Enhanced low-cost lipopeptide biosurfactant production by Bacillus velezensis from residual glycerin.

Biosurfactants (BSFs) are molecules produced by microorganisms from various carbon sources, with applications in bioremediation and petroleum recovery. However, the production cost limits large-scale applications. This study optimized BSFs production by Bacillus velezensis (strain MO13) using residual glycerin as a substrate. The spherical quadratic central composite design (CCD) model was used to standardize carbon source concentration (30 g/L), temperature (34 °C), pH (7.2), stirring (239 rpm), and aeration (0.775 vvm) in a 5-L bioreactor. Maximum BSFs production reached 1527.6 mg/L of surfactins and 176.88 mg/L of iturins, a threefold increase through optimization. Microbial development, substrate consumption, concentration of BSFs, and surface tension were also evaluated on the bioprocess dynamics. Mass spectrometry Q-TOF-MS identified five surfactin and two iturin isoforms produced by B. velezensis MO13. This study demonstrates significant progress on BSF production using industrial waste as a microbial substrate, surpassing reported concentrations in the literature.

Clioquinol and 8-hydroxyquinoline-5-sulfonamide derivatives damage the cell wall of Pythium insidiosum.

AIMS: To evaluate the antimicrobial activity and to determine the pharmacodynamic characteristics of three 8-hydroxyquinoline derivatives (8-HQs) against Pythium insidiosum, the causative agent of pythiosis. METHODS AND RESULTS: Antimicrobial activity was tested by broth microdilution and MTT assays. The antimicrobial mode of action was investigated using sorbitol protection assay, ergosterol binding assay, and scanning electron microscopy. Clioquinol, PH151, and PH153 were active against all isolates, with MIC values ranging from 0.25 to 2 µg ml-1. They also showed a time- and dose-dependent antimicrobial effect, damaging the P. insidiosum cell wall. CONCLUSIONS: Together, these results reinforce the potential of 8-HQs for developing new drugs to treat pythiosis.

Transcriptomic analysis reveals that mTOR pathway can be modulated in macrophage cells by the presence of cryptococcal cells.

Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3ß was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.

Penicillium oxalicum secretomic analysis identify plant cell wall degrading enzymes important for fruit juice extraction.

Pectinases and other carbohydrate-active enzymes are important for the food industry, mainly for juice processing. In addition, the use of peels to produce enzymes can aggregate value to these agro-industrial residues and at the end of the process enhance qualitatively and quantitatively the juice production. In this work, three different extracts produced by Penicillium oxalicum LS09 using agro-industrial residues were optimized and analyzed by mass spectrometry. It was observed an increased production of pectinases in the medium containing orange peel and optimized for production of pectin lyase and pectinesterase (PE). Interestingly, not only pectinases, but also different plant cell wall degrading enzymes (i.e. glucanases, xylanases, arabinases), with a higher ratio (42/73) was identified in the medium optimized for PE. The crude extracts produced by P. oxalicum also reveal the potential for application in the fruit juice industry, showing an increased yield and qualitative characteristics of extracted juices. The presence of other cell wall-degrading enzymes identified by proteomics, reinforce the combination for obtaining clarified and depectinized juice in a single step.

Updating the application of Metarhizium anisopliae to control cattle tick Rhipicephalus microplus (Acari: Ixodidae).

The bovine tick, Rhipicephalus microplus, is the main ectoparasite of cattle and causes loss of billions of dollars worldwide in lost meat, milk, and leather production, as well as control expenses. In addition to systemically impacting the host during the parasitic act, this parasite is also an important disease vector. Traditionally, the main commercial control of the tick is achieved through application of chemical acaricides, which can leave residues in the meat and milk. Moreover, ticks can become resistant to these chemicals due to their massive and incorrect use. Many alternative methods have been tested including vaccines and natural products from plant origin. However, the efficacy of these treatments is variable and limited, especially when used alone. Arthropod-pathogenic fungi, such as Metarhizium anisopliae, are among the natural microbial agents with promising potential to be used alone or in association with other products, for example with chemical acaricides. This article discusses several aspects of bovine tick control related to the use of M. anisopliae, which is one of the most studied and viable alternative tools for effective tick control.

Antifungal activity of Allamanda polyantha seed extract and its iridoids promote morphological alterations in Cryptococcus spp.

