Double Gene Deletion Research Articles

The investigation of distribution of hereditary alpha-thalassemia mutations in Isparta reservoir

Thalassemia is an autosomal recessive disease characterized by the absence or decreased production of the alpha globin chain. The aim of this study was to make the valuable contributions to the map of thalassemia in Turkey and to help the clinicians who plan to cure the patients in our region. Among the individuals who applied to Hematology Department of SDU Medical Faculty with a suspected alpha thalassemia mutation, 25 were selected for present study. Mutations in alpha globin gene were detected by using α-globin StripAssayTM commercial kit. The principle of the assay is based on reverse hybridization. As a result, the most common alpha thalassemi mutations were MED double gene deletion, -20.5 kb single gene deletion, -3.7 single gene deletion and α2IVS 1-5 nt respectively, in Isparta reservoir.

Cyclin Dependent Kinase Inhibitors Differentially Modulate Synergistic Cytokine Responsiveness of Hematopoietic Progenitor Cells

Cyclin dependent kinase inhibitors (CDKIs) influence proliferation of hematopoietic progenitor cells (HPCs), but little is known of how they influence proliferative responsiveness of HPCs to colony stimulating factors (CSFs), alone and in combination with other hematopoietically active factors, such as the potent co-stimulating cytokine stem cell factor (SCF), or inhibition by myelosuppressive chemokines. Using mice with deletions in p18(INK4c), p21(CIP1/WAF1), or p27(KIP1) genes, and in mice with double gene deletions for either p18/p21 or p18/p27, we determined effects of absence of these CDKIs and their interactions on functional HPC numbers in vivo, and HPC proliferative responsiveness in vitro. There is a decrease in bone marrow HPC proliferation in p18(-/-) mice commensurate with decreased numbers of HPC, suggesting a positive role for p18 on HPC in vivo, similar to that for p21. These positive effects of p18 dominate negative effects of p27 gene deletion. Moreover, the CDKIs differentially regulate responsiveness of granulocyte macrophage (GM) progenitors to synergistic cell proliferation in response to GM-CSF plus SCF, which is considered important for normal hematopoiesis. Responsiveness of HPCs to inhibition by myelosuppressive chemokines is directly related to the capacity of HPCs to respond to synergistic stimulation, and their cell cycle status. P18(INK4c) gene deletion rescued the loss of chemokine suppression of synergistic proliferation due to deletion of p21(CIP1/WAF1). These findings underscore the complex interplay of cell cycle regulators in HPC, and demonstrate that loss of one can sometimes be compensated by loss of another CDKI in both, a pro- or anti-proliferative context.

A Non-Marker Mutagenesis Strategy to Generate Poly- hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzicola (Xoc), the critical pathogen causing bacterial leaf streak in rice, possesses a hrp cluster that is responsible for triggering hypersensitive response (HR) in non-host tobacco and pathogenicity in host rice, and is considered to be one of the model pathogens in the rice model plant. Here, we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1. It was used to generate single or poly-gene mutants of hpa1, hpa2, hrcV, hrpE, hpaB, and hrpF gene for functional analysis. In total, five single, four double, and two triple hrp gene mutants were constructed. The double and triple hrp gene deletion mutants triggered novel phenotypes in planta. Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

Quantitative epistasis analysis and pathway inference from genetic interaction data.

Inferring regulatory and metabolic network models from quantitative genetic interaction data remains a major challenge in systems biology. Here, we present a novel quantitative model for interpreting epistasis within pathways responding to an external signal. The model provides the basis of an experimental method to determine the architecture of such pathways, and establishes a new set of rules to infer the order of genes within them. The method also allows the extraction of quantitative parameters enabling a new level of information to be added to genetic network models. It is applicable to any system where the impact of combinatorial loss-of-function mutations can be quantified with sufficient accuracy. We test the method by conducting a systematic analysis of a thoroughly characterized eukaryotic gene network, the galactose utilization pathway in Saccharomyces cerevisiae. For this purpose, we quantify the effects of single and double gene deletions on two phenotypic traits, fitness and reporter gene expression. We show that applying our method to fitness traits reveals the order of metabolic enzymes and the effects of accumulating metabolic intermediates. Conversely, the analysis of expression traits reveals the order of transcriptional regulatory genes, secondary regulatory signals and their relative strength. Strikingly, when the analyses of the two traits are combined, the method correctly infers ∼80% of the known relationships without any false positives.

