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Team:Grinnell/Project/Esp
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| + | Esp is a type of serine protease, secreted by ''Staphylococcus epidermidis'', a non-pathogenic bacterium that is usually found in human nasal and oral cavities. ''S. epidermis'' is closely related to ''Staphylococcus aureus'', a bacterium found on the skin and in the nasal passages of up to 25% of healthy people and animals. Some strains of ''S. aureus'' cause illness by producing a heat stable toxin and it is difficult to get rid of because it can form a biofilm. | ||
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| + | Moon et al. first isolated and biochemically characterized the Esp in 2001, discovering this “novel extracellular protease from ''S. epidermidis''” can degrade fibrinogen, complement protein C5, and several other proteins with a strong preference for cleavage after glutamic acid residues. This mechanism was claimed, therefore, to enable ''S. epidermidis'' to evade the complement defense system. As a matter of fact, this claim was supported by another group of scientists (Moon et al. 2001). Dubin et al. noticed that the gene structure of ''esp'' and the amino acid sequence of its mature form showed close similarity with one of the ''S. aureus'' serine proteases, which have been long regarded as important virulence factors that destroy tissue and blood proteins and thus contribute to enhanced invasiveness (2001). | ||
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| + | However, Iwase et al. found out Esp, in reality, hindered ''S. aureus'' colonization and infection by inhibiting biofilm formation. Although the mechanism of how Esp breaks the ''S. aureus'' biofilm is still unknown and does not resemble any bacterial interference mechanisms such as growth inhibition and bactericidal activity, Esp was observed to degrade ''S. aureus'' biofilm by changing ''S. aureus'' from the sessile to the planktonic form. Sixteen hours incubation with supernatant containing Esp resulted in an astonishing four-fold decrease of ''S. aureus'' biofilm. One thing that distinguishes Esp from the other biofilm breakers is the fact that Esp does not break biofilm by killing the bacteria that are in the biofilm, but rather releasing the bacteria from the biofilm. Therefore, it will be hard for ''S. aureus'' to build resistance to Esp (Iwase et al. 2010) | ||
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| + | We tested two versions of the ''esp'' gene, the wildtype gene and a version with codon sequence optimized for expression in ''Caulobacter crescentus''. The wildtype gene was acquired by PCR amplification of the S''. epidermis'' genome using primers designed for the ''esp'' gene. Because the ''esp'' gene only has a G/C content of around 30% while our model organism ''Caulobacter crecentus'' has a G/C rich genome (60%) and presumably expresses G/C rich DNA more accurately and efficiently, we decided to have the coding sequence optimized for expression in Caulobacter without any changes to the amino acids sequence. This gene was synthesized by Integrated DNA Technologies. BioBrick part BBa_K531003 and Bba_K531006. | ||
Latest revision as of 03:50, 29 September 2011
Esp
Esp is a type of serine protease, secreted by Staphylococcus epidermidis, a non-pathogenic bacterium that is usually found in human nasal and oral cavities. S. epidermis is closely related to Staphylococcus aureus, a bacterium found on the skin and in the nasal passages of up to 25% of healthy people and animals. Some strains of S. aureus cause illness by producing a heat stable toxin and it is difficult to get rid of because it can form a biofilm.
Moon et al. first isolated and biochemically characterized the Esp in 2001, discovering this “novel extracellular protease from S. epidermidis” can degrade fibrinogen, complement protein C5, and several other proteins with a strong preference for cleavage after glutamic acid residues. This mechanism was claimed, therefore, to enable S. epidermidis to evade the complement defense system. As a matter of fact, this claim was supported by another group of scientists (Moon et al. 2001). Dubin et al. noticed that the gene structure of esp and the amino acid sequence of its mature form showed close similarity with one of the S. aureus serine proteases, which have been long regarded as important virulence factors that destroy tissue and blood proteins and thus contribute to enhanced invasiveness (2001).
However, Iwase et al. found out Esp, in reality, hindered S. aureus colonization and infection by inhibiting biofilm formation. Although the mechanism of how Esp breaks the S. aureus biofilm is still unknown and does not resemble any bacterial interference mechanisms such as growth inhibition and bactericidal activity, Esp was observed to degrade S. aureus biofilm by changing S. aureus from the sessile to the planktonic form. Sixteen hours incubation with supernatant containing Esp resulted in an astonishing four-fold decrease of S. aureus biofilm. One thing that distinguishes Esp from the other biofilm breakers is the fact that Esp does not break biofilm by killing the bacteria that are in the biofilm, but rather releasing the bacteria from the biofilm. Therefore, it will be hard for S. aureus to build resistance to Esp (Iwase et al. 2010)
We tested two versions of the esp gene, the wildtype gene and a version with codon sequence optimized for expression in Caulobacter crescentus. The wildtype gene was acquired by PCR amplification of the S. epidermis genome using primers designed for the esp gene. Because the esp gene only has a G/C content of around 30% while our model organism Caulobacter crecentus has a G/C rich genome (60%) and presumably expresses G/C rich DNA more accurately and efficiently, we decided to have the coding sequence optimized for expression in Caulobacter without any changes to the amino acids sequence. This gene was synthesized by Integrated DNA Technologies. BioBrick part BBa_K531003 and Bba_K531006.
