Redox profile and mediators of the unfolded protein response (UPR) in the placenta of rats during pregnancy.

CONTEXT: Proliferation, differentiation, migration and apoptosis of trophoblastic cells are influenced by hypoxia, as well as adequate modulation of oxidative stress and the unfolded protein response (UPR) pathway. AIMS: We aimed to evaluate the expression profile of redox and UPR mediators in the placenta of rats throughout pregnancy. METHODS: Placental expression of hypoxia-inducible factor 1α (HIF1α), 8-Hydroxy-2'-deoxyguanosine (8-OHdG), superoxide dismutase 1 (SOD1), glutathione peroxidase (GPX), catalase (Cat), activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), 78 kD glucose-regulated protein (GRP78) and C/EBP-homologous protein (CHOP), as well as reactive oxygen species (ROS) and peroxynitrite production, were evaluated in Wistar rats on the 10th, 12th, 14th, 16th and 18th day of pregnancy (DP). KEY RESULTS: Increased immunostaining of HIF1α was observed on the 16th and 18th DP, while 8-OHdG and ROS production were greater on the 14th DP. SOD1 and Cat had increased immunostaining on the 14th and 18th DP, while staining of GPX1/2, GRP78 and CHOP was greater on the 18th DP. With regard to gene expression, Hif1α and Sod1 showed increased mRNA expression on the 12th and 16th DP, while Gpx1 had increased expression on the 10th and 16th DP. Cat , Perk and Grp78 gene expression was greater on the 14th DP, unlike Atf6 , which showed greater expression on the 12th DP. In contrast, Chop maintained increased expression from the 12th to the 18th DP. CONCLUSIONS: The placental expression of redox and UPR mediators in rats is influenced by gestational age, with greater expression in periods of greater HIF1α and 8-OHdG expression and at the end of the pregnancy. IMPLICATIONS: This study provides data on the physiological modulation of redox and UPR mediators during placental development in rats.

Staphylococcus aureus triggers a protective inflammatory response against secondary Cryptococcus gattii infection in a murine model.

Prior infections can provide protection or enhance susceptibility to a subsequent infection through microorganism's interaction or host immunomodulation. Staphylococcus aureus (SA) and Cryptococcus gattii (CG) cause lungs infection, but it is unclear how they interact in vivo. This study aimed to study the effects of the primary SA lung infection on secondary cryptococcosis caused by CG in a murine model. The mice's survival, fungal burden, behavior, immune cells, cytokines, and chemokines were quantified to evaluate murine cryptococcosis under the influence of a previous SA infection. Further, fungal-bacterial in vitro interaction was studied in a culture medium and a phagocytosis assay. The primary infection with SA protects animals from the subsequent CG infection by reducing lethality, improving behavior, and impairing the fungal proliferation within the host. This phenotype was associated with the proinflammatory antifungal host response elicited by the bacteria in the early stage of cryptococcosis. There was no direct inhibition of CG by SA, although the phagocytic activity of macrophages was reduced. Identifying mechanisms involved in this protection may lead to new approaches for preventing and treating cryptococcosis.

The Antimicrobial Peptide MK58911-NH2 Acts on Planktonic, Biofilm, and Intramacrophage Cells of Cryptococcus neoformans.

Cryptococcosis is associated with high rates of morbidity and mortality, especially in AIDS patients. Its treatment is carried out by combining amphotericin B and azoles or flucytosine, which causes unavoidable toxicity issues in the host. Thus, the urgency in obtaining new antifungals drives the search for antimicrobial peptides (AMPs). This study aimed to extend the understanding of the mechanism of action of an AMP analog from wasp peptide toxins, MK58911-NH2, on Cryptococcus neoformans. We also evaluated if MK58911-NH2 can act on cryptococcal cells in macrophages, biofilms, and an immersion zebrafish model of infection. Finally, we investigated the structure-antifungal action and the toxicity relationship of MK58911-NH2 fragments and a derivative of this peptide (MH58911-NH2). The results demonstrated that MK58911-NH2 did not alter the fluorescence intensity of the cell wall-binding dye calcofluor white or the capsule-binding dye 18b7 antibody-fluorescein isothiocyanate (FITC) in C. neoformans but rather reduced the number and size of fungal cells. This activity reduced the fungal burden of C. neoformans in both macrophages and zebrafish embryos as well as within biofilms. Three fragments of the MK58911-NH2 peptide showed no activity against Cryptococcus and not toxicity in lung cells. The derivative peptide MH58911-NH2, in which the lysine residues of MK58911-NH2 were replaced by histidines, reduced the activity against extracellular and intracellular C. neoformans. On the other hand, it was active against biofilms and showed reduced toxicity. In summary, these results showed that peptide MK58911-NH2 could be a promising agent against cryptococcosis. This work also opens a perspective for the verification of the antifungal activity of other derivatives.