The Essential Ubc4/Ubc5 Function in Yeast Is HECT E3-dependent, and RING E3-dependent Pathways Require Only Monoubiquitin Transfer by Ubc4

The ubiquitin (Ub)-conjugating enzymes Ubc4 and Ubc5 are involved in a variety of ubiquitination pathways in yeast, including Rsp5- and anaphase-promoting complex (APC)-mediated pathways. We have found the double deletion of UBC4 and UBC5 genes in yeast to be lethal. To investigate the essential pathway disrupted by the ubc4/ubc5 deletion, several point mutations were inserted in Ubc4. The Ubc4 active site mutation C86A and the E3-binding mutations A97D and F63A were both unable to rescue the lethal phenotype, indicating that an active E3/E2∼Ub complex is required for the essential function of Ubc4/Ubc5. A mutation that specifically eliminates RING E3-catalyzed isopeptide formation but not HECT E3 transthiolation (N78S-Ubc4) rescued the lethal phenotype. Thus, the essential redundant function performed by Ubc4 and Ubc5 in yeast is with a HECT-type E3, likely the only essential HECT in yeast, Rsp5. Our results also suggest that Ubc1 can weakly replace Ubc4 to transfer mono-Ub with APC, but Ubc4 cannot replace Ubc1 for poly-Ub chain extension on APC substrates. Finally, the backside Ub-binding mutant S23R-Ubc4 has no observable effect in yeast. Together, our results are consistent with a model in which Ubc4 and Ubc5 are 1) the primary E2s for Rsp5 in yeast and 2) act as monoubiquitinating E2s in RING E3-catalyzed pathways, in contrast to the processive human ortholog UbcH5.

Sequential deletion of Pichia pastoris genes by a self-excisable cassette

A rapid and convenient method is presented for unmarked gene deletions in Pichia pastoris. Cre/mutated lox system, Zeocin(®) (Invitrogen) resistance marker and homologous arms were spliced together by fusion PCR to generate the gene disruption cassettes (homologous region-lox71-Cre-ZeoR-lox66-homologous region), which could be integrated into the P. pastoris genome via homologous recombination. After transferring double-cross-over recombinants to methanol induction medium, transient expression of Cre recombinase caused the recombination of lox71-Cre-ZeoR-lox66 fragment into a double-mutant lox72 site, thus excising the Cre-ZeoR cassette from the P. pastoris genome. As the double-mutant lox72 site displays strongly reduced binding affinity for Cre recombinase, this method could be used sequentially to disrupt P. pastoris genes without introducing selectable markers. The effectiveness of this strategy was verified by introducing both single and double gene deletions into the P. pastoris genome.

Application of Cre-loxP(*) system in constructing the markerless double-gene-deletion strain in Streptococcus mutans

To construct double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans (Sm) and to remove the antibiotic resistance markers with the Cre-loxP(*) site-specific recombination system. The htrA gene was cloned into the pGEM-T-Easy TA cloning vector and then inactivated via the insertion of a kanamycin resistance cassette (lox71-Km-lox66), yielding pGEM-T-ΔhtrA/Km for deleting the htrA gene. Using the same method, the pGEM-T-ΔclpP/Sp was constructed for deleting the clpP gene. Following the transformation of pGEM-T-ΔhtrA/Km in Sm, the homologous recombination event was selected. One such mutant was transformed with a cre expression plasmid (pCrePA). The kanamycin resistance gene was then excised. The pCrePA was then easily eliminated at nonpermissive temperatures, resulting in a mutant strain (MSΔhtrA) carrying a deletion at the htrA loci without a selectable marker. This mutant was verified by PCR and DNA sequencing. Then, the clpP and spectinomycin resistance gene were deleted from MSΔhtrA, yielding markerless mutant strain lacking clpP and htrA. The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing. A mutant of Sm was constructed successfully which contained a deletion of the htrA and clpP gene without selectable marker. The Cre-loxP(*) system can be applied to Sm, which provides experimental evidence for generating markerless multiple gene deletion mutants.

