gluconeogenesis. Semiquantitative RT-PCR analyses revealed that these genes were upregulated when P. brasiliensis was recovered from murine macrophages, without any additional in vitro growth. The induction of this cycle, in response to macrophage microenvironments, was shown to be coordinated with the upregulation of the gluconeogenic phosphoenolpyruvate carboxykinase gene. In addition, assays employing RNA extracted from P. brasiliensis grown in a medium with acetate instead of glucose also showed increased levels of glyoxylate cycle transcripts. Our main results suggest that P. brasiliensis uses the glyoxylate cycle as an important adaptive metabolic pathway.

The transcriptional response of Cryptococcus neoformans to ingestion by Acanthamoeba castellanii and macrophages provides insights into the evolutionary adaptation to the mammalian host.

ABSTRACT Virulence of Cryptococcus neoformans for mammals, and in particular its intracellular style, was proposed to emerge from evolutionary pressures on its natural environment by protozoan predation, which promoted the selection of strategies that allow intracellular survival in macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then can replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least 2-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes that were found in both groups, we focused on open reading frame (ORF) CNAG_05662, which was potentially related to sugar transport but had no determined biological function. To characterize its function, we constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (polyol transporter protein 1), is involved in the transport of 5- and 6-carbon polyols such as mannitol and sorbitol, but its presence or absence had no effect on cryptococcal virulence for mice or moth larvae. Overall, these results are consistent with the hypothesis that the capacity for mammalian virulence originated from fungus-protozoan interactions in the environment and provide a better understanding of how C. neoformans adapts to the mammalian host.

NLRP3 inflammasome activation by Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1β that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1β in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL-1 receptor, we demonstrated that IL-1β signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus.

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.

Modulation of the immune response by Fonsecaea pedrosoi morphotypes in the course of experimental chromoblastomycosis and their role on inflammatory response chronicity

A common theme across multiple fungal pathogens is their ability to impair the establishment of a protective immune response. Although early inflammation is beneficial in containing the infection, an uncontrolled inflammatory response is detrimental and may eventually oppose disease eradication. Chromoblastomycosis (CBM), a cutaneous and subcutaneous mycosis, caused by dematiaceous fungi, is capable of inducing a chronic inflammatory response. Muriform cells, the parasitic form of Fonsecaea pedrosoi, are highly prevalent in infected tissues, especially in long-standing lesions. In this study we show that hyphae and muriform cells are able to establish a murine CBM with skin lesions and histopathological aspects similar to that found in humans, with muriform cells being the most persistent fungal form, whereas mice infected with conidia do not reach the chronic phase of the disease. Moreover, in injured tissue the presence of hyphae and especially muriform cells, but not conidia, is correlated with intense production of pro-inflammatory cytokines in vivo. High-throughput RNA sequencing analysis (RNA-Seq) performed at early time points showed a strong up-regulation of genes related to fungal recognition, cell migration, inflammation, apoptosis and phagocytosis in macrophages exposed in vitro to muriform cells, but not conidia. We also demonstrate that only muriform cells required FcγR and Dectin-1 recognition to be internalized in vitro, and this is the main fungal form responsible for the intense inflammatory pattern observed in CBM, clarifying the chronic inflammatory reaction observed in most patients. Furthermore, our findings reveal two different fungal-host interaction strategies according to fungal morphotype, highlighting fungal dimorphism as an important key in understanding the bipolar nature of inflammatory response in fungal infections.

Turning Up the Heat: Inflammasome Activation by Fungal Pathogens.

