Ildinete Silva-Pereira

Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance.

Antimicrobial peptides (AMPs) are natural antibiotics produced by various organisms such as mammals, arthropods, plants, and bacteria. In addition to antimicrobial activity, AMPs can induce chemokine production, accelerate angiogenesis, and wound healing and modulate apoptosis in multicellular organisms. Originally, their antimicrobial mechanism of action was thought to consist solely of an increase in pathogen cell membrane permeability, but it has already been shown that several AMPs do not modulate membrane permeability in the minimal lethal concentration. Instead, they exert their effects by inhibiting processes such as protein and cell wall synthesis, as well as enzyme activity, among others. Although resistance to these molecules is uncommon several pathogens developed different strategies to overcome AMPs killing such as surface modification, expression of efflux pumps, and secretion of proteases among others. This review describes the various mechanisms of action of AMPs and how pathogens evolve resistance to them.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature‐dependent cell morphology change from mycelium (22° C) to yeast (36° C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3938 (Y = 1654 and M = 2274) ESTs were sequenced and clustered into 597 contigs and 1563 singlets, making up a total of 2160 genes, which possibly represent one‐quarter of the complete gene repertoire in P. brasiliensis. From this total, 1040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes—cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage‐specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis. All EST sequences have been deposited in GenBank under Accession Nos CA580326–CA584263. Copyright

Cloning and characterization of cDNA sequences encoding for new venom peptides of the Brazilian scorpion Opisthacanthus cayaporum

Scorpion venom glands produce a large variety of bioactive peptides. This communication reports the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared with venomous glands of the Brazilian scorpion Opisthacanthus cayaporum (Ischnuridae). Two main types of components were identified: peptides with toxin-like sequences and proteins involved in cellular processes. Using the expressed sequence tag (EST) strategy 118 clones were identified, from which 61 code for unique sequences (17 contigs and 44 singlets) with an average length of 531 base-pairs (bp). These results were compared with those previously obtained by the proteomic analysis of the same venom, showing a considerable degree of similarity in terms of the molecular masses expected and DNA sequences found. About 36% of the ESTs correspond to toxin-like peptides and proteins with identifiable open reading frames (ORFs). The cDNA sequencing results also show the presence of sequences whose putative products correspond to a scorpine-like component; three short antimicrobial peptides; three K(+)-channel blockers; and an additional peptide containing 78 amino acid residues, whose sequence resembles peptide La1 from another Ischnuridae scorpion Liocheles australiasiae, thus far with unknown function.

Quorum Sensing-Mediated, Cell Density-Dependent Regulation of Growth and Virulence in Cryptococcus neoformans

ABSTRACT Quorum sensing (QS) is a cell density-dependent mechanism of communication between microorganisms, characterized by the release of signaling molecules that affect microbial metabolism and gene expression in a synchronized way. In this study, we investigated cell density-dependent behaviors mediated by conditioned medium (CM) in the pathogenic encapsulated fungus Cryptococcus neoformans. CM produced dose-dependent increases in the growth of planktonic and biofilm cells, glucuronoxylomannan release, and melanin synthesis, important virulence attributes of this organism. Mass spectrometry revealed the presence of pantothenic acid (PA) in our samples, and commercial PA was able to increase growth and melanization, although not to the same extent as CM. Additionally, we found four mutants that were either unable to produce active CM or failed to respond with increased growth in the presence of wild-type CM, providing genetic evidence for the existence of intercellular communication in C. neoformans. C. neoformans CM also increased the growth of Cryptococcus albidus, Candida albicans, and Saccharomyces cerevisiae. Conversely, CM from Cryptococcus albidus, C. albicans, S. cerevisiae, and Sporothrix schenckii increased C. neoformans growth. In summary, we report the existence of a new QS system regulating the growth and virulence factor expression of C. neoformans in vitro and, possibly, also able to regulate growth in other fungi. IMPORTANCE Quorum sensing is a strategy of communication used by pathogenic microorganisms to coordinate the expression of attributes necessary to cause disease. In this work, we describe a quorum sensing system in Cryptococcus neoformans, a yeast that can cause severe central nervous system infections. Adding conditioned medium—culture medium in which C. neoformans has previously grown—to fresh cultures resulted in faster growth of C. neoformans both as isolated cells and in microbial communities called biofilms. The addition of conditioned medium also increased the secretion of capsule carbohydrates and the formation of melanin pigment, two tools used by this microorganism to thrive in the host. This remarkable example of microbial communication shows that C. neoformans cells can act in unison when expressing attributes necessary to survive in the host, a finding that could point the way to improvements in the treatment of cryptococcosis. Quorum sensing is a strategy of communication used by pathogenic microorganisms to coordinate the expression of attributes necessary to cause disease. In this work, we describe a quorum sensing system in Cryptococcus neoformans, a yeast that can cause severe central nervous system infections. Adding conditioned medium—culture medium in which C. neoformans has previously grown—to fresh cultures resulted in faster growth of C. neoformans both as isolated cells and in microbial communities called biofilms. The addition of conditioned medium also increased the secretion of capsule carbohydrates and the formation of melanin pigment, two tools used by this microorganism to thrive in the host. This remarkable example of microbial communication shows that C. neoformans cells can act in unison when expressing attributes necessary to survive in the host, a finding that could point the way to improvements in the treatment of cryptococcosis.

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.

The stress responsive and morphologically regulated hsp90 gene from Paracoccidioides brasiliensis is essential to cell viability.

BackgroundParacoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target.ResultsIn this work, we decided to further study the Pbhsp90 gene, its expression and role in cell viability because it plays important roles in fungal physiology and pathogenesis. Thus, we have sequenced a Pbhsp90 cDNA and shown that this gene is present on the genome as a single copy. We have also confirmed its preferential expression in the yeast phase and its overexpression during dimorphic transition and oxidative stress. Treatment of the yeast with the specific HSP90 inhibitors geldanamycin and radicicol inhibited growth at 2 and 10 μM, respectively.ConclusionThe data confirm that the Pbhsp90 gene encodes a morphologically regulated and stress-responsive protein whose function is essential to cell viability of this pathogen. This work also enforces the potential of HSP90 as a target for antifungal therapies, since the use of HSP90 inhibitors is lethal to the P. brasiliensis yeast cells in a dose-responsive manner.

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.

Cloning, characterization and heterologous expression of the first Penicillium echinulatum cellulase gene

Aims:  Penicillium echinulatum is effective for bioconversion processes. However, nothing is known about the molecular biology of its cellulolytic system. We describe for the first time the isolation, cloning and expression of a P. echinulatum cellulase cDNA (Pe‐egl1) encoding a putative endoglucanase.

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.