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Volume 14 Supplement 2

Sepsis 2010

Identification of immune modulators using a phage library displaying Staphylococcus aureus secreted proteins

Understanding the mechanisms of bacterial immune modulation will teach us more about pathogenesis of infection and could lead to new strategies in anti-inflammatory therapy during sepsis. Various approaches are already used to identify bacterial immune modulating proteins. However, these are inefficient and time consuming. Immune modulating proteins need to be secreted in order to act on their targets outside the bacterial cell. In the present study, phage display technology was used to specifically identify secreted immune modulating proteins with high efficacy.

Phage display technology is a technique to express a protein fused to a coat protein of a filamentous phage. The most widely used coat protein is pIII encoded in the phage genome by gIII. This gene contains a signal sequence that is essential for production of stable phage particles. When a bacterial genome is randomly fragmented and these fragments are inserted into a phage vector containing gIII lacking the signal sequence, intact phage particles are formed only when the inserted bacterial genomic fragment contains a signal sequence. This allows for selective expression of a bacterial secretome since secreted proteins also contain a signal sequence. The resulting secretome phage library can be used to select displayed proteins that specifically bind to various components of the immune system. This powerful technique has several advantages: it can be used for different Gram-positive and Gram-negative bacterial genomes. There is no need for extensive culturing of bacteria so it can be used for difficult or slow-growing bacteria. Expressed proteins are not hampered by solubility problems.

There is a direct relation between expressed protein and coding gene that allows for rapid identification of selected proteins. As a proof of principle, a Staphylococcus aureus library was constructed. The goal is to evaluate the proportion of previously described secreted immune modulators that can be recovered and to identify new immune modulators. In order to express most of the 300 secreted proteins encoded by this microorganism, the library reached a diversity of 108 clones. The secretome phage library was screened for interaction with leukocytes and for modulation of the coagulation and complement pathways, which are highly activated in sepsis process.

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Correspondence to C Fevre.

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Fevre, C., van Strijp, J. & Haas, PJ. Identification of immune modulators using a phage library displaying Staphylococcus aureus secreted proteins. Crit Care 14, P50 (2010). https://doi.org/10.1186/cc9153

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Keywords

  • Staphylococcus Aureus
  • Coat Protein
  • Signal Sequence
  • Bacterial Genome
  • Immune Modulate
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