Cryptococcosis, caused by Cryptococcus spp., is an invasive fungal infection of the central nervous system, associated with high mortality, affecting mainly immunocompromised patients. Due to the development of resistance to the current therapy, there is an urgent need for less toxic and more effective antifungal agents. In this study, we describe the antifungal activity against Cryptococcus spp. of an aqueous seed extract from Allamanda polyantha (ASEAP) and two iridoids, plumieride and plumieridine, isolated from this extract with an antifungal activity. The capsule formation and the morphological alterations were evaluated using fluorescent microscopy. The cytotoxic activity was also investigated. The minimal inhibitory concentration (MIC) values of ASEAP for Cryptococcus gattii were 70 and 36 µg/ml (for the R265 and R272 strains, respectively) and 563 µg/ml for Cryptococcus neoformans H99. ASEAP inhibited C. neoformans H99 capsule formation, an important virulence factor, and decreased the cell body size for both the C. gattii strains. H99 cells also presented morphological alterations, with defects in bud detachment and nuclear fragmentation. Plumieride and plumieridine presented higher MIC values than ASEAP, indicating that other compounds might contribute to antifungal activity and/or that combination of the compounds results in a higher antifungal activity.

Proteomics of Rat Lungs Infected by Cryptococcus gattii Reveals a Potential Warburg-like Effect.

Cryptococcus gattii is the causative agent of cryptococcosis infection that can lead to pneumonia and meningitis in immunocompetent individuals. The molecular basis of the pathogenic process and impact on the host biochemistry are poorly understood and remain largely unknown. In this context, a comparative proteomic analysis was performed to investigate the response of the host during an infection caused by C. gattii. Lungs of experimentally infected rats were analyzed by shotgun proteomics to identify differentially expressed proteins induced by C. gattii clinical strain. The proteomic results were characterized using bioinformatic tools, and subsequently, the molecular findings were validated in cell culture and lungs of infected animals. A dramatic change was observed in protein expression triggered by C. gattii infection, especially related to energy metabolism. The main pathways affected include aerobic glycolysis cycle, TCA cycle, and pyrimidine and purine metabolism. Analyses in human lung fibroblast cells confirmed the altered metabolic status found in infected lungs. Thus, it is clear that C. gattii infection triggers important changes in energy metabolism leading to the activation of glycolysis and lactate accumulation in lung cells, culminating in a cancerlike metabolic status known as the Warburg effect. The results presented here provide important insights to better understand C. gattii molecular pathogenesis.

Pharmacological inhibition of pigmentation in Cryptococcus.

Melanin formation is a promising target for antifungal development. We screened a collection of 727 compounds that were previously approved for clinical use in humans for inhibition of pigmentation in Cryptococcus gattii, a lethal fungal pathogen that causes damage to both immunocompetent and immunocompromised hosts. The pyrimidine analogues flucytosine (5-fluorocytosine [5-FC]), 5-fluorouracil (5-FU) and carmofur were identified as efficient inhibitors of pigmentation in the C. gattii model. Since melanin synthesis is enzymatically catalyzed by laccase in Cryptococcus, we investigated whether inhibition of pigmentation by the pyrimidine analogues was laccase-mediated. Enzyme activity and expression of LAC genes were not involved in the effects of the pyrimidine analogues, suggesting alternative cellular targets for inhibition of pigmentation. To address this hypothesis, we screened a collection of approximately 8000 mutants of C. gattii that were produced by insertional mutation after incubation with Agrobacterium tumefaciens and identified a gene product required for the anti-pigmentation activity of 5-FC as a beta-DNA polymerase. Reduced expression of this gene affected capsule formation and urease activity, suggesting essential roles in the cryptococcal physiology. These results demonstrate a previously unknown antifungal activity of 5-FC and reveal a promising target for the development of novel antifungals.

Cryptococcal dissemination to the central nervous system requires the vacuolar calcium transporter Pmc1.