Gentamicin encapsulated within a biopolymer for the treatment of Staphylococcus aureus and Escherichia coli infected skin ulcers.

Skin wound infection requires carefully long-term treatment with an immense financial burden to healthcare systems worldwide. Various strategies such as drug delivery systems using polymer matrix from natural source have been used to enhance wound healing. Natural rubber latex (NRL) from Hevea brasiliensis has shown angiogenic and tissue repair properties. Gentamicin sulfate (GS) is a broad-spectrum antibiotic which inhibits the growth of a wide variety of microorganisms and, because of this, it has also been applied topically for treatment of local infections. The aim of this study was to develop a GS release system using NRL as matrix for Staphylococcus aureus and Escherichia coli infected skin ulcers treatment, without changing drug antibiotic properties. The matrix did not change the GS antimicrobial activity against S. aureus and E. coli strains. Moreover, the NRL-GS biomembrane did not exhibit hemolytic activity, being non-toxic to red blood cells. The eluates of NRL-GS biomembranes and GS solutions did not significantly reduce the survival of Caenorhabditis elegans worms for 24 h at any of the tested concentrations. Thus, these results emphasize that the NRL-GS biomembrane proved to be a promising biomaterial for future studies on the development of dressings for topical uses, inexpensive and practicable, keeping drug antibiotic properties against pathogens and to reduce the side effects.

Dynamics of Mono- and Dual-Species Biofilm Formation and Interactions Between Paracoccidioides brasiliensis and Candida albicans.

The oral cavity is a highly diverse microbial environment in which microorganisms interact with each other, growing as biofilms on biotic and abiotic surfaces. Understanding the interaction among oral microbiota counterparts is pivotal for clarifying the pathogenesis of oral diseases. Candida spp. is one of the most abundant fungi in the oral mycobiome with the ability to cause severe soft tissue lesions under certain conditions. Paracoccidioides spp., the causative agent of paracoccidioidomycosis, may also colonize the oral cavity leading to soft tissue damage. It was hypothesized that both fungi can interact with each other, increasing the growth of the biofilm and its virulence, which in turn can lead to a more aggressive infectivity. Therefore, this study aimed to evaluate the dynamics of mono- and dual-species biofilm growth of Paracoccidioides brasiliensis and Candida albicans and their infectivity using the Galleria mellonella model. Biomass and fungi metabolic activity were determined by the crystal violet and the tetrazolium salt reduction tests (XTT), respectively, and the colony-forming unit (CFU) was obtained by plating. Biofilm structure was characterized by both scanning electronic- and confocal laser scanning- microscopy techniques. Survival analysis of G. mellonella was evaluated to assess infectivity. Our results showed that dual-species biofilm with P. brasiliensis plus C. albicans presented a higher biomass, higher metabolic activity and CFU than their mono-species biofilms. Furthermore, G. mellonella larvae infected with P. brasiliensis plus C. albicans presented a decrease in the survival rate compared to those infected with P. brasiliensis or C. albicans, mainly in the form of biofilms. Our data indicate that P. brasiliensis and C. albicans co-existence is likely to occur on oral mucosal biofilms, as per in vitro and in vivo analysis. These data further widen the knowledge associated with the dynamics of fungal biofilm growth that can potentially lead to the discovery of new therapeutic strategies for these infections.

Fungal-host interactions: insights into microRNA in response to Paracoccidioides species.

