Jacek Puchałka

Genome-Scale Reconstruction and Analysis of the Pseudomonas putida KT2440 Metabolic Network Facilitates Applications in Biotechnology

A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, 13C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype–phenotype relationships and provides a sound framework to explore this versatile bacterium and to capitalize on its vast biotechnological potential.

Genome-Scale Metabolic Network Analysis of the Opportunistic Pathogen Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a major life-threatening opportunistic pathogen that commonly infects immunocompromised patients. This bacterium owes its success as a pathogen largely to its metabolic versatility and flexibility. A thorough understanding of P. aeruginosas metabolism is thus pivotal for the design of effective intervention strategies. Here we aim to provide, through systems analysis, a basis for the characterization of the genome-scale properties of this pathogens versatile metabolic network. To this end, we reconstructed a genome-scale metabolic network of Pseudomonas aeruginosa PAO1. This reconstruction accounts for 1,056 genes (19% of the genome), 1,030 proteins, and 883 reactions. Flux balance analysis was used to identify key features of P. aeruginosa metabolism, such as growth yield, under defined conditions and with defined knowledge gaps within the network. BIOLOG substrate oxidation data were used in model expansion, and a genome-scale transposon knockout set was compared against in silico knockout predictions to validate the model. Ultimately, this genome-scale model provides a basic modeling framework with which to explore the metabolism of P. aeruginosa in the context of its environmental and genetic constraints, thereby contributing to a more thorough understanding of the genotype-phenotype relationships in this resourceful and dangerous pathogen.

Loss-of-function mutations in the IL-21 receptor gene cause a primary immunodeficiency syndrome

A primary immunodeficiency syndrome caused by loss-of-function mutations in the IL-21 receptor exhibits impaired B, T, and NK cell function.

Reconciliation of genome-scale metabolic reconstructions for comparative systems analysis.

In the past decade, over 50 genome-scale metabolic reconstructions have been built for a variety of single- and multi- cellular organisms. These reconstructions have enabled a host of computational methods to be leveraged for systems-analysis of metabolism, leading to greater understanding of observed phenotypes. These methods have been sparsely applied to comparisons between multiple organisms, however, due mainly to the existence of differences between reconstructions that are inherited from the respective reconstruction processes of the organisms to be compared. To circumvent this obstacle, we developed a novel process, termed metabolic network reconciliation, whereby non-biological differences are removed from genome-scale reconstructions while keeping the reconstructions as true as possible to the underlying biological data on which they are based. This process was applied to two organisms of great importance to disease and biotechnological applications, Pseudomonas aeruginosa and Pseudomonas putida, respectively. The result is a pair of revised genome-scale reconstructions for these organisms that can be analyzed at a systems level with confidence that differences are indicative of true biological differences (to the degree that is currently known), rather than artifacts of the reconstruction process. The reconstructions were re-validated with various experimental data after reconciliation. With the reconciled and validated reconstructions, we performed a genome-wide comparison of metabolic flexibility between P. aeruginosa and P. putida that generated significant new insight into the underlying biology of these important organisms. Through this work, we provide a novel methodology for reconciling models, present new genome-scale reconstructions of P. aeruginosa and P. putida that can be directly compared at a network level, and perform a network-wide comparison of the two species. These reconstructions provide fresh insights into the metabolic similarities and differences between these important Pseudomonads, and pave the way towards full comparative analysis of genome-scale metabolic reconstructions of multiple species.

A congenital neutrophil defect syndrome associated with mutations in VPS45

BACKGROUND Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. METHODS We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. RESULTS All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the childs ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of β1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. CONCLUSIONS Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).

The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single-and multiple-nutrient-limited growth: Highlights from a multi-level omics approach

BackgroundPseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.ResultsWe found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system.ConclusionThis is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.

JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia.

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor–mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.

Dissecting the energy metabolism in Mycoplasma pneumoniae through genome-scale metabolic modeling

Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint‐based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time‐dependent changes, albeit using a static model. By performing an in silico knock‐out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.

In-vivo expression profiling of Pseudomonas aeruginosa infections reveals niche-specific and strain-independent transcriptional programs.

Pseudomonas aeruginosa is a threatening, opportunistic pathogen causing disease in immunocompromised individuals. The hallmark of P. aeruginosa virulence is its multi-factorial and combinatorial nature. It renders such bacteria infectious for many organisms and it is often resistant to antibiotics. To gain insights into the physiology of P. aeruginosa during infection, we assessed the transcriptional programs of three different P. aeruginosa strains directly after isolation from burn wounds of humans. We compared the programs to those of the same strains using two infection models: a plant model, which consisted of the infection of the midrib of lettuce leaves, and a murine tumor model, which was obtained by infection of mice with an induced tumor in the abdomen. All control conditions of P. aeruginosa cells growing in suspension and as a biofilm were added to the analysis. We found that these different P. aeruginosa strains express a pool of distinct genetic traits that are activated under particular infection conditions regardless of their genetic variability. The knowledge herein generated will advance our understanding of P. aeruginosa virulence and provide valuable cues for the definition of prospective targets to develop novel intervention strategies.

Very Early Onset Inflammatory Bowel Disease Associated with Aberrant Trafficking of IL-10R1 and Cure by T Cell Replete Haploidentical Bone Marrow Transplantation

PurposeLoss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT).MethodsClinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry.ResultsWe identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3′ splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient’s cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients.ConclusionsOur findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.