Cryptococcus neoformans is a basidiomycetous yeast and the cause of cryptococcosis in immunocompromised individuals. The most severe form of the disease is meningoencephalitis, which is one of the leading causes of death in HIV/AIDS patients. In order to access the central nervous system, C. neoformans relies on the activity of certain virulence factors such as urease, which allows transmigration through the blood-brain barrier. In this study, we demonstrate that the calcium transporter Pmc1 enables C. neoformans to penetrate the central nervous system, because the pmc1 null mutant failed to infect and to survive within the brain parenchyma in a murine systemic infection model. To investigate potential alterations in transmigration pathways in these mutants, global expression profiling of the pmc1 mutant strain was undertaken, and genes associated with urease, the Ca2+ -calcineurin pathway, and capsule assembly were identified as being differentially expressed. Also, a decrease in urease activity was observed in the calcium transporter null mutants. Finally, we demonstrate that the transcription factor Crz1 regulates urease activity and that the Ca2+ -calcineurin signalling pathway positively controls the transcription of calcium transporter genes and factors related to transmigration.

Bullera vrieseae sp. nov., a tremellaceous yeast species isolated from bromeliads.

Two independent surveys of yeasts associated with different bromeliads in different Brazilian regions led to the proposal of a novel yeast species, Bullera vrieseae sp. nov., belonging to the Tremellales clade (Agaricomycotina, Basidiomycota). Analysis of the sequences in the internal transcribed spacer (ITS) region and D1/D2 domain of the LSU rRNA gene suggested affinity to a phylogenetic lineage that includes Bullera miyagiana and Bullera sakaeratica. Six isolates of the novel species were obtained from different bromeliads and regions in Brazil. Sequence analysis of the D1/D2 domains of the large subunit of the rRNA gene showed that the novel species differs from B. miyagiana and B. sakaeratica by 85 and 64 nt substitutions, respectively and by more than 75 nt substitutions in the ITS region. Phenotypically, Bullera vrieseae sp. nov. can be distinguished from both species based on the assimilation of meso-erythritol, which was negative for B. vrieseae sp. nov. but positive for the others, assimilation of d-glucosamine, which was positive for B. vrieseae sp. nov. but negative for B. miyagiana and of l-sorbose, which was negative for B. vrieseae sp. nov. but positive for B. sakaeratica. The novel species Bullera vrieseae sp. nov. is proposed to accommodate these isolates. The type strain of Bullera vrieseae sp. nov. is UFMG-CM-Y379T (BRO443T; ex-type CBS 13870T).

Hannaella pagnoccae sp. nov., a tremellaceous yeast species isolated from plants and soil.

Several independent surveys of yeasts associated with different plant materials and soil led to the proposal of a novel yeast species belonging to the Tremellales clade (Agaricomycotina, Basidiomycota). Analysis of the sequences of the D1/D2 domains and internal transcribed spacer region of the large subunit of the rRNA gene suggested affinity to a phylogenetic lineage that includes Hannaella coprosmaensis, Hannaella oryzae and Hannaella sinensis. Thirty-two isolates were obtained from different sources, including bromeliads, nectar of Heliconia psittacorum (Heliconiaceae), flowers of Pimenta dioica (Myrtaceae), roots and leaves of sugar cane (Saccharum spp.) in Brazil, leaves of Cratoxylum maingayi, Arundinaria pusilla and Vitis vinifera in Thailand, soil samples in Taiwan, and prairie soil in the USA. Sequence analysis of the D1/D2 domains of the large subunit of the rRNA gene showed that the novel species differs from Hannaella coprosmaensis and Hannaella oryzae by 36 and 46 nt substitutions, respectively. A novel species is suggested to accommodate these isolates, for which the name Hannaella pagnoccae sp. nov. is proposed. The type strain is BI118(T) ( = CBS 11142(T) = ATCC MYA-4530(T)).

Proteomic profile of Cryptococcus gattii biofilm: Metabolic shift and the potential activation of electron chain transport.

Cryptococcus gattii is a primary pathogenic fungus that causes pneumonia. This species is also responsible for an outbreak in Vancouver, Canada, and spreading to the mainland and United States. The use of medical devices is often complicated by infections with biofilm-forming microbes with increased resistance to antimicrobial agents and host defense mechanisms. This study investigated the comparative proteome of C. gattii R265 (VGIIa) grown under planktonic and biofilm conditions. A brief comparison with C. neoformans H99 biofilm and the use of different culture medium and surface were also evaluated. Using Multidimensional Protein Identification Technology (MudPIT), 1819 proteins were identified for both conditions, where 150 (8.2%) were considered differentially regulated (up- or down-regulated and unique in biofilm cells). Overall, the proteomic approach suggests that C. gattii R265 biofilm cells are maintained by the induction of electron transport chain for reoxidation, and by alternative energy metabolites, such as succinate and acetate. SIGNIFICANCE: Since C. gattii is considered a primary pathogen and is one of the most virulent and less susceptible to antifungals, understanding how biofilms are maintained is fundamental to search for new targets to control this important mode of growth that is difficult to eradicate.