BACKGROUND Paracoccidioides spp. causes paracoccidioidomycosis (PCM), an important and frequent systemic mycosis that occurs in Latin America. The infectious process begins with contact between the fungus and lung cells, and the molecular pattern of this interaction is currently poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the gene expression in many biological processes, including in the infections. OBJECTIVE This study aimed to analyse the expression of miRNAs in lung cells as response to infection by Paracoccidioides spp. METHODS A quantitative real-time polymerase chain reaction (RT-qPCR) based screening was employed to verify differentially expressed miRNAs in human lung cells infected with three different species; Paracoccidioides lutzii, Paracoccidioides americana, and Paracoccidioides brasiliensis. Furthermore, the in silico predictions of target genes and pathways for miRNAs were obtained. FINDINGS The results showed that miRNAs identified in the lung cells were different according to the species studied. However, based on the predicted targets, the potential signaling pathways regulated by miRNAs are common and related to adhesion, actin cytoskeleton rearrangement, apoptosis, and immune response mediated by T cells and TGF-ß. MAIN CONCLUSIONS In summary, this study showed the miRNAs pattern of epithelial cells in response to infection by Paracoccidioides species and the potential role of these molecules in the regulation of key pathogenesis mechanisms of PCM.

Incorporation of Nonyl 3,4-Dihydroxybenzoate Into Nanostructured Lipid Systems: Effective Alternative for Maintaining Anti-Dermatophytic and Antibiofilm Activities and Reducing Toxicity at High Concentrations.

Dermatophytosis is the most common mycosis worldwide, affecting approximately 20 to 25% of the population, regardless of gender, race, color, and age. Most antifungal agents used for the treatment of dermatophytosis belong to the azole and allylamine classes. Dermatophytes are reported to be resistant to most commercial drugs, especially microbial biofilms, in addition to their considerable toxicity. It should be emphasized the importance of looking for new molecules with reduced toxicity, as well as new targets and mechanisms of action. This work aims to incorporate nonyl 3,4-dihydroxybenzoate, a potent fungicide compound against planktonic cells and dermatophyte biofilms in nanostructured lipid systems (NLS), in order to reduce toxicity in high concentrations, improve its solubility and maintain its effectiveness. The compound was incorporated into NLS constituted by cholesterol, mixture of polyoxyethylene (23) lauryl ether (Brij®98) and soybean phosphatidylcholine (Epikuron® 200)], 2: 1 ratio and PBS (phosphate-buffered saline). The characterization of the incorporation was performed. Susceptibility tests were conducted according to document M38-A2 by CLSI (2008). The toxicity of the NLS compound was evaluated in HaCaT cell lines by the sulforhodamine B method and in alternative models Caenorhabditis elegans and zebrafish. Finally, its efficacy was evaluated against the mature Trichophyton rubrum and Trichophyton mentagrophytes biofilms. NLS and nonyl 3,4-dihydroxybenzoate loaded into NLS displayed sizes ranging from 137.8 ± 1.815 to 167.9 ± 4.070 nm; the polydispersity index (PDI) varying from 0.331 ± 0.020 to 0.377 ± 0.004 and zeta potential ranging from -1.46 ± 0.157 to -4.63 ± 0.398 mV, respectively. Polarized light microscopy results confirmed the formation of NLS of the microemulsion type. Nonyl incorporated into NLS showed minimum inhibitory concentration (MIC) values, ranging from 2 to 15.6 mg/L. The toxicity tests presented cell viability higher than 80% in all tested concentrations, as well as, a significantly increased of the survival of Caenorhabditis elegans and zebrafish models. Anti-biofilm tests proved the efficacy of the incorporation. These findings contribute significantly to the search for new antifungals and allow the systemic administration of the compound, since the incorporation can increase the solubility of non-polar compounds, improve bioavailability, effectiveness and reduce toxicity.

Heat Shock Protein 60, Insights to Its Importance in Histoplasma capsulatum: From Biofilm Formation to Host-Interaction.

