Exploring the metabolic network of the epidemic pathogen Burkholderia cenocepacia J2315 via genome-scale reconstruction

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia cenocepacia </it>is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF) or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain <it>B. cenocepacia </it>J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of <it>B. cenocepacia </it>J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets.</p> <p>Results</p> <p>We reconstructed the genome-scale metabolic network of <it>B. cenocepacia </it>J2315. An iterative reconstruction process led to the establishment of a robust model, <it>i</it>KF1028, which accounts for 1,028 genes, 859 internal reactions, and 834 metabolites. The model <it>i</it>KF1028 captures important metabolic capabilities of <it>B. cenocepacia </it>J2315 with a particular focus on the biosyntheses of key metabolic virulence factors to assist in understanding the mechanism of disease infection and identifying potential drug targets. The model was tested through BIOLOG assays. Based on the model, the genome annotation of <it>B. cenocepacia </it>J2315 was refined and 24 genes were properly re-annotated. Gene and enzyme essentiality were analyzed to provide further insights into the genome function and architecture. A total of 45 essential enzymes were identified as potential therapeutic targets.</p> <p>Conclusions</p> <p>As the first genome-scale metabolic network of <it>B. cenocepacia </it>J2315, <it>i</it>KF1028 allows a systematic study of the metabolic properties of <it>B. cenocepacia </it>and its key metabolic virulence factors affecting the CF community. The model can be used as a discovery tool to design novel drugs against diseases caused by this notorious pathogen.</p

The intracellular behaviour of Burkholderia cenocepacia in murine macrophages

Burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in cystic fibrosis and other immunocompromised patients. The bacterium survives within macrophages by interfering with typical endocytic trafficking, resulting in delayed maturation of a B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia alters gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation. Furthermore, we hypothesize that specialized bacterial secretion systems are involved in the interactions between intracellular bacteria and macrophages. In this work, we characterize later-stage infection of macrophages by B. cenocepacia, showing replication within an acidified endosomal compartment suggestive of a phagolysosome. We examine differential gene expression by intracellular B. cenocepacia using selective capture of transcribed sequences (SCOTS) with both competitive enrichment and microarray analysis. We identified 766 genes differentially regulated in intracellular bacteria, of which 329 were induced and 437 repressed. Affected genes are involved in all aspects of cellular life, including information storage and processing, cellular processes and signalling, and metabolism; in general, intracellular gene expression demonstrates a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. Deletion of various SCOTS-identified genes affects B. cenocepacia entry into macrophages and intracellular replication, as well as host-directed cytotoxicity and spread to neighbouring cells. Expression of secretion system genes is differentially-regulated by intracellular B. cenocepacia. Although none of the five major secretion systems are essential for growth in culture, we show that bacterial secretion systems are involved in macrophage entry, intracellular replication, and host-directed cytotoxicity. Type IV secretion systems play a role in early interactions with macrophages, while type II and IV secretion systems contribute to post-internalization intracellular replication and host-directed cytotoxicity. As a whole, secretion systems appear to increase pathogenicity in macrophages while limiting the spread of B. cenocepacia infection. Together, these studies advance our understanding of the intracellular behaviour of B. cenocepacia in macrophages. Further investigation into the remaining SCOTS-identified genes, as well as putative secreted effectors, will provide a better understanding of the adaptive responses of intracellular B. cenocepacia, leading to life in a phagosomal niche and host cell cytotoxicity

Death of a bacterium: exploring the inhibition of Staphylococcus aureus by Burkholderia cenocepacia.