The heat shock protein (Hsp) 70 of Cryptococcus neoformans is associated with the fungal cell surface and influences the interaction between yeast and host cells.

The pathogenic yeast Cryptococcus neoformans secretes numerous proteins, such as heat shock proteins, by unconventional mechanisms during its interaction with host cells. Hsp70 is a conserved chaperone that plays important roles in various cellular processes, including the interaction of fungi with host immune cells. Here, we report that sera from individuals with cryptococcosis infection recognize a recombinant C. neoformans Hsp70 (Cn_rHsp70). Moreover, immunofluorescence assays using antibodies against Cn_rHsp70 revealed the localization of this protein at the cell surface mainly in association with the capsular network. We found that the addition of Cn_rHsp70 positively modulated the interaction of C. neoformans with human alveolar epithelial cells and decreased fungal killing by mouse macrophages, without affecting phagocytosis rates. Immunofluorescence analysis showed that there was a competitive association among the receptor, GXM and Cn_rHsp70, indicating that the Hsp70-binding sites in host cells appear to be shared by glucuronoxylomannan (GXM), the major capsular antigen in C. neoformans. Our observations suggest additional mechanisms by which Hsp70 influences the interaction of C. neoformans with host cells.

Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers affects multiple mechanisms required for fungal pathogenesis.

The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.

Inhibition of ecto-phosphatase activity in conidia reduces adhesion and virulence of Metarhizium anisopliae on the host insect Dysdercus peruvianus.

Metarhizium anisopliae is an entomopathogenic fungus with the ability to infect a broad range of arthropods, and have evolved distinct strategies for their attachment to hosts. Here, we describe the characterisation of ecto-phosphatase activity on the conidia surface of M. anisopliae and its relevance in the host interaction process. Ecto-phosphatase activity was linear for 60 min and during this time, was linear with the increase of cell density. The optimum pH was in the acidic range and some divalent metals, such as Cu(2+), Cd(2+) and Zn(2+), inhibited ecto-phosphatase activity. The activity was also reduced by phosphatase inhibitors. Importantly, the inhibition of phosphatase activity in conidia reduced the adhesion to Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) integument and, consequently and indirectly, M. anisopliae infection. The results herein presented show, for the first time, the importance of ecto-phosphatase activity in M. anisopliae conidia and provide the first evidence of its direct involvement in adhesion and host infection.

Role for Golgi reassembly and stacking protein (GRASP) in polysaccharide secretion and fungal virulence.

Secretion of virulence factors is a critical mechanism for the establishment of cryptococcosis, a disease caused by the yeast pathogen Cryptococcus neoformans. One key virulence strategy of C. neoformans is the release of glucuronoxylomannan (GXM), a capsule-associated immune-modulatory polysaccharide that reaches the extracellular space through secretory vesicles. Golgi reassembly and stacking protein (GRASP) is required for unconventional protein secretion mechanisms in different eukaryotic cells, but its role in polysaccharide secretion is unknown. This study demonstrates that a C. neoformans functional mutant of a GRASP orthologue had attenuated virulence in an animal model of cryptococcosis, in comparison with wild-type (WT) and reconstituted cells. Mutant cells manifested altered Golgi morphology, failed to produce typical polysaccharide capsules and showed a reduced ability to secrete GXM both in vitro and during animal infection. Isolation of GXM from cultures of WT, reconstituted or mutant strains revealed that the GRASP orthologue mutant produced polysaccharides with reduced dimensions. The mutant was also more efficiently associated to and killed by macrophages than WT and reconstituted cells. These results demonstrate that GRASP, a protein involved in unconventional protein secretion, is also required for polysaccharide secretion and virulence in C. neoformans.

Evaluation of the origin of a sample of Sporothrix schenckii that caused contamination of a researcher in Southern Brazil.