Heat shock proteins (Hsps) are among the most widely distributed and evolutionary conserved proteins, acting as essential regulators of diverse constitutive metabolic processes. The Hsp60 of the dimorphic fungal Histoplasma capsulatum is the major surface adhesin to mammalian macrophages and studies of antibody-mediated protection against H. capsulatum have provided insight into the complexity involving Hsp60. However, nothing is known about the role of Hsp60 regarding biofilms, a mechanism of virulence exhibited by H. capsulatum. Considering this, the present study aimed to investigate the influence of the Hsp60 on biofilm features of H. capsulatum. Also, the non-conventional model Galleria mellonella was used to verify the effect of this protein during in vivo interaction. The use of invertebrate models such as G. mellonella is highly proposed for the evaluation of pathogenesis, immune response, virulence mechanisms, and antimicrobial compounds. For that purpose, we used a monoclonal antibody (7B6) against Hsp60 and characterized the biofilm of two H. capsulatum strains by metabolic activity, biomass content, and images from scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). We also evaluated the survival rate of G. mellonella infected with both strains under blockage of Hsp60. The results showed that mAb 7B6 was effective to reduce the metabolic activity and biomass of both H. capsulatum strains. Furthermore, the biofilms of cells treated with the antibody were thinner as well as presented a lower amount of cells and extracellular polymeric matrix compared to its non-treated controls. The blockage of Hsp60 before fungal infection of G. mellonella larvae also resulted in a significant increase of the larvae survival compared to controls. Our results highlight for the first time the importance of the Hsp60 protein to the establishment of the H. capsulatum biofilms and the G. mellonella larvae infection. Interestingly, the results with Hsp60 mAb 7B6 in this invertebrate model suggest a pattern of fungus-host interaction different from those previously found in a murine model, which can be due to the different features between insect and mammalian immune cells such as the absence of Fc receptors in hemocytes. However further studies are needed to support this hypothesis.

Fungal-host interactions: insights into microRNA in response to Paracoccidioides species

BACKGROUND Paracoccidioides spp. causes paracoccidioidomycosis (PCM), an important and frequent systemic mycosis that occurs in Latin America. The infectious process begins with contact between the fungus and lung cells, and the molecular pattern of this interaction is currently poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the gene expression in many biological processes, including in the infections. OBJECTIVE This study aimed to analyse the expression of miRNAs in lung cells as response to infection by Paracoccidioides spp. METHODS A quantitative real-time polymerase chain reaction (RT-qPCR) based screening was employed to verify differentially expressed miRNAs in human lung cells infected with three different species; Paracoccidioides lutzii, Paracoccidioides americana, and Paracoccidioides brasiliensis. Furthermore, the in silico predictions of target genes and pathways for miRNAs were obtained. FINDINGS The results showed that miRNAs identified in the lung cells were different according to the species studied. However, based on the predicted targets, the potential signaling pathways regulated by miRNAs are common and related to adhesion, actin cytoskeleton rearrangement, apoptosis, and immune response mediated by T cells and TGF-β. MAIN CONCLUSIONS In summary, this study showed the miRNAs pattern of epithelial cells in response to infection by Paracoccidioides species and the potential role of these molecules in the regulation of key pathogenesis mechanisms of PCM.

Antifungal Activity, Toxicity, and Membranolytic Action of a Mastoparan Analog Peptide.

Invasive fungal infections, such as cryptococcosis and paracoccidioidomycosis are associated with significant rates of morbidity and mortality. Cryptococcosis, caused by Cryptococcus neoformans, is distributed worldwide and has received much attention as a common complication in patients with HIV. Invasive fungal infections are usually treated with a combination of amphotericin B and azoles. In addition, 5-fluorocytosine (5-FC) is applied in cryptococcosis, specifically to treat central nervous system infection. However, host toxicity, high cost, emerging number of resistant strains, and difficulty in developing new selective antifungals pose challenges. The need for new antifungals has therefore prompted a screen for inhibitory peptides, which have multiple mechanisms of action. The honeycomb moth Galleria mellonella has been widely used as a model system for evaluating efficacy of antifungal agents. In this study, a peptide analog from the mastoparan class of wasps (MK58911) was tested against Cryptococcus spp. and Paracoccidioides spp. In addition, peptide toxicity tests on lung fibroblasts (MRC5) and glioblastoma cells (U87) were performed. Subsequent tests related to drug interaction and mechanism of action were also performed, and efficacy and toxicity of the peptide were evaluated in vivo using the G. mellonella model. Our results reveal promising activity of the peptide, with an MIC in the range of 7.8-31.2 µg/mL, and low toxicity in MRC and U87 cells (IC50 > 500 µg/mL). Taken together, these results demonstrate that MK58911 is highly toxic in fungal cells, but not mammalian cells (SI > 16). The mechanism of toxicity involved disruption of the plasma membrane, leading to death of the fungus mainly by necrosis. In addition, no interaction with the drugs amphotericin B and fluconazole was found either in vitro or in vivo. Finally, the peptide showed no toxic effects on G. mellonella, and significantly enhanced survival rates of larvae infected with C. neoformans. Although not statistically significant, treatment of larvae with all doses of MK58911 showed a similar trend in decreasing the fungal burden of larvae. These effects were independent of any immunomodulatory activity. Overall, these results present a peptide with potential for use as a new antifungal drug to treat systemic mycoses.