Antimicrobial resistance is a phenomenon of increasing concern as antimicrobial overuse and misuse eliminate current therapeutic options, ushering society into a post-antimicrobial era. Antibiotic discovery and synthesis efforts are urgently needed to counter the increasing burden of antimicrobial resistance. Staphylococcus aureus is a causative agent of a variety of clinical manifestations including bacteremia, endocarditis, soft tissue infection, osteomyelitis, and device-related infections. S. aureus infection presents additional treatment challenges due to its capacity for biofilm formation, which is a mode of growth that confers protection from antibiotics and physical elimination, and the emergence of antibiotic resistant strains, including methicillin-resistant S. aureus and vancomycin-resistant S. aureus. Infection with antibiotic-resistant strains occurs within both nosocomial and community settings, broadening the potential impact of this organism. Bacteria within the genus Burkholderia hold vast potential as sources of antimicrobial agents. Our analysis of patient culture data, provided by the Cystic Fibrosis Foundation, suggests a negative relationship between members of the Burkholderia cepacia complex and Staphylococcus aureus. An in vitro screen for activity against S. aureus indicated several clinical strains of Burkholderia cenocepacia confer potent anti-Staphylococcus activity. This dissertation characterizes the deleterious effect of the presence of B. cenocepacia J2315 and H111, two clinical isolates from cystic fibrosis patients, against S. aureus. Co-culture biofilm-associated survival of both methicillin-sensitive and methicillin-resistant strains was, overall, decreased with both B. cenocepacia J2315 and H111. I further established the breadth of antibiotic activity of these two strains in co-culture with multiple Staphylococcus and other Gram-positive species, including Enterococcus, Bacillus and Listeria. While both B. cenocepacia strains demonstrated detrimental effects against survival of co-inoculated Staphylococcus species, the extent of inhibition of other Gram-positive species differed. Antagonistic activity against the Enterococcus and Bacillus strains assessed in co-culture with B. cenocepacia H111 was profound, with reduction of many co-cultured strains to below the limit of detection. Co-culture survival of the same Gram-positive species with B. cenocepacia J2315 indicated no significant reduction versus cognate mono-culture. Inhibition of S. aureus by both B. cenocepacia strains occurs via a secreted compound, as evidenced by reduction in survival of S. aureus when exposed to B. cenocepacia sterile biofilm supernatants. The inhibitory substance, at least for B. cenocepacia J2315 is secreted in larger quantities in response to the presence of S. aureus. Enzymatic treatment of the supernatants suggests that a protein and an RNA, or a nucleoprotein, are involved in the B. cenocepacia J2315-mediated antagonism of S. aureus, but that inhibition by B. cenocepacia H111 involves a different mechanism. The inhibitory effect is largely dependent upon culture medium, and B. cenocepacia J2315 is more sensitive to differences in nutrient composition of the growth medium than B. cenocepacia H111. Further, this decrease in viable S. aureus is not simply due to B. cenocepacia causing a release of S. aureus from biofilms but is due to killing of S. aureus. Collectively, these data confirm the biotechnological potential of two B. cenocepacia strains and serve to optimize conditions for observation and analysis of this phenomenon

A Systems Biology Approach to Drug Targets in Pseudomonas aeruginosa Biofilm

Antibiotic resistance is an increasing problem in the health care system and we are in a constant race with evolving bacteria. Biofilm-associated growth is thought to play a key role in bacterial adaptability and antibiotic resistance. We employed a systems biology approach to identify candidate drug targets for biofilm-associated bacteria by imitating specific microenvironments found in microbial communities associated with biofilm formation. A previously reconstructed metabolic model of Pseudomonas aeruginosa (PA) was used to study the effect of gene deletion on bacterial growth in planktonic and biofilm-like environmental conditions. A set of 26 genes essential in both conditions was identified. Moreover, these genes have no homology with any human gene. While none of these genes were essential in only one of the conditions, we found condition-dependent genes, which could be used to slow growth specifically in biofilm-associated PA. Furthermore, we performed a double gene deletion study and obtained 17 combinations consisting of 21 different genes, which were conditionally essential. While most of the difference in double essential gene sets could be explained by different medium composition found in biofilm-like and planktonic conditions, we observed a clear effect of changes in oxygen availability on the growth performance. Eight gene pairs were found to be synthetic lethal in oxygen-limited conditions. These gene sets may serve as novel metabolic drug targets to combat particularly biofilm-associated PA. Taken together, this study demonstrates that metabolic modeling of human pathogens can be used to identify oxygen-sensitive drug targets and thus, that this systems biology approach represents a powerful tool to identify novel candidate antibiotic targets