We report a case of a researcher from a laboratory of Mycology in Rio Grande do Sul, Brazil that presented a clinical evidence of sporotrichosis. The researcher had an accident while manipulating the microculture slides of chromoblastomycosis agents and presented a clinical evidence of sporotrichosis. As the laboratory has some cultures of Sporothrix schenckii, it was suggested that it might be a laboratory contamination. In order to test this hypothesis, the genotypic characterization of the samples was performed by means of the random amplified polymorphic DNA (RAPD) analysis method. In addition, we evaluated the in vitro antifungal activity of four antifungal agents against the isolated fungus. The sample obtained from the researcher was not genetically similar to any of the samples kept in the laboratory and showed the minimum inhibitory concentrations of 0.5 µg/mL for itraconazole and ketoconazole, > 64 µg/mL for fluconazole and 0.125 µg/mL for terbinafine. It is suggested that the contamination had an environmental origin.

Lonomia obliqua venomous secretion induces human platelet adhesion and aggregation.

The caterpillar Lonomia obliqua is a venomous animal that causes numerous accidents, especially in southern Brazil, where it is considered a public health problem. The clinical manifestations include several haemostatic disturbances that lead to a hemorrhagic syndrome. Considering that platelets play a central role in hemostasis, in this work we investigate the effects of L. obliqua venomous secretion upon blood platelets responses in vitro. Results obtained shows that L. obliqua venom directly induces aggregation and ATP secretion in human washed platelets in a dose-dependent manner. Electron microscopy studies clearly showed that the venomous bristle extract was also able to produce direct platelets shape change and adhesion as well as activation and formation of platelet aggregates. Differently from other enzyme inhibitors, the venom-induced platelet aggregation was significatively inhibited by p-bromophenacyl bromide, a specific inhibitor of phospholipases A2. Additional experiments with different pharmacological antagonists indicate that the aggregation response triggered by the venom active components occurs through a calcium-dependent mechanism involving arachidonic acid metabolite(s) of the cyclooxygenase pathway and activation of phosphodiesterase 3A, an enzyme that leads to the consumption of intracellular cAMP content. It was additionally found that L. obliqua-induced platelet aggregation was independent of ADP release. Altogether, these findings are in line with the need for a better understanding of the complex hemorrhagic syndrome resulting from the envenomation caused by L. obliqua caterpillars, and can also give new insights into the management of its clinical profile.

The contest of microbial pigeon neighbors: Interspecies competition between Serratia marcescens and the human pathogen Cryptococcus neoformans.

In nature, microorganisms often exhibit competitive behavior for nutrients and limited space, allowing them to alter the virulence determinants of pathogens. The human pathogenic yeast Cryptococcus neoformans can be found organized in biofilms, a complex community composed of an extracellular matrix which confers protection against predation. The aim of this study was to evaluate and characterize antagonistic interactions between two cohabiting microorganisms: C. neoformans and the bacteria Serratia marcescens. The interaction of S. marcescens with C. neoformans expressed a negative effect on biofilm formation, polysaccharide capsule, production of urease, and melanization of the yeast. These findings evidence that competition in mixed communities can result in dominance by one species, with direct impact on the physiological modulation of virulence determinants. Such an approach is key for understating the response of communities to the presence of competitors and, ultimately, rationally designing communities to prevent and treat certain diseases.

Scanning electron microscopy and machine learning reveal heterogeneity in capsular morphotypes of the human pathogen Cryptococcus spp.

Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread worldwide. Here, we have applied scanning electron microscopy (SEM) to define four Cryptococcus spp. capsule morphotypes, namely Regular, Spiky, Bald, and Phantom. These morphotypes were persistently observed in varying proportions among yeast isolates. To assess the distribution of such morphotypes we implemented an automated pipeline capable of (1) identifying potentially cell-associated objects in the SEM-derived images; (2) computing object-level features; and (3) classifying these objects into their corresponding classes. The machine learning approach used a Random Forest (RF) classifier whose overall accuracy reached 85% on the test dataset, with per-class specificity above 90%, and sensitivity between 66 and 94%. Additionally, the RF model indicates that structural and texture features, e.g., object area, eccentricity, and contrast, are most relevant for classification. The RF results agree with the observed variation in these features, consistently also with visual inspection of SEM images. Finally, our work introduces morphological variants of Cryptococcus spp. capsule. These can be promptly identified and characterized using computational models so that future work may unveil morphological associations with yeast virulence.