Difference between revisions of "Part:BBa K1659000"
Line 2: Line 2:
  
  
−
This part contains the sequence for the ''Pseudomonas''-selective microbial lysis protein Art-175.
+
This part contains the sequence for the antimicrobial protein Art-175.
  
−
We have also made several parts with Art-175 fused to different secretion signal sequences:
+
Our Art-175 part collection comes in a family of four parts, one being Art-175 by itself and three others being Art-175 fused to different secretion signal sequences:
  
  
Line 38: Line 38:
 
Artilysins are an exciting class of enzyme-based antibacterials. Their name is derived from "artificial endolysin" and they exploit the lytic power of bacteriophage-encoded endolyins. Endolysins are peptidoglycan hydrolases produced at the end of the lytic cycle that pass through the cytoplasmic membrane, degrade the peptidoglycan layer and cause the osmotic lysis of the infected bacterial cell, thus liberating the progeny. Endolysins have a degree of specificity in terms of of the peptidoglycan chemotype which they can break down by means of the structural selectivity of their enzymatically-active domain (EAD) or cell wall binding domain (CBD).
 
Artilysins are an exciting class of enzyme-based antibacterials. Their name is derived from "artificial endolysin" and they exploit the lytic power of bacteriophage-encoded endolyins. Endolysins are peptidoglycan hydrolases produced at the end of the lytic cycle that pass through the cytoplasmic membrane, degrade the peptidoglycan layer and cause the osmotic lysis of the infected bacterial cell, thus liberating the progeny. Endolysins have a degree of specificity in terms of of the peptidoglycan chemotype which they can break down by means of the structural selectivity of their enzymatically-active domain (EAD) or cell wall binding domain (CBD).
  
−
Purified endolysins have been used to kill Gram-positive pathogens. Gram-negative bacteria, however, have a protective outer membrane containing lipopolysaccharide (LPS) that serves as a barrier against the peptidoglycan hydrolytic activity of endolysins from the outside. To overcome this problem, selected polycationic or amphipathic peptides that locally destabilize the LPS layer can be covalently fused to endolysins to transport them past the outer membrane to reach the peptidoglycan layer.  
+
Purified endolysins have been used to kill Gram-positive pathogens. Gram-negative bacteria, however, have a protective outer membrane containing lipopolysaccharide (LPS) that serves as a barrier against the peptidoglycan hydrolytic activity of endolysins from the outside. To overcome this problem, selected polycationic or amphipathic peptides that locally destabilize the LPS layer can be covalently fused to endolysins to transport them past the outer membrane to reach the peptidoglycan layer.
  
−
Biers et al. fused the sheep myeloid antimicrobial peptide (SMAP-29), which introduces transient cracks in the outer membrane by means of interaction with cationic binding sites combined with hydrophobic disruption of barrier function, to the N-terminus of the endolysin KZ144 to create Artilysin Art-175. Art-175 has been shown to be a highly potent antibacterial against pathogenic ''P. aeruginosa'' strains PAO1 and PA14, being able to kill even persister cells effectively as it does not require active bacterial metabolism to exert its lytic activity [1].
+
Biers et al. fused the sheep myeloid antimicrobial peptide SMAP-29 to the N-terminus of the endolysin KZ144 to create Artilysin Art-175 [1]. Endolysin KZ144 has previously been shown to selectively exert cell wall lytic activity on the peptidoglycan chemotype A1γ (which Gram-negative bacteria such as ''P. aeruginosa'', ''E. coli'', and ''Salmonella typhimurium'' belong to), where the bacterial outer membranes have already been separately permeabilized, by targeting the fully N-acetylated glucosamine units present in that peptidoglycan chemotype [2]. On the other hand, SMAP-29 on its own exhibits broad antimicrobial activity by means of using its N-terminal ampiphathic α-helical region in conjunction with its C-terminal hydrophobic region to disrupt of the outer and inner membranes of bacteria. SMAP-29 on its own, however, is unsuitable for clinical applications because it is also hemolytic towards human erythrocytes [3][4].
 +
 
 +
Art-175, the product of their linkage, is able to use its SMAP-29 moiety to transport itself past the bacterial outer membrane and exert lytic activity on the peptidoglycan layer. It exhibits strong antibacterial activity against pathogenic ''P. aeruginosa'' strains PAO1 and PA14, being able to kill even persister cells effectively as it does not require active bacterial metabolism to exert its lytic activity. However, unlike SMAP-29 by itself, Art-175 cannot kill bacteria using SMAP-29's cell membrane disruption mechanism, and owing to the chemotype selectivity of KZ144 is hence ineffective against bacteria of other peptidoglycan chemotypes, such as ''S. aureus'' [1].
  
