Comparative Evolutionary Patterns of Burkholderia cenocepacia and B. multivorans During Chronic Co-infection of a Cystic Fibrosis Patient Lung.

During chronic respiratory infections of cystic fibrosis (CF) patients, bacteria adaptively evolve in response to the nutritional and immune environment as well as influence other infecting microbes. The present study was designed to gain insights into the genetic mechanisms underlying adaptation and diversification by the two most prevalent pathogenic species of the Burkholderia cepacia complex (Bcc), B. cenocepacia and B. multivorans. Herein, we study the evolution of both of these species during coinfection of a CF patient for 4.4 years using genome sequences of 9 B. multivorans and 11 B. cenocepacia. This co-infection spanned at least 3 years following initial infection by B. multivorans and ultimately ended in the patient's death by cepacia syndrome. Both species acquired several mutations with accumulation rates of 2.08 (B. cenocepacia) and 2.27 (B. multivorans) SNPs/year. Many of the mutated genes are associated with oxidative stress response, transition metal metabolism, defense mechanisms against antibiotics, and other metabolic alterations consistent with the idea that positive selection might be driven by the action of the host immune system, antibiotic therapy and low oxygen and iron concentrations. Two orthologous genes shared by B. cenocepacia and B. multivorans were found to be under strong selection and accumulated mutations associated with lineage diversification. One gene encodes a nucleotide sugar dehydratase involved in lipopolysaccharide O-antigen (OAg) biosynthesis (wbiI). The other gene encodes a putative two-component regulatory sensor kinase protein required to sense and adapt to oxidative- and heavy metal- inducing stresses. This study contributes to understanding of shared and species-specific evolutionary patterns of B. cenocepacia and B. multivorans evolving in the same CF lung environment.

Structure of O-Antigen and Hybrid Biosynthetic Locus in Burkholderia cenocepacia Clonal Variants Recovered from a Cystic Fibrosis Patient.

Burkholderia cenocepacia is an opportunistic pathogen associated with chronic lung infections and increased risk of death in patients with cystic fibrosis (CF). In this work, we investigated the lipopolysaccharide (LPS) of clinical variants of B. cenocepacia that were collected from a CF patient over a period of 3.5 years, from the onset of infection until death by necrotizing pneumonia (cepacia syndrome). We report the chemical structure of the LPS molecule of various sequential isolates and the identification of a novel hybrid O-antigen (OAg) biosynthetic cluster. The OAg repeating unit of the LPS from IST439, the initial isolate, is a [→2)-ß-D-Ribf-(1→4)-α-D-GalpNAc-(1→] disaccharide, which was not previously described in B. cenocepacia. The IST439 OAg biosynthetic gene cluster contains 7 of 23 genes that are closely homologous to genes found in B. multivorans, another member of the Burkholderia cepacia complex. None of the subsequent isolates expressed OAg. Genomic sequencing of these isolates enabled the identification of mutations within the OAg cluster, but none of these mutations could be associated with the loss of OAg. This study provides support to the notion that OAg LPS modifications are an important factor in the adaptation of B. cenocepacia to chronic infection and that the heterogeneity of OAgs relates to variation within the OAg gene cluster, indicating that the gene cluster might have been assembled through multiple horizontal transmission events.

Variation of Burkholderia cenocepacia virulence potential during cystic fibrosis chronic lung infection.

During long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces multiple selective pressures in this highly stressful and fluctuating environment. As a consequence, the initial infecting strain undergoes genetic changes that result in the diversification of genotypes and phenotypes. Whether this clonal expansion influences the pathogenic potential is unclear. The virulence potential of 39 sequential B. cenocepacia (recA lineage IIIA) isolates, corresponding to 3 different clones retrieved from 3 chronically infected CF patients was compared in this study using the non-mammalian infection hosts Galleria mellonella and Caenorhabditis elegans. The isolates used in this retrospective study were picked randomly from selective agar plates as part of a CF Center routine, from the onset of infection until patients' death after 3.5 and 7.5 y or the more recent isolation date after 12.5 y of chronic infection. The infection models proved useful to assess virulence potential diversification, but for some isolates the relative values diverged in C. elegans and G. mellonella. Results also reinforce the concept of the occurrence of clonal diversification and co-existence of multiple phenotypes within the CF lungs, also with respect to pathogenicity. No clear trend of decrease (or increase) of the virulence potential throughout long-term infection was found but there is an apparent tendency for a clone/patient-dependent decrease of virulence when the G. mellonella model was used. The sole avirulent variant in both infection hosts was found to lack the small third replicon previously associated to virulence. Although possible, the in vivo loss of this nonessential megaplasmid was found to be a rare event (1 among a total of 64 isolates examined).

