Pathway Driven Target Selection in Klebsiella pneumoniae: Insights Into Carbapenem Exposure

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) represents an emerging threat to public health. CR-KP infections result in elevated morbidity and mortality. This fact, coupled with their global dissemination and increasingly limited number of therapeutic options, highlights the urgency of novel antimicrobials. Innovative strategies linking genome-wide interrogation with multi-layered metabolic data integration can accelerate the early steps of drug development, particularly target selection. Using the BioCyc ontology, we generated and manually refined a metabolic network for a CR-KP, K. pneumoniae Kp13. Converted into a reaction graph, we conducted topological-based analyses in this network to prioritize pathways exhibiting druggable features and fragile metabolic points likely exploitable to develop novel antimicrobials. Our results point to the aptness of previously recognized pathways, such as lipopolysaccharide and peptidoglycan synthesis, and casts light on the possibility of targeting less explored cellular functions. These functions include the production of lipoate, trehalose, glycine betaine, and flavin, as well as the salvaging of methionine. Energy metabolism pathways emerged as attractive targets in the context of carbapenem exposure, targeted either alone or in conjunction with current therapeutic options. These results prompt further experimental investigation aimed at controlling this highly relevant pathogen.Fil: Serral, Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Pardo, Agustin Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sosa, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Palomino, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Nicolás, Marisa F.. Laboratório Nacional de Computação Científica; BrasilFil: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Ramos, Pablo Ivan P.. Fundación Oswaldo Cruz; BrasilFil: Fernández Do Porto, Darío Augusto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

The polymyxin B-induced transcriptomic response of a clinical, multidrug-resistant Klebsiella pneumoniae involves multiple regulatory elements and intracellular targets

Background: The emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling. Results: We sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13(pH)), magnesium deprivation (Kp13(Mg)), high concentrations of calcium (Kp13(Ca)) and iron (Kp13(Fe)), and a control condition with PB (Kp13(PolB)). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic. Conclusions: We obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets.Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Lab Nacl Comp Cient, Petropolis, RJ, BrazilFiocruz MS, Ctr Pesquisas Goncalo Moniz, Salvador, BA, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Internal Med, Lab Alerta,Div Infect Dis, Sao Paulo, SP, BrazilUniv Catolica Cordoba, Fac Ingn, CONICET, Cordoba, ArgentinaUniv Fed Sao Paulo, Escola Paulista Med, Dept Internal Med, Lab Alerta,Div Infect Dis, Sao Paulo, SP, BrazilFAPERJ: E-26/110.315/2014FAPESP: 2010/12891-9CAPES: 23038.010041/2013-13Web of Scienc

Functional genomics, analysis of adaptation in and applications of models to the metabolism of engineered Escherichia coli

In order to examine the metabolism of bacteria in the genus Enterobacteriaceae tools for gene complement comparison and stoichiometric model building have been developed to take advantage of both the number of complete bacterial genome sequences currently available and the relationship between genes and metabolism. A functional genomic approach to improving knowledge of the metabolism of Escherichia coli CFT073 (a uropathogen) has been undertaken taking into account not only its genome sequence, but its close relationship to E. coli MG1655. A fresh comparison of E. coli CFT073 has been done with E. coli MG1655 to identify all those genes in CFT073 that are not present in MG1655 and may have metabolic characteristics. These genes have further been bioinformatically assessed to determine whether they might encode enzymes for the metabolism of chemicals commonly found in human urine, and one set of such genes has been experimentally confirmed to encode an L-sorbose utilisation pathway. Little experimental work has been done as yet to elucidate how bacteria adaptively respond to the introduction of heterologous metabolic genes. To investigate how bacteria respond to such DNA, genes encoding the L-sorbose utilisation and uptake operon from CFT073 have been cloned and transformed into DH5 and a selective pressure (minimal medium with L-sorbose as sole carbon source) has been applied over 100 generations of growth of this strain in serial passage to investigate the change in its behaviour. The availability of large numbers of completely sequenced genomes, along with the development of a stoichiometric metabolic model with very high coverage of E. coli metabolism (iAF1260 [1]) have made possible the analysis of the core metabolism of large numbers of bacteria to investigate gene essentiality in these bacteria. A novel way of assessing gene complement has been developed using BLAST and DiagHunter to improve reliability of gene synteny comparisons with contextual information about the genes and to extend work by others to cover all E. coli and Shigella genome sequences with available sequences on GanBank (as of 1st June 2009) in order to bioinformatically investigate essential genes in these bacteria and the heterogeneity of their metabolic networks. Further to this a metabolic model has been constructed for DH5 with an added L-sorbose pathway and for CFT073 and these models have been used to investigate behavioural changes during adaptation of bacteria to novel heterologous genes