Using next generation sequencing approaches to define the population biology of the neglected cystic fibrosis lung pathogen Burkholderia multivorans

Burkholderia multivorans is the most frequently isolated Burkholderia cepacia complex species recovered from cystic fibrosis lung infection. However, its pathogenesis and species population biology remain elusive. Understanding adaptational factors of B. multivorans to the CF lung microenvironment is important for predicting its pathogenesis and disease outcome. B. multivorans population biology was explored using pan genome analysis, average nucleotide identity and phylogenomic analysis (n = 283). The population split into two major genomic lineages, designated 1 and 2, and four B. multivorans model strains were selected to represent them: the soil strain ATCC 17616 (lineage 2a), BCC1272 (lineage 2a), BCC0033 (lineage 2b), and BCC0084 (lineage 1). The latter 3 CF strains were completely genome sequenced to add to the readily available reference genome ATCC 17616. Using gene presence-absence analysis, unique B. multivorans lineage-specific genes were identified. This enabled diagnostic PCR design with genes ghrB_1 and glnM_2 selected as the lineage 1 and lineage 2 targets, respectively. The PCRs showed 100% lineage-specificity against 48 B. multivorans strains. Phenotypic analysis was performed on a subset of 49 B. multivorans strains evaluating their morphology, growth kinetics, motility, biofilm formation, and exopolysaccharide production. The B. multivorans phenotype was variable amongst the strains, with no link to genomic lineage. Phenotypic comparison was also performed when B. multivorans were mixed with a secondary CF pathogen. The suppression of P. aeruginosa LESB58 protease production, when cultured with B. multivorans, was identified as an interesting interaction based on an unknown mechanism. Three of the B. multivorans model strains (BCC0033, BCC0084, and ATCC 17616) were also evaluated in a murine respiratory infection model and all showed good persistence over 5-days. Overall, this work has built a foundation of knowledge on the B. multivorans phenotype and genotype, enabling associations between lineage, therapeutics testing, and clinical outcome to be studie

Predicting new molecular targets for rhein using network pharmacology

<p>Abstract</p> <p>Background</p> <p>Drugs can influence the whole biological system by targeting interaction reactions. The existence of interactions between drugs and network reactions suggests a potential way to discover targets. The in silico prediction of potential interactions between drugs and target proteins is of core importance for the identification of new drugs or novel targets for existing drugs. However, only a tiny portion of drug-targets in current datasets are validated interactions. This motivates the need for developing computational methods that predict true interaction pairs with high accuracy. Currently, network pharmacology has used in identifying potential drug targets to predicting the spread of drug activity and greatly contributed toward the analysis of biological systems on a much larger scale than ever before.</p> <p>Methods</p> <p>In this article, we present a computational method to predict targets for rhein by exploring drug-reaction interactions. We have implemented a computational platform that integrates pathway, protein-protein interaction, differentially expressed genome and literature mining data to result in comprehensive networks for drug-target interaction. We used Cytoscape software for prediction rhein-target interactions, to facilitate the drug discovery pipeline.</p> <p>Results</p> <p>Results showed that 3 differentially expressed genes confirmed by Cytoscape as the central nodes of the complicated interaction network (99 nodes, 153 edges). Of note, we further observed that the identified targets were found to encompass a variety of biological processes related to immunity, cellular apoptosis, transport, signal transduction, cell growth and proliferation and metabolism.</p> <p>Conclusions</p> <p>Our findings demonstrate that network pharmacology can not only speed the wide identification of drug targets but also find new applications for the existing drugs. It also implies the significant contribution of network pharmacology to predict drug targets.</p

Genomic insights into bacterial adaptation during infection

Bacteria evolve during the colonization of human hosts, yet little is known about the selective pressures and evolutionary forces that shape this evolution. Illumination of these processes may inspire new therapeutic directions for combating bacterial infections and promoting healthy bacteria-host interactions. The advent of high-throughput sequencing has enabled the identification of mutations that occur within the human host, and various tools from computational and evolutionary biology can aid in creating biological understanding from these mutations. Chapter 1 describes recent progress in understanding within-patient bacterial adaption, focusing on insights made from genomic studies