You are currently browsing the monthly archive for January 2014.

Hey everyone,

For the upcoming session (21st of January, 8pm), we will be diving into bacterial metabolism and its regulation in Bacillus subtilis. I have chosen this paper because it is looking at the whole system and not only a small detail. The paper can be found here: http://onlinelibrary.wiley.com/doi/10.1038/msb.2013.66/full.

Title: Transcriptional regulation is insufficient to explain substrate-induced flux changes in Bacillus subtilis

Authors: Victor Chubukov,Markus Uhr, Ludovic Le Chat, Roelco J Kleijn, Matthieu Jules, Hannes Link, Stephane Aymerich, Jörg Stelling, Uwe Sauer

 

I have copied the abstract and the synopsis from the link here:

Abstract

One of the key ways in which microbes are thought to regulate their metabolism is by modulating the availability of enzymes through transcriptional regulation. However, the limited success of efforts to manipulate metabolic fluxes by rewiring the transcriptional network has cast doubt on the idea that transcript abundance controls metabolic fluxes. In this study, we investigate control of metabolic flux in the model bacterium Bacillus subtilis by quantifying fluxes, transcripts, and metabolites in eight metabolic states enforced by different environmental conditions. We find that most enzymes whose flux switches between on and off states, such as those involved in substrate uptake, exhibit large corresponding transcriptional changes. However, for the majority of enzymes in central metabolism, enzyme concentrations were insufficient to explain the observed fluxes—only for a number of reactions in the tricarboxylic acid cycle were enzyme changes approximately proportional to flux changes. Surprisingly, substrate changes revealed by metabolomics were also insufficient to explain observed fluxes, leaving a large role for allosteric regulation and enzyme modification in the control of metabolic fluxes.

Synopsis

Regulation of enzyme expression is one key mechanism by which cells control their metabolic programs. In this work, a quantitative analysis of metabolism in a model bacterium under different conditions shows that expression alone cannot explain the majority of the observed metabolic changes.

  • Most enzymes are indeed highly expressed in conditions where they are more active.
  • Quantitatively, however, the observed changes in expression between conditions do not match the changes in activity for most enzymes.
  • A good quantitative match is only observed for enzymes involved in the TCA cycle.
  • Metabolomics reveals that increased substrate availability explains only a few instances of changes in activity.

Discussion points

  • Was the paper clearly written?
  • Were the methods chosen appropriate?
  • Do the experiments show sufficient proof for the claim that transcriptional regulation is not sufficient to explain flux changes?
  • What experiments could be done next?

So, see you all on the 21st of January, 8 pm! Looking forward to it,

Janitensen

Advertisements

Hi everyone

The 1st #microtwjc of 2014 will take place on Tues 14th Jan at 8pm GMT and we will be looking at this paper

Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

Ya-Ping Ko, Annemarie Kuipers, Claudia M. Freitag, Ilse Jongerius, Eva Medina, Willemien J. van Rooijen, András N. Spaan, Kok P. M. van Kessel, Magnus Höök, Suzan H. M. Rooijakkers

Abstract

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureussecretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.

Author Summary

Staphylococcus aureus is a leading cause of severe bacterial infections in both hospital and community settings. Due to its increasing resistance to antibiotics, development of additional therapeutic strategies like vaccination is required to control this pathogen. Vaccination attempts against S. aureus have not been successful so far and an important reason may be the pathogen’s elaborate repertoire of molecules that dampen the immune response. These evasion molecules not only suppress natural immunity but also hamper the current attempts to create effective vaccines. In this paper, we describe a novel mechanism by which S. aureus can prevent uptake by phagocytic immune cells. We discover that the secreted S. aureusprotein Extracellular fibrinogen binding protein (Efb) generates a ‘capsule’-like shield around the bacterial surface through a dual interaction with the plasma proteins complement C3b and fibrinogen. The Efb-dependent fibrinogen shield masks important opsonic molecules like C3b and antibodies from binding to phagocyte receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of this anti-phagocytic shield.

Discussion points

  1. Was the paper clearly written, did everything make sense etc.?
  2. Were the methods appropriate?
  3. What do the results tell us and how important/significant are they?
  4. What work would you do next?

Looking forward to seeing you there

 

Zoo