Yeast toxicogenomics: lessons from a eukaryotic cell model and cell factory.

The yeast Saccharomyces cerevisiae remains a highly relevant experimental model in the field of toxicogenomics and is an important microbial cell factory for the production of added-value chemicals and biofuels. Its deep functional characterization coupled with the straightforward exploitation of Omic approaches and metabolic engineering, at the frontline of systems and synthetic biology, is instrumental to obtain mechanistic insights into the response to multiple toxicants and for the development of robust industrial strains. This critical review focuses on the current field, ranging from the identification of toxicological outcomes of exposure to environmental toxicants, with impact in risk assessment, bioremediation and plant biotechnology, to the improvement of biomass-based biorefinery processes, with applications in pharmacology and in the food and beverages industry.

MFS transporters required for multidrug/multixenobiotic (MD/MX) resistance in the model yeast: understanding their physiological function through post-genomic approaches.

Multidrug/Multixenobiotic resistance (MDR/MXR) is a widespread phenomenon with clinical, agricultural and biotechnological implications, where MDR/MXR transporters that are presumably able to catalyze the efflux of multiple cytotoxic compounds play a key role in the acquisition of resistance. However, although these proteins have been traditionally considered drug exporters, the physiological function of MDR/MXR transporters and the exact mechanism of their involvement in resistance to cytotoxic compounds are still open to debate. In fact, the wide range of structurally and functionally unrelated substrates that these transporters are presumably able to export has puzzled researchers for years. The discussion has now shifted toward the possibility of at least some MDR/MXR transporters exerting their effect as the result of a natural physiological role in the cell, rather than through the direct export of cytotoxic compounds, while the hypothesis that MDR/MXR transporters may have evolved in nature for other purposes than conferring chemoprotection has been gaining momentum in recent years. This review focuses on the drug transporters of the Major Facilitator Superfamily (MFS; drug:H(+) antiporters) in the model yeast Saccharomyces cerevisiae. New insights into the natural roles of these transporters are described and discussed, focusing on the knowledge obtained or suggested by post-genomic research. The new information reviewed here provides clues into the unexpectedly complex roles of these transporters, including a proposed indirect regulation of the stress response machinery and control of membrane potential and/or internal pH, with a special emphasis on a genome-wide view of the regulation and evolution of MDR/MXR-MFS transporters.

Proteomic profiling of Burkholderia cenocepacia clonal isolates with different virulence potential retrieved from a cystic fibrosis patient during chronic lung infection.

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient's death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.

Quantitative- and phospho-proteomic analysis of the yeast response to the tyrosine kinase inhibitor imatinib to pharmacoproteomics-guided drug line extension.

Imatinib mesylate (IM) is a potent tyrosine kinase inhibitor used as front-line therapy in chronic myeloid leukemia, a disease caused by the oncogenic kinase Bcr-Abl. Although the clinical success of IM set a new paradigm in molecular-targeted therapy, the emergence of IM resistance is a clinically significant problem. In an effort to obtain new insights into the mechanisms of adaptation and tolerance to IM, as well as the signaling pathways potentially affected by this drug, we performed a two-dimensional electrophoresis-based quantitative- and phospho-proteomic analysis in the eukaryotic model Saccharomyces cerevisiae. We singled out proteins that were either differentially expressed or differentially phosphorylated in response to IM, using the phosphoselective dye Pro-Q(®) Diamond, and identified 18 proteins in total. Ten were altered only at the content level (mostly decreased), while the remaining 8 possessed IM-repressed phosphorylation. These 18 proteins are mainly involved in cellular carbohydrate processes (glycolysis/gluconeogenesis), translation, protein folding, ion homeostasis, and nucleotide and amino acid metabolism. Remarkably, all 18 proteins have human functional homologs. A role for HSP70 proteins in the response to IM, as well as decreased glycolysis as a metabolic marker of IM action are suggested, consistent with findings from studies in human cell lines. The previously-proposed effect of IM as an inhibitor of vacuolar H(+)-ATPase function was supported by the identification of an underexpressed protein subunit of this complex. Taken together, these findings reinforce the role of yeast as a valuable eukaryotic model for pharmacological studies and identification of new drug targets, with potential clinical implications in drug reassignment or line extension under a personalized medicine perspective.

Yeast toxicogenomics: genome-wide responses to chemical stresses with impact in environmental health, pharmacology, and biotechnology.

