Analysis of mouse faecal dysbiosis, during the development of cachexia, induced by transplantation with Lewis lung carcinoma cells.

Cachexia (CC) is a complex wasting syndrome that significantly affects life quality and life expectancy among cancer patients. Original studies, in which CC was induced in mouse models through inoculation with BaF and C26 tumour cells, demonstrated that CC development correlates with bacterial gut dysbiosis in these animals. In both cases, a common microbial signature was observed, based on the expansion of Enterobacteriaceae in the gut of CC animals. However, these two types of tumours induce unique microbial profiles, suggesting that different CC induction mechanisms significantly impact the outcome of gut dysbiosis. The present study sought to expand the scope of such analyses by characterizing the CC-associated dysbiosis that develops when mice are inoculated with Lewis lung carcinoma (LLC) cells, which constitutes one of the most widely employed mechanisms for CC induction. Interestingly, Enterobacteriaceae expansion is also observed in LLC-induced CC. However, the dysbiosis identified herein displays a more complex pattern, involving representatives from seven different bacterial phyla, which were consistently identified across successive levels of taxonomic hierarchy. These results are supported by a predictive analysis of gene content, which identified a series of functional/structural changes that potentially occur in the gut bacterial population of these animals, providing a complementary and alternative approach to microbiome analyses based solely on taxonomic classification.

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Transcriptomic profiling identifies novel transcripts, isomorphs, and noncoding RNAs in Paracoccidioides brasiliensis.

This paper describes a transcriptomic profiling of Paracoccidioides brasiliensis (Pb) performed with the aid of an RNA-seq-based approach, aimed at characterizing the general transcriptome in this human pathogenic fungus, responsible for paracoccidioidomycosis (PCM). Results confirm that ∼75% of the genes currently annotated in the P. brasiliensis genome are, in fact, transcribed in vivo and that ∼19% of them may display alternative isomorphs. Moreover, we identified 627 transcripts that do not match any gene currently mapped in the genome, represented by 114 coding transcripts (probably derived from previously unmapped protein-coding genes) and 513 noncoding RNAs (ncRNAs), including 203 long-noncoding RNAs (lncRNAs).

Fungal Dysbiosis Correlates with the Development of Tumor-Induced Cachexia in Mice.

Cachexia (CC) is a devastating metabolic syndrome associated with a series of underlying diseases that greatly affects life quality and expectancy among cancer patients. Studies involving mouse models, in which CC was induced through inoculation with tumor cells, originally suggested the existence of a direct correlation between the development of this syndrome and changes in the relative proportions of several bacterial groups present in the digestive tract. However, these analyses have focus solely on the characterization of bacterial dysbiosis, ignoring the possible existence of changes in the relative populations of fungi, during the development of CC. Thus, the present study sought to expand such analyses, by characterizing changes that occur in the gut fungal population (mycobiota) of mice, during the development of cancer-induced cachexia. Our results confirm that cachectic animals, submitted to Lewis lung carcinoma (LLC) transplantation, display significant differences in their gut mycobiota, when compared to healthy controls. Moreover, identification of dysbiotic fungi showed remarkable consistency across successive levels of taxonomic hierarchy. Many of these fungi have also been associated with dysbioses observed in a series of gut inflammatory diseases, such as obesity, colorectal cancer (CRC), myalgic encephalomyelitis (ME) and inflammatory bowel disease (IBD). Nonetheless, the dysbiosis verified in the LLC model of cancer cachexia seems to be unique, presenting features observed in both obesity (reduced proportion of Mucoromycota) and CRC/ME/IBD (increased proportions of Sordariomycetes, Saccharomycetaceae and Malassezia). One species of Mucoromycota (Rhyzopus oryzae) stands out as a promising probiotic candidate in adjuvant therapies, aimed at treating and/or preventing the development of CC.

ParaDB: A manually curated database containing genomic annotation for the human pathogenic fungi Paracoccidioides spp.