Stoichiometric network reconstruction and analysis of yeast sphingolipid metabolism incorporating different states of hydroxylation

The first elaborate metabolic model of Saccharomyces cerevisiae sphingolipid metabolism was reconstructed in silico. The model considers five different states of sphingolipid hydroxylation, rendering it unique among other models. It is aimed to clarify the significance of hydroxylation on sphingolipids and hence to interpret the preferences of the cell between different metabolic pathway branches under different stress conditions. The newly constructed model was validated by single, double and triple gene deletions with experimentally verified phenotypes. Calcium sensitivity and deletion mutations that may suppress calcium sensitivity were examined by CSG1 and CSG2 related deletions. The model enabled the analysis of complex sphingolipid content of the plasma membrane coupled with diacylglycerol and phosphatidic acid biosynthesis and ATP consumption in in silico cell. The flux data belonging to these critically important key metabolites are integrated with the fact of phytoceramide induced cell death to propose novel potential drug targets for cancer therapeutics. In conclusion, we propose that IPT1, GDA1, CSG and AUR1 gene deletions may be novel candidates of drug targets for cancer therapy according to the results of flux balance and variability analyses coupled with robustness analysis.

Development and analysis of an in vivo-compatible metabolic network of Mycobacterium tuberculosis

BackgroundDuring infection, Mycobacterium tuberculosis confronts a generally hostile and nutrient-poor in vivo host environment. Existing models and analyses of M. tuberculosis metabolic networks are able to reproduce experimentally measured cellular growth rates and identify genes required for growth in a range of different in vitro media. However, these models, under in vitro conditions, do not provide an adequate description of the metabolic processes required by the pathogen to infect and persist in a host.ResultsTo better account for the metabolic activity of M. tuberculosis in the host environment, we developed a set of procedures to systematically modify an existing in vitro metabolic network by enhancing the agreement between calculated and in vivo-measured gene essentiality data. After our modifications, the new in vivo network contained 663 genes, 838 metabolites, and 1,049 reactions and had a significantly increased sensitivity (0.81) in predicted gene essentiality than the in vitro network (0.31). We verified the modifications generated from the purely computational analysis through a review of the literature and found, for example, that, as the analysis suggested, lipids are used as the main source for carbon metabolism and oxygen must be available for the pathogen under in vivo conditions. Moreover, we used the developed in vivo network to predict the effects of double-gene deletions on M. tuberculosis growth in the host environment, explore metabolic adaptations to life in an acidic environment, highlight the importance of different enzymes in the tricarboxylic acid-cycle under different limiting nutrient conditions, investigate the effects of inhibiting multiple reactions, and look at the importance of both aerobic and anaerobic cellular respiration during infection.ConclusionsThe network modifications we implemented suggest a distinctive set of metabolic conditions and requirements faced by M. tuberculosis during host infection compared with in vitro growth. Likewise, the double-gene deletion calculations highlight the importance of specific metabolic pathways used by the pathogen in the host environment. The newly constructed network provides a quantitative model to study the metabolism and associated drug targets of M. tuberculosis under in vivo conditions.

Prediction of quantitative phenotypes based on genetic networks: a case study in yeast sporulation

BackgroundAn exciting application of genetic network is to predict phenotypic consequences for environmental cues or genetic perturbations. However, de novo prediction for quantitative phenotypes based on network topology is always a challenging task.ResultsUsing yeast sporulation as a model system, we have assembled a genetic network from literature and exploited Boolean network to predict sporulation efficiency change upon deleting individual genes. We observe that predictions based on the curated network correlate well with the experimentally measured values. In addition, computational analysis reveals the robustness and hysteresis of the yeast sporulation network and uncovers several patterns of sporulation efficiency change caused by double gene deletion. These discoveries may guide future investigation of underlying mechanisms. We have also shown that a hybridized genetic network reconstructed from both temporal microarray data and literature is able to achieve a satisfactory prediction accuracy of the same quantitative phenotypes.ConclusionsThis case study illustrates the value of predicting quantitative phenotypes based on genetic network and provides a generic approach.

Simplified PGD of common determinants of haemoglobin Bart’s hydrops fetalis syndrome using multiplex-microsatellite PCR

The high incidence of double-gene deletions in α-thalassaemia increases the risk of having pregnancies with homozygous α(0)-thalassaemia, the cause of the lethal haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. However, the current gap-PCR based PGD protocol for deletional α-thalassaemia requires specific primer design for each specific deletion. A universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome has been developed. Microsatellite markers 16PTEL05 and 16PTEL06 within the α-globin gene cluster were co-amplified with a third microsatellite marker outside the affected region in a multiplex-PCR reaction and analysed by capillary electrophoresis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. A total of 47 embryos were analysed. Three pregnancies were achieved from three couples, with the births of two healthy babies and one ongoing pregnancy. This work has successfully adapted an earlier protocol and developed a simple and reliable single-cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of type of deletion. Alpha-thalassaemia is one of the most common inheritable disorders worldwide. It is a blood disorder that, in its lethal form caused by deletion of all four copies of the α-globin gene, results in the demise of the affected fetus, a condition referred to as haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. Current PGD protocols for deletional α-thalassaemia utilize a strategy called gap-PCR, which requires the different assays for different deletion types. We have developed a universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome based on microsatellite marker analysis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. Forty-five embryos were analysed in total. Three pregnancies were achieved from three couples, with the births of two healthy babies and one pregnancy still ongoing. We have successfully adapted our earlier protocol and developed a simple and reliable single cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of the type of deletion.

BioMet Toolbox: genome-wide analysis of metabolism

The rapid progress of molecular biology tools for directed genetic modifications, accurate quantitative experimental approaches, high-throughput measurements, together with development of genome sequencing has made the foundation for a new area of metabolic engineering that is driven by metabolic models. Systematic analysis of biological processes by means of modelling and simulations has made the identification of metabolic networks and prediction of metabolic capabilities under different conditions possible. For facilitating such systemic analysis, we have developed the BioMet Toolbox, a web-based resource for stoichiometric analysis and for integration of transcriptome and interactome data, thereby exploiting the capabilities of genome-scale metabolic models. The BioMet Toolbox provides an effective user-friendly way to perform linear programming simulations towards maximized or minimized growth rates, substrate uptake rates and metabolic production rates by detecting relevant fluxes, simulate single and double gene deletions or detect metabolites around which major transcriptional changes are concentrated. These tools can be used for high-throughput in silico screening and allows fully standardized simulations. Model files for various model organisms (fungi and bacteria) are included. Overall, the BioMet Toolbox serves as a valuable resource for exploring the capabilities of these metabolic networks. BioMet Toolbox is freely available at www.sysbio.se/BioMet/.

Association of G6PD −202/−376 with Lower Hemoglobin Concentration but Not Increased Hemolysis in Patients with Sickle Cell Anemia.

Abstract 1511Poster Board I-534 BackgroundWhat determines the degree of hemolysis and of anemia in patients with hemoglobin SS is not fully known. The rate of hemolysis and severity of anemia are ameliorated by the presence of alpha thalassemia and by higher hemoglobin F percentage. Mild G6PD deficiency in the form of G6PD-202/-376 may be associated with episodic hemolysis in individuals of African descent, but past studies indicated little influence of G6PD-202/-376 on the degree of hemolysis and anemia in sickle cell disease patients (1,2). In this study we examined the roles of single and double α-globin deletions and G6PD-202/-376 on the degree of hemolysis and the hemoglobin concentration in hemoglobin SS patients. MethodsTwo hundred sixty two children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Principal component analysis was used to develop a hemolytic component from concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of α-thalassemia and G6PD-202/-376. Multivariate models were employed to determine the independent effects of these genotypes on hemoglobin concentration and degree of hemolysis. ResultsSingle a-globin deletion was associated with an estimated 0.4 g/dL increase in steady-state hemoglobin concentration and double α-globin gene deletion with a 0.8 g/dL increase (P = 0.005 for trend) due to, progressively lower degrees of hemolysis (P = 0.004). G6PD-202/-376 was associated with an estimated 0.7 g/dL decrease in the hemoglobin concentration (P = 0.003) (Figure 1a), but this observation could not be explained by increased hemolysis. Rather, the reticulocyte count was an estimated 22% lower with G6PD-202/-376 (P = 0.032) (Figure 1b). DiscussionG6PD-202/-376 may be associated with lower hemoglobin concentration in sickle cell anemia and the mechanism is probably impaired erythropoiesis rather than hemolysis. A recent study (3) indicates that G6PD is needed for definitive erythropoiesis as well as for normal survival of red blood cells in the periphery. Our present findings raise the possibility that, in the setting of the markedly increased erythropoiesis of sickle cell anemia, G6PD-202/-376 may result in impairment in erythropoiesis that is discernible in the peripheral blood hemoglobin concentration. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Frequency of Positive XmnIGγ polymorphism and coinheritance of common alpha thalassemia mutations do not show statistically significant difference between thalassemia major and intermedia cases with homozygous IVSII-1 mutation

From 362 thalassemia cases referred to adult thalassemia clinic of the Iranian blood transfusion organization (IBTO) for genotyping, 103 cases (28.5%) had a common primary disease factor, IVSII-1 mutation in homozygous state. 61 (59.2%) of these individuals represented thalassemia major and 42 (40.8%) thalassemia intermedia clinical phenotype. To re-evaluate our current diagnostic criteria, XmnI Gγ polymorphism and coexistence of alpha thalassemia mutations, frequently recalled as important factors modifying the clinical phenotype of homozygous beta zero thalassemia cases in our country, were examined in both groups. No statistically significant difference in the frequency of positive XmnI Gγ polymorphism was observed between thalassemia intermedia and thalassemia major patients. Double gene deletion -- Med was observed in only one thalassemia major case, while − a 3.7 in heterozygous state (− a 3.7/aa) was identified in 6 (9.8%) of thalassemia major and 8 (19%) of thalassemia intermedia patients. − a 4.2 was observed in only one thalassemia major case. No statistically significant difference in the frequency of coinheritance of alpha thalassemia was observed between the two groups. These results imply that other interacting mechanisms which modify the phenotype of thalassemia patients is still in the dark in our current diagnostic criteria of thalassemia.

Mutations and Lethality in Simulated Prebiotic Networks

The Graded Autocatalysis Replication Domain (GARD) model describes an origin of life scenario which involves non-covalent compositional assemblies, made of monomeric mutually catalytic molecules. GARD constitutes an alternative to informational biopolymers as a mechanism of primordial inheritance. In the present work, we examined the effect of mutations, one of the most fundamental mechanisms for evolution, in the context of the networks of mutual interaction within GARD prebiotic assemblies. We performed a systematic analysis analogous to single and double gene deletions within GARD. While most deletions have only a small effect on both growth rate and molecular composition of the assemblies, ~10% of the deletions caused lethality, or sometimes showed enhanced fitness. Analysis of 14 different network properties on 2,000 different GARD networks indicated that lethality usually takes place when the deleted node has a high molecular count, or when it is a catalyst for such node. A correlation was also found between lethality and node degree centrality, similar to what is seen in real biological networks. Addressing double knockout mutations, our results demonstrate the occurrence of both synthetic lethality and extragenic suppression within GARD networks, and convey an attempt to correlate synthetic lethality to network node-pair properties. The analyses presented help establish GARD as a workable alternative prebiotic scenario, suggesting that life may have begun with large molecular networks of low fidelity, that later underwent evolutionary compaction and fidelity augmentation.

Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.

A Severe α Thalassemia Case Compound Heterozygous for Hb Adana in α1 Gene and 20.5 kb Double Gene Deletion

We report a 6-year-old boy diagnosed as transfusion dependent chronic nonspherocytic hemolytic anemia since 40 days old. Hemoglobin H inclusions were detected with brilliant cresyl blue preparation. His parents were found to be normal on physical examination. His mother had hemoglobin level of 9.34 g/dL, accompanied by typical thalassemic changes of the red cells, and inclusion bodies were also detected with brilliant cresyl blue staining. His father had normal hemoglobin level and borderline red cell indices. Mutation analysis using strip assay capable of detecting 22 mutations within the alpha genes was performed for the proband and the parents which revealed that the case was compound heterozygous for Hb Adana in alpha1 gene and 20.5 kb double gene deletion. The father was found to be heterozygous for Hb Adana alpha1 gene whereas the mother was found to be compound heterozygous for 20.5 kb double gene deletion and 3.7 kb single gene deletion. It is well known that non deletional forms of HbH disease are more severe than the deletional forms. This case represents another example of the nondeletional mutation underlying Hb Adana, which is rarely seen in alpha1 gene, and illustrates the distinctive phenotypes of both the deletional and nondeletional forms of hemoglobin H disease within the same family.

Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering

With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief hurdle for the generation of efficient production platforms. As such, we employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. Specifically, two different model objectives were used to identify possible genotypes that exhibited either improved overall NADPH production or an improved flux through an artificial reaction coupling NADPH yield to biomass. The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin. Each is derived from their common precursor dihydroquercetin using two recombinant NADPH-dependent enzymes: dihydroflavonol 4-reductase and leucoanthocyanidin reductase. The best engineered strain carrying Δ pgi, Δ ppc and Δ pldA deletions accumulated up to 817 mg/L of leucocyanidin and 39 mg/L (+)-catechin in batch culture with 10 g/L glucose in modified M9 medium, a 4-fold and 2-fold increase, respectively, compared to the wild-type control.

Comparative Genome-Scale Metabolic Reconstruction and Flux Balance Analysis of Multiple Staphylococcus aureus Genomes Identify Novel Antimicrobial Drug Targets

Mortality due to multidrug-resistant Staphylococcus aureus infection is predicted to surpass that of human immunodeficiency virus/AIDS in the United States. Despite the various treatment options for S. aureus infections, it remains a major hospital- and community-acquired opportunistic pathogen. With the emergence of multidrug-resistant S. aureus strains, there is an urgent need for the discovery of new antimicrobial drug targets in the organism. To this end, we reconstructed the metabolic networks of multidrug-resistant S. aureus strains using genome annotation, functional-pathway analysis, and comparative genomic approaches, followed by flux balance analysis-based in silico single and double gene deletion experiments. We identified 70 single enzymes and 54 pairs of enzymes whose corresponding metabolic reactions are predicted to be unconditionally essential for growth. Of these, 44 single enzymes and 10 enzyme pairs proved to be common to all 13 S. aureus strains, including many that had not been previously identified as being essential for growth by gene deletion experiments in S. aureus. We thus conclude that metabolic reconstruction and in silico analyses of multiple strains of the same bacterial species provide a novel approach for potential antibiotic target identification.

Redundancy in the requirement for the glycolytic enzymes phosphofructokinase (Pfk) 1 and 2 in the in vivo fitness of Salmonella enterica serovar Typhimurium

To assess the role of the glycolytic enzyme phosphofructokinase (Pfk) in the in vivo fitness of the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) we have generated single and double gene deletion mutants of the two known isoforms of this enzyme, pfkA and pfkB. In a mouse model of typhoid fever, bacterial counts in the spleen and liver were similar between wild type and single pfkA and pfkB mutant-infected mice. However, a double pfkAB mutant was significantly attenuated for growth in vivo. This defect was complemented by provision of either pfkA or pfkB on pBR322. Together these data show that Pfk activity is required for the full in vivo fitness of S. Typhimurium with functional redundancy between pfkA and pfkB. The level of attenuation of the pfkAB double mutant was not sufficient for its consideration as a live attenuated vaccine strain.