The emerging transdisciplinary field of Toxicogenomics aims to study the cell response to a given toxicant at the genome, transcriptome, proteome, and metabolome levels. This approach is expected to provide earlier and more sensitive biomarkers of toxicological responses and help in the delineation of regulatory risk assessment. The use of model organisms to gather such genomic information, through the exploitation of Omics and Bioinformatics approaches and tools, together with more focused molecular and cellular biology studies are rapidly increasing our understanding and providing an integrative view on how cells interact with their environment. The use of the model eukaryote Saccharomyces cerevisiae in the field of Toxicogenomics is discussed in this review. Despite the limitations intrinsic to the use of such a simple single cell experimental model, S. cerevisiae appears to be very useful as a first screening tool, limiting the use of animal models. Moreover, it is also one of the most interesting systems to obtain a truly global understanding of the toxicological response and resistance mechanisms, being in the frontline of systems biology research and developments. The impact of the knowledge gathered in the yeast model, through the use of Toxicogenomics approaches, is highlighted here by its use in prediction of toxicological outcomes of exposure to pesticides and pharmaceutical drugs, but also by its impact in biotechnology, namely in the development of more robust crops and in the improvement of yeast strains as cell factories.

A genome-wide screen identifies yeast genes required for protection against or enhanced cytotoxicity of the antimalarial drug quinine.

Quinine is used in the treatment of Plasmodium falciparum severe malaria. However, both the drug's mode of action and mechanisms of resistance are still poorly understood and subject to debate. In an effort to clarify these questions, we used the yeast Saccharomyces cerevisiae as a model for pharmacological studies with quinine. Following on a previous work that examined the yeast genomic expression program in response to quinine, we now explore a genome-wide screen for altered susceptibility to quinine using the EUROSCARF collection of yeast deletion strains. We identified 279 quinine-susceptible strains, among which 112 conferred a hyper-susceptibility phenotype. The expression of these genes, mainly involved in carbohydrate metabolism, iron uptake and ion homeostasis functions, is required for quinine resistance in yeast. Sixty-two genes whose deletion leads to increased quinine resistance were also identified in this screen, including several genes encoding ribosome protein subunits. These well-known potential drug targets in Plasmodium are associated with quinine action for the first time in this study. The suggested involvement of phosphate signaling and transport in quinine tolerance was also studied, and activation of phosphate starvation-responsive genes was observed under a mild-induced quinine stress. Finally, P. falciparum homology searches were performed for a selected group of 41 genes. Thirty-two encoded proteins possess homologs in the parasite, including subunits of a parasitic vacuolar H(+)-ATPase complex, ion and phosphate importers, and several ribosome protein subunits, suggesting that the results obtained in yeast are good candidates to be transposed and explored in a P. falciparum context.

Long-term colonization of the cystic fibrosis lung by Burkholderia cepacia complex bacteria: epidemiology, clonal variation, and genome-wide expression alterations.

Long-term respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) patients generally lead to a more rapid decline in lung function and, in some cases, to a fatal necrotizing pneumonia known as the "cepacia syndrome." Bcc bacteria are ubiquitous in the environment and are recognized as serious opportunistic pathogens that are virtually impossible to eradicate from the CF lung, posing a serious clinical threat. The epidemiological survey of Bcc bacteria involved in respiratory infections at the major Portuguese CF Treatment Center at Santa Maria Hospital, in Lisbon, has been carried out by our research group for the past 16 years, covering over 500 clinical isolates where B. cepacia and B. cenocepacia are the predominant species, with B. stabilis, B. contaminans, B. dolosa, and B. multivorans also represented. The systematic and longitudinal study of this CF population during such an extended period of time represents a unique case-study, comprehending 41 Bcc-infected patients (29 pediatric and 12 adult) of whom around 70% have been persistently colonized between 7 months and 9 years. During chronic infection, the CF airways represent an evolving ecosystem, with multiple phenotypic variants emerging from the clonal population and becoming established in the patients' airways as the result of genetic adaptation. Understanding the evolutionary mechanisms involved is crucial for an improved therapeutic outcome of chronic infections in CF. This review focuses on our contribution to the understanding of these adaptive mechanisms based on extensive phenotypic, genotypic, and genome-wide expression approaches of selected Bcc clonal variants obtained during long-term colonization of the CF airways.

Transcriptomic profiling of the Saccharomyces cerevisiae response to quinine reveals a glucose limitation response attributable to drug-induced inhibition of glucose uptake.