BACKGROUND: The genus Paracoccidioides consists of thermodymorphic fungi responsible for Paracoccidioidomycosis (PCM), a systemic mycosis that has been registered to affect ~10 million people in Latin America. Biogeographical data subdivided the genus Paracoccidioides in five divergent subgroups, which have been recently classified as different species. Genomic sequencing of five Paracoccidioides isolates, representing each of these subgroups/species provided an important framework for the development of post-genomic studies with these fungi. However, functional annotations of these genomes have not been submitted to manual curation and, as a result, ~60-90% of the Paracoccidioides protein-coding genes (depending on isolate/annotation) are currently described as responsible for hypothetical proteins, without any further functional/structural description. PRINCIPAL FINDINGS: The present work reviews the functional assignment of Paracoccidioides genes, reducing the number of hypothetical proteins to ~25-28%. These results were compiled in a relational database called ParaDB, dedicated to the main representatives of Paracoccidioides spp. ParaDB can be accessed through a friendly graphical interface, which offers search tools based on keywords or protein/DNA sequences. All data contained in ParaDB can be partially or completely downloaded through spreadsheet, multi-fasta and GFF3-formatted files, which can be subsequently used in a variety of downstream functional analyses. Moreover, the entire ParaDB environment has been configured in a Docker service, which has been submitted to the GitHub repository, ensuring long-term data availability to researchers. This service can be downloaded and used to perform fully functional local installations of the database in alternative computing ecosystems, allowing users to conduct their data mining and analyses in a personal and stable working environment. CONCLUSIONS: These new annotations greatly reduce the number of genes identified solely as hypothetical proteins and are integrated into a dedicated database, providing resources to assist researchers in this field to conduct post-genomic studies with this group of human pathogenic fungi.

Bioportainer Workbench: a versatile and user-friendly system that integrates implementation, management, and use of bioinformatics resources in Docker environments.

BACKGROUND: The Docker project is providing a promising strategy for the development of virtualization systems in bioinformatics. However, implementation, management, and launching of Docker containers is not entirely trivial for users not fully familiarized with command line interfaces. This has prompted the development of graphical user interfaces to facilitate the interaction of inexperienced users with Docker environments. RESULTS: We describe the BioPortainer Workbench, an integrated Docker system that assists inexperienced users in interacting with a bioinformatics-dedicated Docker environment at 3 main levels: (i) infrastructure, (ii) platform, and (iii) application. CONCLUSIONS: The BioPortainer Workbench represents a pioneering effort in developing a comprehensive and easy-to-use Docker platform focused on bioinformatics, which may greatly assist in the dissemination of Docker virtualization technology in this complex field of research.

Dugong: a Docker image, based on Ubuntu Linux, focused on reproducibility and replicability for bioinformatics analyses.

Summary: This manuscript introduces and describes Dugong, a Docker image based on Ubuntu 16.04, which automates installation of more than 3500 bioinformatics tools (along with their respective libraries and dependencies), in alternative computational environments. The software operates through a user-friendly XFCE4 graphic interface that allows software management and installation by users not fully familiarized with the Linux command line and provides the Jupyter Notebook to assist in the delivery and exchange of consistent and reproducible protocols and results across laboratories, assisting in the development of open science projects. Availability and implementation: Source code and instructions for local installation are available at https://github.com/DugongBioinformatics, under the MIT open source license. Contact: Luiz.nunes@ufabc.edu.br.