The polymyxin B-induced transcriptomic response of a clinical, multidrug-resistant Klebsiella pneumoniae involves multiple regulatory elements and intracellular targets

Background: The emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling. Results: We sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13pH), magnesium deprivation (Kp13Mg), high concentrations of calcium (Kp13Ca) and iron (Kp13Fe), and a control condition with PB (Kp13PolB). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic. Conclusions: We obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets.Fil: Pereira Ramos, Pablo Ivan. Fundación Oswaldo Cruz; BrasilFil: Flores Custodio, Gregori Marlon. No especifíca;Fil: Quispe Saji, Guadalupe del Rosario. No especifíca;Fil: Cardoso, Thiago. No especifíca;Fil: Luchetti da Silva, Gisele. No especifíca;Fil: Braun, Graziela. Universidade Federal de Sao Paulo; BrasilFil: Martins, Williams. Universidade Federal de Sao Paulo; BrasilFil: Girardello, Raquel. Universidade Federal de Sao Paulo; BrasilFil: Ribeiro de Vasconcellos, Ana Teresa. No especifíca;Fil: Fernandez, Elmer Andres. Universidad Católica de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Gales, Cristina. Universidade Federal de Sao Paulo; BrasilFil: Nicolas, Marisa. No especifíca

Costless metabolic secretions as drivers of interspecies interactions in microbial ecosystems

Metabolic exchange mediates interactions among microbes, helping explain diversity in microbial communities. As these interactions often involve a fitness cost, it is unclear how stable cooperation can emerge. Here we use genome-scale metabolic models to investigate whether the release of “costless” metabolites (i.e. those that cause no fitness cost to the producer), can be a prominent driver of intermicrobial interactions. By performing over 2 million pairwise growth simulations of 24 species in a combinatorial assortment of environments, we identify a large space of metabolites that can be secreted without cost, thus generating ample cross-feeding opportunities. In addition to providing an atlas of putative interactions, we show that anoxic conditions can promote mutualisms by providing more opportunities for exchange of costless metabolites, resulting in an overrepresentation of stable ecological network motifs. These results may help identify interaction patterns in natural communities and inform the design of synthetic microbial consortia.We thank Dr. Niels Klitgord for pioneering ideas that inspired launch of this work. We are also grateful to David Bernstein, Joshua E. Goldford, Meghan Thommes, Demetrius DiMucci, and all members of the Segre Lab for helpful discussions. A.R.P. is supported by a National Academies of Sciences, Engineering, and Medicine Ford Foundation Predoctoral Fellowship and a Howard Hughes Medical Institute Gilliam Fellowship. This work was supported by funding from the Defense Advanced Research Projects Agency (purchase request no. HR0011515303, contract no. HR0011-15-C-0091), the U.S. Department of Energy (grants DE-SC0004962 and DE-SC0012627), the NIH (grants 5R01DE024468, R01GM121950, and Sub_P30DK036836_P&F), the National Science Foundation (grants 1457695 and NSFOCE-BSF 1635070), MURI Grant W911NF-12-1-0390, the Human Frontiers Science Program (grant RGP0020/2016), and the Boston University Inter-disciplinary Biomedical Research Office. (National Academies of Sciences, Engineering, and Medicine Ford Foundation Predoctoral Fellowship; Howard Hughes Medical Institute Gilliam Fellowship; HR0011515303 - Defense Advanced Research Projects Agency; HR0011-15-C-0091 - Defense Advanced Research Projects Agency; DE-SC0004962 - U.S. Department of Energy; DE-SC0012627 - U.S. Department of Energy; 5R01DE024468 - NIH; R01GM121950 - NIH; Sub_P30DK036836_PF - NIH; 1457695 - National Science Foundation; NSFOCE-BSF 1635070 - National Science Foundation; W911NF-12-1-0390 - MURI Grant; RGP0020/2016 - Human Frontiers Science Program; Boston University Inter-disciplinary Biomedical Research Office)Published versio