Evaluation of cytotoxicity features of antimicrobial peptides with potential to control bacterial diseases of citrus.

Antimicrobial peptides (AMPs) can be found in various organisms, and could be considered an alternative for pesticides used to control plant pathogens, including those affecting citrus. Brazil is the largest producer and exporter of frozen concentrated orange juice in the world. However, the citrus industry has been affected by several diseases such as citrus canker and huanglongbing (HLB), caused by the bacteria Xanthomonas citri subsp. citri (X.citri) and Candidatus Liberibacter asiaticus (CaLas), respectively. In order to control these pathogens, putative AMPs were prospected in databases containing citrus sequences. Furthermore, AMPs already reported in the literature were also used for in vitro and in vivo assays against X.citri. Since CaLas cannot be cultivated in vitro, surrogates as Sinorhizobium meliloti and Agrobacterium tumefaciens were used. This study reports the evaluation of six AMPs obtained from different sources, two of them from Citrus spp. (citrus-amp1 and citrus-amp2), three from amphibians (Hylin-a1, K0-W6-Hy-a1 and Ocellatin 4-analogue) and one from porcine (Tritrpticin). Peptides K0-W6-Hy-a1, Ocellatin 4-analogue, and citrus-amp1 showed bactericidal activity against X.citri and S. meliloti and bacteriostatic effect on A. tumefaciens. These results were confirmed for X.citri in planta. In addition cytotoxicity evaluations of these molecules were performed. The AMPs that showed the lowest hemolytic activities were Triptrpticin, citrus-amp1 and citrus-amp2. Citrus-amp1 and citrus-amp2 not presented toxicity in experiments using in vivo model, G. mellonella and U87 MG cells. To verify the interaction of these AMPs with bacteria and erythrocyte cell membranes, vesicles mimicking these cells were built. Citrus-amp1 and Tritrpticin exhibited higher affinity to bacterial membranes, while Ocellatin 4-analogue and Hylin-a1 showed higher affinity to erythrocyte membranes; exclude their use in citrus. This work demonstrates an essential alternative, trough AMPs obtained from Citrus spp., which can be feasibly used to control bacterial pathogens.

Potential of the association of dodecyl gallate with nanostructured lipid system as a treatment for paracoccidioidomycosis: In vitro and in vivo efficacy and toxicity.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, caused by Paracoccidioides spp. A limited number of antifungal agents are available and the search for new compounds has increased. Additionally, nanostructured lipid system (NLS) has emmerged as an interesting strategy to carrier compounds for the treatment of mycosis. In this work, the antifungal efficacy and toxicity of dodecyl gallate (DOD) associated with a NLS was evaluated through in vitro and in vivo tests. DOD showed good in vitro antifungal activity and low toxicity in lung fibroblasts and zebrafish embryos, but no antifungal efficacy in infected mice, which may have been a result of low bioavailability. On the other hand, the association of DOD + NLS was beneficial and resulted in lower toxicity in lung fibroblasts and zebrafish embryos. In addition, NLS + DOD promoted a significant reduction in the fungal burden of mice lungs and could be a potential therapeutic option against PCM.

Evaluation of Caenorhabditis elegans as a host model for Paracoccidioides brasiliensis and Paracoccidioides lutzii.