Since its first description in 2002 [1], the inflammasome has been implicated in the mechanisms underlying a growing number of infectious, autoimmune, and metabolic diseases [2]. Regarding infectious processes, several studies have shown the involvement of this critical component of innate immunity in the outcome of infection with nearly every class of microbe, including fungi [3]. Innate immunity is the frontline of defense against infection and relies on the ability of its main players (phagocytes and epithelial barriers) to detect conserved components of microbes or pathogen-associated molecular patterns (PAMPs). In fungi, the carbohydrate polymers of the cell wall, such as chitin, β-glucan, and mannan are the major PAMPs recognized by the host’s innate immune cells; this recognition occurs via germline-encoded receptors termed pattern recognition receptors (PRRs) [4]. In addition to PAMPs, endogenous molecules associated with damaged host cells, or damage-associated molecular patterns (DAMPs), are released during tissue injury and activate PRRs. This innate detection system includes the Toll-like receptors (TLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and AIM2-like receptors (ALRs). Although the main fungal- recognition PRRs (CLRs and TLRs) are bound to the cytoplasmic membrane of innate immune cells [4], fungal sensing by PRRs located in the cytosol, such as the NLRs and ALRs, is becoming increasingly evident. A number of NLRs and ALRs can assemble into the inflammasome, a multiprotein complex consisted of PRRs such as NLRP3 (NLR family, pyrin domain-containing 3), NLRC4, or AIM2, adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD), and procaspase-1 [3]. Upon formation of the complex, procaspase-1 is cleaved into an active cysteine protease, which further cleaves the proinflammatory cytokines IL-1β and IL-18 into their mature forms, followed by unconventional secretion. IL-1β and IL-18 mediate several innate antimicrobial responses and are critical to direct adaptive Th17/Th1 cellular responses [5]. In addition, inflammasome activation causes pyroptosis, a lytic inflammatory form of cell death [2,5].

Transcriptomic reprogramming of genus Paracoccidioides in dimorphism and host niches

The thermodimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic systemic mycosis in Latin America. Paracoccidioides grows as saprophytic mycelia that produce infective conidia propagules, which are inhaled into the lungs where the fungus converts to the pathogenic yeast form. From the lungs, Paracoccidioides may disseminate through blood and lymphatics to several other organs and tissues. During the last decade we have witnessed the generation of a large amount of transcriptomic data regarding the events leading to the morphological transition and host niche adaptation. In this review we summarize those findings and discuss the consequence of gene expression plasticity in the persistence and survival of this pathogen. In addition, we discuss the future trends on the host-pathogen studies and how new molecular strategies, such as RNA-seq, dual RNA-seq and Chip-Seq can be powerful tools to improve our understanding on the pathobiology of this systemic mycosis in Latin America.

Distinct patterns of yeast cell morphology and host responses induced by representative strains of Paracoccidioides brasiliensis (Pb18) and Paracoccidioides lutzii (Pb01)

Paracoccidioidomycosis (PCM) is a systemic mycosis, widespread in Latin America. PCM is a granulomatous disease characterized by a polymorphism of lesions depending on the pathogens virulence, the immune status of the host and its genetic susceptibility. The thermodimorphic fungus Paracoccidioides brasiliensis was considered the only etiologic agent of PCM, yet recent works have shown significant genetic diversity among different strains of P. brasiliensis. Therefore, it has been proposed for a new species within the Paracoccidioides genus, named Paracoccidioides lutzii. To better understand the fungus-host interactions elicited by strains Pb01 and Pb18 as key representatives of P. lutzii and P. brasiliensis, respectively, we carried out studies to investigate differences in morphology, induced immune response, virulence and pathology between these two Paracoccidioides species. Our results demonstrate distinct patterns of host-parasite interaction and pathology caused by Pb18 and Pb01. These results open up new fronts for NEW: clinical studies, which may result in significant consequences for the diagnosis and treatment of PCM. Considering that our results cannot be extended to all strains of both species, more studies about the virulence among Paracoccioides must be explored in the future.

Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms

The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 μM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals.

Dectin-1 is required for miR155 upregulation in murine macrophages in response to Candida albicans

ABSTRACT The commensal fungal pathogen Candida albicans is a leading cause of lethal systemic infections in immunocompromised patients. One of the main mechanisms of host immune evasion and virulence by this pathogen is the switch from yeast form to hyphal growth morphologies. Micro RNAs (miRNAs), a small regulatory non-coding RNA, has been identified as an important part of the immune response to a wide variety of pathogens. In general, miRNAs act by modulating the intensity of inflammatory responses. miRNAs act by base-paring binding to specific sequences of target mRNAs, generally causing their silencing through mRNA degradation or translational repression. To study the impact of C. albicans cell morphology upon host miRNA expression, we investigated the differential modulation of 9 different immune response-related miRNAs in primary murine bone marrow-derived macrophages (BMDMs) exposed to either yeasts or hyphal forms of Candida albicans. Here, we show that the different growth morphologies induce distinct miRNA expression patterns in BMDMs. Interestingly, our data suggest that the C-Type lectin receptor Dectin-1 is a major PRR that orchestrates miR155 upregulation in a Syk-dependent manner. Our results suggest that PRR-mediating signaling events are key drivers of miRNA-mediated gene regulation during fungal pathogenesis.