broth-microdilution-vitro-screening-an-easy-fast-method-to-detect-new

Broth Microdilution <em>In Vitro</em> Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

University of Brasília

Paracoccidioides 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.

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

Time-course expression profiles of one xylanase and eight cellulase encoding genes, as well as of two transcription factor encoding genes of Humicola grisea var. thermoidea were established in different culture media pHs and carbon sources (glucose and sugarcane bagasse). Quantitative real-time RT-PCR analysis revealed a remarkable and parallel increase in mRNA accumulation for cbh1.1, cbh1.2, egl1, egl2, egl3, bgl4 and xyn1 at alkaline pH and with sugarcane bagasse employed as the sole carbon source. Glucose utilization led to a higher creA mRNA accumulation compared to the other genes. A distinct pattern was observed for egl4, whose mRNA preferably accumulated in acidic conditions. The transcriptional profile data combined with the analysis of the in vitro binding of PacC and CreA transcription factors to the promoters support the CreA-mediated carbon repression and the PacC-related pH regulation of H. grisea cellulase and xylanase encoding genes. Moreover, EMSA analyses suggest a role for CreA on pacC transcriptional regulation. These data will be useful to H. grisea hydrolytic enzymes production improvement, as well as to the design of optimized promoters aiming industrial heterologous proteins production.

Carbon source and pH-dependent transcriptional regulation of cellulase genes of Humicola grisea var. thermoidea grown on sugarcane bagasse.

Mycobacterium massiliense is a rapidly growing bacterium associated with opportunistic infections. The genome of a representative isolate (strain GO 06) recovered from wound samples from patients who underwent arthroscopic or laparoscopic surgery was sequenced. To the best of our knowledge, this is the first announcement of the complete genome sequence of an M. massiliense strain.

Complete genome sequence of Mycobacterium massiliense.

Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as K m = 27.5 ± 4.33 mg/mL, V max = 1.185 ± 0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol.

An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues.

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.

Quorum sensing-mediated, cell density-dependent regulation of growth and virulence in Cryptococcus neoformans.

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.

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

Mastoparans, a class of peptides found in wasp venom, have significant effects following a sting as well as useful applications in clinical practice. Among these is their potential use in the control of micro-organisms that cause infectious diseases with a significant impact on society. Thus, the present study describes the isolation and identification of a mastoparan peptide from the venom of the social wasp Pseudopolybia vespiceps and evaluated its antimicrobial profile against bacteria (Staphylococcus aureus and Mycobacterium abscessus subsp. massiliense), fungi (Candida albicans and Cryptococcus neoformans) and in vivo S. aureus infection. The membrane pore-forming ability was also assessed. The mastoparan reduced in vitro and ex vivo mycobacterial growth by 80% at 12.5 µM in infected peritoneal macrophages but did not affect the shape of bacterial cells at the dose tested (6.25 µM). The peptide also showed potent action against S. aureus in vitro (EC50 and EC90 values of 1.83 µM and 2.90 µM, respectively) and reduced the in vivo bacterial load after 6 days of topical treatment (5 mg/kg). Antifungal activity was significant, with EC50 and EC90 values of 12.9 µM and 15.3 µM, respectively, for C. albicans, and 11 µM and 22.70 µM, respectively, for C. neoformans. Peptides are currently attracting interest for their potential in the design of antimicrobial drugs, particularly due to the difficulty of micro-organisms in developing resistance to them. In this respect, Polybia-MPII proved to be highly effective, with a lower haemolysis rate compared with peptides of the same family.

Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera).

Article	Year
The stress responsive and morphologically regulated hsp90 gene from Paracoccidioides brasiliensis is essential to cell viability. BMC microbiology\| PubMed ID: 18808717	2008
Carbon source and pH-dependent transcriptional regulation of cellulase genes of Humicola grisea var. thermoidea grown on sugarcane bagasse. Enzyme and microbial technology\| PubMed ID: 22112766	2011
Complete genome sequence of Mycobacterium massiliense. Journal of bacteriology\| PubMed ID: 22965084	2012
An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues. Enzyme research\| PubMed ID: 23936633	2013
Quorum sensing-mediated, cell density-dependent regulation of growth and virulence in Cryptococcus neoformans. mBio\| PubMed ID: 24381301	2014
Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms. Frontiers in microbiology\| PubMed ID: 27917162	2016
Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera). International journal of antimicrobial agents\| PubMed ID: 28108242	2017
Intracellular Eukaryotic Pathogens' Virulence Attributes and Their Interplay with Host Immune Defenses. Mediators of inflammation\| PubMed ID: 29269994	2017

Ildinete Silva-Pereira

Biography

JoVE Journal Publications

Other Publications