Paracoccidioidomycosis is a systemic fungal infection affecting mainly Latin American countries that is caused by Paracoccidioides brasiliensis and Paracoccidioides lutzii. During the study of fungal pathogenesis, in vivo studies are crucial to understand the overall mechanisms involving the infection as well as to search for new therapeutic treatments and diagnosis. Caenorhabditis elegans is described as an infection model for different fungi species and a well-characterized organism to study the innate immune response. This study evaluates C. elegans as an infection model for Paracoccidioides spp. It was observed that both species do not cause infection in C. elegans, as occurs with Candida albicans, and one possible explanation is that the irregular size and shape of Paracoccidioides spp. difficult the ingestion of these fungi by the nematode. Besides this difficulty in the infection, we could observe that the simple exposition of C. elegans to Paracoccidioides species was able to trigger a distinct pattern of expression of antimicrobial peptide genes. The expression of cnc-4, nlpl-27 and nlp-31 was superior after the exposure to P. brasiliensis in comparison to P. lutzii (P < 0.05), and these findings demonstrate important differences regarding innate immune response activation caused by the two species of the Paracoccidioides genus.

Can passage in Galleria mellonella activate virulence factors of Paracoccidioides brasiliensis as in the murine model?

Paracoccidioidomycosis (PCM) is a fungal disease restricted to Latin countries, and its etiologic agents derive from the Paracoccidioides genus. Attenuation or loss of virulence in Paracoccidioides spp. following successive subculturing has been described. However, virulence can be recovered by passage in mammalian host. In this study, the recovery of adhesion of P. brasiliensis through passage in mice was compared to that in the insect Galleria mellonella. Analysis of in vitro fungal-host cell interaction, gene expression of adhesins, and analysis of the survival curves revealed that Galleria mellonella is useful for the reactivation of P. brasiliensis adhesion.

Activity of gallic acid and its ester derivatives in Caenorhabditis elegans and zebrafish (Danio rerio) models.

AIM: Gallic acid and its ester derivatives have shown antifungal activity in vitro. This study was performed to investigate their activity against Candida albicans and their toxicity in the animal models Caenorhabditis elegans and zebrafish embryos. RESULTS: The compounds protected worms from C. albicans infection. The dodecyl gallate was the most effective. In zebrafish embryo, gallic acid and dodecyl gallate were the least toxic. CONCLUSION: Gallic acid and its ester derivatives have potential for in vivo use against C. albicans infection. The antifungal effects and toxicity of gallate esters in these alternative animal models were dependent on carbon chain length.

Candida/Candida biofilms. First description of dual-species Candida albicans/C. rugosa biofilm.

Denture liners have physical properties that favour plaque accumulation and colonization by Candida species, irritating oral tissues and causing denture stomatitis. To isolate and determine the incidence of oral Candida species in dental prostheses, oral swabs were collected from the dental prostheses of 66 patients. All the strains were screened for their ability to form biofilms; both monospecies and dual-species combinations were tested. Candida albicans (63 %) was the most frequently isolated microorganism; Candida tropicalis (14 %), Candida glabrata (13 %), Candida rugosa (5 %), Candida parapsilosis (3 %), and Candida krusei (2 %) were also detected. The XTT assay showed that C. albicans SC5314 possessed a biofilm-forming ability significantly higher (p < 0.001) than non-albicans Candida strains, after 6 h 37 °C. The total C. albicans CFU from a dual-species biofilm was less than the total CFU of a monospecies C. albicans biofilm. In contrast to the profuse hyphae verified in monospecies C. albicans biofilms, micrographies showed that the C. albicans/non-albicans Candida biofilms consisted of sparse yeast forms and profuse budding yeast cells that generated a network. These results suggested that C. albicans and the tested Candida species could co-exist in biofilms displaying apparent antagonism. The study provide the first description of C. albicans/C. rugosa mixed biofilm.

Decreased expression of 14-3-3 in Paracoccidioides brasiliensis confirms its involvement in fungal pathogenesis.