Role of mobile genetic elements in the global network of bacterial horizontal gene transfer

Many bacteria can exchange genetic material through horizontal gene transfer (HGT) mediated by plasmids and plasmid-borne transposable elements. One grave consequence of this exchange is the rapid spread of antibiotic resistance determinants among bacterial communities across the world. In this thesis, I make use of large datasets of publicly available bacterial genomes and various analytical approaches to improve our understanding of the nature and the impact of HGT at a global scale. In the first part, I study the population structure and dynamics of over 10,000 bacterial plasmids. By reconstructing and analysing a network of plasmids based on their shared k-mer content, I was able to sort them into biologically meaningful clusters. This network-based analysis allowed me to make further inferences into global network of HGT and opened up prospect for a natural and exhaustive classification framework of bacterial plasmids. The second part focuses on global spreading of blaNDM – an important antibiotic resistance gene. To this end, I compiled a dataset of over 6000 bacterial genomes harbouring this element and developed a novel computational approach to track structural variants surrounding blaNDM across bacterial genomes. This facilitated identification of prevalent genomic contexts of blaNDM and reconstruction of key mobile genetic elements and events which led to its global dissemination. Taken together, my results highlight transposable elements as the main drivers of HGT at broad phylogenetic and geographical scales with plasmid exchange being much more spatially restricted due to the adaptation to specific bacterial hosts and evolutionary pressures

Dissimilatory Metabolism of Nitrogen Oxides in Bacteria: Comparative Reconstruction of Transcriptional Networks

Bacterial response to nitric oxide (NO) is of major importance since NO is an obligatory intermediate of the nitrogen cycle. Transcriptional regulation of the dissimilatory nitric oxides metabolism in bacteria is diverse and involves FNR-like transcription factors HcpR, DNR, and NnrR; two-component systems NarXL and NarQP; NO-responsive activator NorR; and nitrite-sensitive repressor NsrR. Using comparative genomics approaches, we predict DNA-binding motifs for these transcriptional factors and describe corresponding regulons in available bacterial genomes. Within the FNR family of regulators, we observed a correlation of two specificity-determining amino acids and contacting bases in corresponding DNA recognition motif. Highly conserved regulon HcpR for the hybrid cluster protein and some other redox enzymes is present in diverse anaerobic bacteria, including Clostridia, Thermotogales, and delta-proteobacteria. NnrR and DNR control denitrification in alpha- and beta-proteobacteria, respectively. Sigma-54-dependent NorR regulon found in some gamma- and beta-proteobacteria contains various enzymes involved in the NO detoxification. Repressor NsrR, which was previously known to control only nitrite reductase operon in Nitrosomonas spp., appears to be the master regulator of the nitric oxides' metabolism, not only in most gamma- and beta-proteobacteria (including well-studied species such as Escherichia coli), but also in Gram-positive Bacillus and Streptomyces species. Positional analysis and comparison of regulatory regions of NO detoxification genes allows us to propose the candidate NsrR-binding motif. The most conserved member of the predicted NsrR regulon is the NO-detoxifying flavohemoglobin Hmp. In enterobacteria, the regulon also includes two nitrite-responsive loci, nipAB (hcp-hcr) and nipC (dnrN), thus confirming the identity of the effector, i.e. nitrite. The proposed NsrR regulons in Neisseria and some other species are extended to include denitrification genes. As the result, we demonstrate considerable interconnection between various nitrogen-oxides-responsive regulatory systems for the denitrification and NO detoxification genes and evolutionary plasticity of this transcriptional network

Prioritisation of potential drug targets against bartonella bacilliformis by an integrative in-silico approach

BACKGROUND Carrion’s disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion’s disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.Fil: Farfán López, Mariella. Universidad Nacional Mayor de San Marcos; PerúFil: Espinoza Culupú, Abraham. Universidad Nacional Mayor de San Marcos; PerúFil: García De la guarda, Ruth. Universidad Nacional Mayor de San Marcos; PerúFil: Serral, Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Sosa, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Palomino, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Fernández Do Porto, Darío Augusto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; Argentin