Thioridazine inhibits gene expression control of the cell wall signaling pathway (CWI) in the human pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), the most common systemic mycosis in Latin America. PCM treatment involves a long-term chemotherapeutic approach and relapses occur at an alarming frequency. Moreover, the emergence of strains with increased drug-resistance phenotypes puts constant pressure on the necessity to develop new alternatives to treat systemic mycoses. In this work, we show that the phenothiazine (PTZ) derivative thioridazine (TR) inhibits in vitro growth of P. brasiliensis yeasts at micromolar concentrations. We employed microarray hybridization to examine how TR affects gene expression in this fungus, identifying ~1800 genes that were modulated in response to this drug. Dataset evaluation showed that TR inhibits the expression of genes that control the onset of the cell wall integrity (CWI) response, hampering production of all major structural polysaccharides of the fungal cell wall (chitin, α-glucan and ß-glucan). Although TR and other PTZs have been shown to display antimicrobial activity by various mechanisms, inhibition of CWI signaling has not yet been reported for these drugs. Thus, TR may provide a novel approach to treat fungal infections by targeting cell wall biogenesis.

Differential gene expression in Xylella fastidiosa 9a5c during co-cultivation with the endophytic bacterium Methylobacterium mesophilicum SR1.6/6.

Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC), colonizes plant xylem, reducing sap flow, and inducing internerval chlorosis, leaf size reduction, necrosis, and harder and smaller fruits. This bacterium may be transmitted from plant to plant by sharpshooter insects, including Bucephalogonia xanthopis. The citrus endophytic bacterium Methylobacterium mesophilicum SR1.6/6 colonizes citrus xylem and previous studies showed that this strain is also transferred from plant to plant by B. xanthopis (Insecta), suggesting that this endophytic bacterium may interact with X. fastidiosa in planta and inside the insect vector during co-transmission by the same insect vector. To better understand the X. fastidiosa behavior in the presence of M. mesophilicum, we evaluated the X. fastidiosa transcriptional profile during in vitro interaction with M. mesophilicum SR1.6/6. The results showed that during co-cultivation, X. fastidiosa down-regulated genes related to growth and up-regulated genes related to energy production, stress, transport, and motility, suggesting the existence of a specific adaptive response to the presence of M. mesophilicum in the culture medium.

Comparative genomic analysis of coffee-infecting Xylella fastidiosa strains isolated from Brazil.

Strains of Xylella fastidiosa constitute a complex group of bacteria that develop within the xylem of many plant hosts, causing diseases of significant economic importance, such as Pierce's disease in North American grapevines and citrus variegated chlorosis in Brazil. X. fastidiosa has also been obtained from other host plants, in direct correlation with the development of diseases, as in the case of coffee leaf scorch (CLS)--a disease with potential to cause severe economic losses to the Brazilian coffee industry. This paper describes a thorough genomic characterization of coffee-infecting X. fastidiosa strains, initially performed through a microarray-based approach, which demonstrated that CLS strains could be subdivided in two phylogenetically distinct subgroups. Whole-genomic sequencing of two of these bacteria (one from each subgroup) allowed identification of ORFs and horizontally transferred elements (HTEs) that were specific to CLS-related X. fastidiosa strains. Such analyses confirmed the size and importance of HTEs as major mediators of chromosomal evolution amongst these bacteria, and allowed identification of differences in gene content, after comparisons were made with previously sequenced X. fastidiosa strains, isolated from alternative hosts. Although direct experimentation still needs to be performed to elucidate the biological consequences associated with such differences, it was interesting to verify that CLS-related bacteria display variations in genes that produce toxins, as well as surface-related factors (such as fimbrial adhesins and LPS) that have been shown to be involved with recognition of specific host factors in different pathogenic bacteria.

Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition.

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.

Competitive hybridization on spotted microarrays as a tool to conduct comparative genomic analyses of Xylella fastidiosa strains.

Xylella fastidiosa strains are responsible for several plant diseases and since such isolates display a broad host range and complex biological behavior, genomic comparisons employing microarray hybridizations may provide an effective method to compare them. Thus, we performed a thorough validation of this type of approach using two recently sequenced strains of this phytopathogen. By matching microarray hybridization results to direct sequence comparisons, we were able to establish precise cutoff ratios for common and exclusive sequences, allowing the identification of exclusive genes involved in important biological traits. This validation will enable the use of microarray-based comparisons across a wide variety of microorganisms