Quinine has been employed in the treatment of malaria for centuries and is still used against severe Plasmodium falciparum malaria. However, its interactions with the parasite remain poorly understood and subject to debate. In this study, we used the Saccharomyces cerevisiae eukaryotic model to better understand quinine's mode of action and the mechanisms underlying the cell response to the drug. We obtained a transcriptomic profile of the yeast's early response to quinine, evidencing a marked activation of genes involved in the low-glucose response (e.g., CAT8, ADR1, MAL33, MTH1, and SNF3). We used a low inhibitory quinine concentration with no detectable effect on plasma membrane function, consistent with the absence of a general nutrient starvation response and suggesting that quinine-induced glucose limitation is a specific response. We have further shown that transport of [(14)C]glucose is inhibited by quinine, with kinetic data indicating competitive inhibition. Also, tested mutant strains deleted for genes encoding high- and low-affinity hexose transporters (HXT1 to HXT5, HXT8, and HXT10) exhibit resistance phenotypes, correlating with reduced levels of quinine accumulation in the mutants examined. These results suggest that the hexose transporters are facilitators of quinine uptake in S. cerevisiae, possibly through a competitive inhibition mechanism. Interestingly, P. falciparum is highly dependent on glucose uptake, which is mediated by the single-copy transporter PfHT1, a protein with high homology to yeast's hexose transporters. We propose that PfHT1 is an interesting candidate quinine target possibly involved in quinine import in P. falciparum, an uptake mechanism postulated in recent studies to occur through a still-unidentified importer(s).

Genome-wide identification of genes required for yeast growth under imatinib stress: vacuolar H+-ATPase function is an important target of this anticancer drug.

Imatinib is a highly selective tyrosine kinase inhibitor of the oncogenic kinase Bcr-Abl, the result of a chromosomal abnormality that is associated with chronic myeloid leukaemia (CML). Despite the success of this target-directed therapy, imatinib resistance is an emerging problem, especially in advanced stages of CML. In this study, we explored the yeast Saccharomyces cerevisiae as a model eukaryotic system to better understand the mode of action of imatinib, as well as potential mechanisms of resistance to this drug. Using a systematic approach, we screened a yeast haploid deletion collection with individual knockouts of most nonessential yeast genes, and identified 51 genes that are required for yeast resistance to imatinib. The genes identified are involved mainly in DNA repair and transcription control, cell cycle control and differentiation, vacuolar pH homeostasis, vesicular transport, and protein trafficking. Remarkably, approximately 80% of the genes identified in our screen have human orthologs. The vacuolar pH homeostasis function is associated to our dataset by 13 genes that encode subunits and assembly factors of the yeast vacuolar proton-translocating ATPase (V-ATPase). Further studies using fluorescence microscopy showed that physiological acidification of the vacuole is severely compromised following imatinib treatment of yeast cells, an effect that was found to be dose dependent. Results suggest that imatinib might act as an effective inhibitor of V-ATPase function in yeast, identifying V-ATPase activity and vacuolar function as novel imatinib targets.

Drug:H+ antiporters in chemical stress response in yeast.

The emergence of widespread multidrug resistance (MDR) is a serious challenge for therapeutics, food-preservation and crop protection. Frequently, MDR is a result of the action of drug-efflux pumps, which are able to catalyze the extrusion of unrelated chemical compounds. This review summarizes the current knowledge on the Saccharomyces cerevisiae drug:H+ antiporters of the major facilitator superfamily (MFS), a group of MDR transporters that is still characterized poorly in eukaryotes. Particular focus is given here to the physiological role and expression regulation of these transporters, while we provide a unified view of new data emerging from functional genomics approaches. Although traditionally described as drug pumps, evidence reviewed here corroborates the hypothesis that several MFS-MDR transporters might have a natural substrate and that drug transport might occur only fortuitously or opportunistically. Their role in MDR might even result from the transport of endogenous metabolites that affect the partition of cytotoxic compounds indirectly. Finally, the extrapolation of the gathered knowledge on the MDR phenomenon in yeast to pathogenic fungi and higher eukaryotes is discussed.

Evaluation of the acoustic intensity of new ultrasound therapy equipment.

International safety standards recommend a limit below 30% variation in acoustic intensity for ultrasound therapy (UST) equipment. In view of this question, the purpose of this work was to evaluate the intensity of new UST equipment in the Brazilian market. An evaluation was performed of eight models manufactured by six different national manufacturers; under continuous and pulsed conditions, at frequencies of 1.0-3.0 MHz, for a total of 48 items of equipment. The intensities were analysed according to the technical standards IEC 601-2-5, in the range 0.01-3.0 Wcm(-2), using a radiation pressure scale UPM-DT-10 (Ohmic Instruments), previously calibrated. The results demonstrated that the models Sonacel, Sonacel plus, Sonacel III, Avatar I, and Sonamed I, although they were new (unused) presented calibration errors of over 30% in more than one intensity checked, and the models SONOPULSE, PRO-SEVEN and SONOMASTER ST. are within the standards proposed. The results show that industry must improve quality control on their production lines, as well as that there is a need for a supervising body at national level. Published by Elsevier Science B.V.