The interaction between the fungal pathogen Paracoccidioides brasiliensis and host cells is usually mediated by specific binding events between adhesins on the fungal surface and receptors on the host extracellular matrix or cell surface. One molecule implicated in the P. brasiliensis-host interaction is the 14-3-3 protein. The 14-3-3 protein belongs to a family of conserved regulatory molecules that are expressed in all eukaryotic cells and are involved in diverse cellular functions. Here, we investigated the relevance of the 14-3-3 protein to the virulence of P. brasiliensis. Using antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated a 14-3-3-silenced strain (expression reduced by ˜55%). This strain allowed us to investigate the interaction between 14-3-3 and the host and to correlate the functions of P. brasiliensis 14-3-3 with cellular features, such as morphological characteristics and virulence, that are important for pathogenesis.

Paracoccidioides-host Interaction: An Overview on Recent Advances in the Paracoccidioidomycosis.

Paracoccidioides brasiliensis and P. lutzii are etiologic agents of paracoccidioidomycosis (PCM), an important endemic mycosis in Latin America. During its evolution, these fungi have developed characteristics and mechanisms that allow their growth in adverse conditions within their host through which they efficiently cause disease. This process is multi-factorial and involves host-pathogen interactions (adaptation, adhesion, and invasion), as well as fungal virulence and host immune response. In this review, we demonstrated the glycoproteins and polysaccharides network, which composes the cell wall of Paracoccidioides spp. These are important for the change of conidia or mycelial (26°C) to parasitic yeast (37°C). The morphological switch, a mechanism for the pathogen to adapt and thrive inside the host, is obligatory for the establishment of the infection and seems to be related to pathogenicity. For these fungi, one of the most important steps during the interaction with the host is the adhesion. Cell surface proteins called adhesins, responsible for the first contact with host cells, contribute to host colonization and invasion by mediating this process. These fungi also present the capacity to form biofilm and through which they may evade the host's immune system. During infection, Paracoccidioides spp. can interact with different host cell types and has the ability to modulate the host's adaptive and/or innate immune response. In addition, it participates and interferes in the coagulation system and phenomena like cytoskeletal rearrangement and apoptosis. In recent years, Paracoccidioides spp. have had their endemic areas expanding in correlation with the expansion of agriculture. In response, several studies were developed to understand the infection using in vitro and in vivo systems, including alternative non-mammal models. Moreover, new advances were made in treating these infections using both well-established and new antifungal agents. These included natural and/or derivate synthetic substances as well as vaccines, peptides, and anti-adhesins sera. Because of all the advances in the PCM study, this review has the objective to summarize all of the recent discoveries on Paracoccidioides-host interaction, with particular emphasis on fungi surface proteins (molecules that play a fundamental role in the adhesion and/or dissemination of the fungi to host-cells), as well as advances in the treatment of PCM with new and well-established antifungal agents and approaches.

Comparison of virulence between Paracoccidioides brasiliensis and Paracoccidioides lutzii using Galleria mellonella as a host model.

Paracoccidioidomycosis is a systemic mycosis, endemic in Latin America. The etiologic agents of this mycosis are composed of 2 species: Paracoccidioides brasiliensis and P. lutzii. Murine animal models are the gold standard for in vivo studies; however, ethical, economical and logistical considerations limit their use. Galleria mellonella is a suitable model for in vivo studies of fungal infections. In this study, we compared the virulence of P. brasiliensis and P. lutzii in G. mellonella model. The deaths of larvae infected with P. brasiliensis or P. lutzii were similar, and both species were able to reduce the number of hemocytes, which were estimated by microscopy and flow cytometer. Additionally, the phagocytosis percentage was similar for both species, but when we analyze hemocyte-Paracoccidioides spp. interaction using flow cytometer, P. lutzii showed higher interactions with hemocytes. The gene expression of gp43 as well as this protein was higher for P. lutzii, and this expression may contribute to a greater adherence to hemocytes. These results helped us evaluate the behavior of Paracoccidioides spp in G. mellonella, which is a convenient model for investigating the host-Paracoccidioides spp. interaction.

Antimicrobial activity of recombinant Pg-AMP1, a glycine-rich peptide from guava seeds.

Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168 bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98 kDa as a monomer and 13.96 kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases.