Dataset Information

Metabolic investigation of host/pathogen interaction using MS2-infected Escherichia coli.

ABSTRACT:

Background

RNA viruses are responsible for a variety of illnesses among people, including but not limited to the common cold, the flu, HIV, and ebola. Developing new drugs and new strategies for treating diseases caused by these viruses can be an expensive and time-consuming process. Mathematical modeling may be used to elucidate host-pathogen interactions and highlight potential targets for drug development, as well providing the basis for optimizing patient treatment strategies. The purpose of this work was to determine whether a genome-scale modeling approach could be used to understand how metabolism is impacted by the host-pathogen interaction during a viral infection. Escherichia coli/MS2 was used as the host-pathogen model system as MS2 is easy to work with, harmless to humans, but shares many features with eukaryotic viruses. In addition, the genome-scale metabolic model of E. coli is the most comprehensive model at this time.

Results

Employing a metabolic modeling strategy known as "flux balance analysis" coupled with experimental studies, we were able to predict how viral infection would alter bacterial metabolism. Based on our simulations, we predicted that cell growth and biosynthesis of the cell wall would be halted. Furthermore, we predicted a substantial increase in metabolic activity of the pentose phosphate pathway as a means to enhance viral biosynthesis, while a break down in the citric acid cycle was predicted. Also, no changes were predicted in the glycolytic pathway.

Conclusions

Through our approach, we have developed a technique of modeling virus-infected host metabolism and have investigated the metabolic effects of viral infection. These studies may provide insight into how to design better drugs. They also illustrate the potential of extending such metabolic analysis to higher order organisms, including humans.

SUBMITTER: Jain R

PROVIDER: S-EPMC2813233 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Publications

Metabolic investigation of host/pathogen interaction using MS2-infected Escherichia coli.

Jain Rishi R Srivastava Ranjan R

BMC systems biology 20091230

<h4>Background</h4>RNA viruses are responsible for a variety of illnesses among people, including but not limited to the common cold, the flu, HIV, and ebola. Developing new drugs and new strategies for treating diseases caused by these viruses can be an expensive and time-consuming process. Mathematical modeling may be used to elucidate host-pathogen interactions and highlight potential targets for drug development, as well providing the basis for optimizing patient treatment strategies. The pu ...[more]

PMID: 20042079

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Project description:The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to Llysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJP330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJP330Q mutant was recessive and could be suppressed by Lovercomes dnaJ (Lodj ) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ?20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ?X174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions.IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

Project description:BackgroundBurkholderia pseudomallei is an intracellular bacteria causing Melioidosis, the disease widely disseminates in Southeast Asia and Northern Australia. B. pseudomallei has ability to invade various types of host cell and to interfere with host defense mechanisms, such as nitric oxide (NO). Due to the cross-talk among alternative killing mechanisms in host immune response against invading microbes, autophagy is the molecular mechanism belonging to intracellular elimination of eukaryotic cells that has been widely discussed. However, bacterial evasion strategy of B. pseudomallei and host-bacterial protein-protein interaction within autophagic machinery remain unknown.MethodsHere, we demonstrated the protein-protein interaction study between different strains of B. pseudomallei, including wild type PP844 and rpoS mutant, with autophagy-related protein LC3 that has been constructed, using the modified immunoaffinity hydrophobic chromatography based-technique. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was utilized for identifying the eluted proteins obtained from the established column. In addition, the expression level of gene encoding candidate protein was predicted prior to verification using real-time quantitative reverse transcription PCR assay (RT-qPCR).ResultsLC3 recombinant proteins could be entrapped inside the column before encountering their bacterial interacting partners. Based on affinity interaction, the binding capacity of LC3 with antibody displayed over 50% readily for hydrophobically binding with bacterial proteins. Following protein identification, bacterial ATP-binding cassette (ABC) transporter periplasmic substrate-binding protein (BPSL2203) was identified as a candidate LC3-interacting protein, which was found only in B. pseudomallei wild type. Gene expression analysis and bioinformatics of BPSL2203 were validated the proteomic result which are suggesting the role of RpoS-dependent gene regulation.ConclusionsRemarkably, utilization of the modified immunoaffinity hydrophobic chromatography with LC-MS/MS is a convenient and reliable approach to a study in B. pseudomallei-LC3 protein-protein interaction.

Dataset Information

Metabolic investigation of host/pathogen interaction using MS2-infected Escherichia coli.

Background

Results

Conclusions

Publications

Metabolic investigation of host/pathogen interaction using MS2-infected Escherichia coli.

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