  
 
===Usage===
 
===Usage===
  
−
By transforming this coding sequence in a commercial pBAD expression vector into our host organisms of choice, ''E. coli'' MG1655, ''E. coli'' RP437 ∆FliC, and ''E. coli'' DH5α, we hope to create an optimized strain of artilysin-producing ''E. coli'' from which we can purify Art-175 following cell lysis. We wish to compare unmodified Art-175 against its secretion-tagged cogeners which we have designed both in terms of antimicrobial activity as well as level of expression.
+
We aim to test this part for antibacterial activity by means of protein purification from host cell lysate after induction of gene expression. The antibacterial potency of this part will serve as a standard which we will compare our secretion-tagged modified Art-175 parts against.
 +
 
  
  
Line 51: Line 54:
  
 
[1] Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J. P., Defraine, V., … Lavigne, R. (2014). Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 58(7), 3774–3784. http://doi.org/10.1128/AAC.02668-14
 
[1] Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J. P., Defraine, V., … Lavigne, R. (2014). Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 58(7), 3774–3784. http://doi.org/10.1128/AAC.02668-14
 +
 +
[2] Briers, Y. et al., 2007. Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL. Molecular Microbiology, 65(5), pp.1334–1344.
 +
 +
[3] Skerlavaj, B. et al., 1999. SMAP-29: A potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Letters, 463(1-2), pp.58–62.
 +
 +
[4] Shin, S.Y. et al., 2001. Structure-activity analysis of SMAP-29, a sheep leukocytes-derived antimicrobial peptide. Biochemical and biophysical research communications, 285(4), pp.1046–1051.
  
  

Revision as of 20:15, 7 September 2015

Artilysin Art-175, an antibacterial fusion protein


This part contains the sequence for the antimicrobial protein Art-175.

Our Art-175 part collection comes in a family of four parts, one being Art-175 by itself and three others being Art-175 fused to different secretion signal sequences:


Secretion Tag Part Number
None BBa_K1659000
Flagellin 26-47 peptide segment BBa_K1659001
DsbA BBa_K1659002
YebF BBa_K1659003


Biology

Artilysins are an exciting class of enzyme-based antibacterials. Their name is derived from "artificial endolysin" and they exploit the lytic power of bacteriophage-encoded endolyins. Endolysins are peptidoglycan hydrolases produced at the end of the lytic cycle that pass through the cytoplasmic membrane, degrade the peptidoglycan layer and cause the osmotic lysis of the infected bacterial cell, thus liberating the progeny. Endolysins have a degree of specificity in terms of of the peptidoglycan chemotype which they can break down by means of the structural selectivity of their enzymatically-active domain (EAD) or cell wall binding domain (CBD).

Purified endolysins have been used to kill Gram-positive pathogens. Gram-negative bacteria, however, have a protective outer membrane containing lipopolysaccharide (LPS) that serves as a barrier against the peptidoglycan hydrolytic activity of endolysins from the outside. To overcome this problem, selected polycationic or amphipathic peptides that locally destabilize the LPS layer can be covalently fused to endolysins to transport them past the outer membrane to reach the peptidoglycan layer.

Biers et al. fused the sheep myeloid antimicrobial peptide SMAP-29 to the N-terminus of the endolysin KZ144 to create Artilysin Art-175 [1]. Endolysin KZ144 has previously been shown to selectively exert cell wall lytic activity on the peptidoglycan chemotype A1γ (which Gram-negative bacteria such as P. aeruginosa, E. coli, and Salmonella typhimurium belong to), where the bacterial outer membranes have already been separately permeabilized, by targeting the fully N-acetylated glucosamine units present in that peptidoglycan chemotype [2]. On the other hand, SMAP-29 on its own exhibits broad antimicrobial activity by means of using its N-terminal ampiphathic α-helical region in conjunction with its C-terminal hydrophobic region to disrupt of the outer and inner membranes of bacteria. SMAP-29 on its own, however, is unsuitable for clinical applications because it is also hemolytic towards human erythrocytes [3][4].

Art-175, the product of their linkage, is able to use its SMAP-29 moiety to transport itself past the bacterial outer membrane and exert lytic activity on the peptidoglycan layer. It exhibits strong antibacterial activity against pathogenic P. aeruginosa strains PAO1 and PA14, being able to kill even persister cells effectively as it does not require active bacterial metabolism to exert its lytic activity. However, unlike SMAP-29 by itself, Art-175 cannot kill bacteria using SMAP-29's cell membrane disruption mechanism, and owing to the chemotype selectivity of KZ144 is hence ineffective against bacteria of other peptidoglycan chemotypes, such as S. aureus [1].


Usage

We aim to test this part for antibacterial activity by means of protein purification from host cell lysate after induction of gene expression. The antibacterial potency of this part will serve as a standard which we will compare our secretion-tagged modified Art-175 parts against.


References

[1] Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J. P., Defraine, V., … Lavigne, R. (2014). Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 58(7), 3774–3784. http://doi.org/10.1128/AAC.02668-14

[2] Briers, Y. et al., 2007. Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL. Molecular Microbiology, 65(5), pp.1334–1344.

[3] Skerlavaj, B. et al., 1999. SMAP-29: A potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Letters, 463(1-2), pp.58–62.

[4] Shin, S.Y. et al., 2001. Structure-activity analysis of SMAP-29, a sheep leukocytes-derived antimicrobial peptide. Biochemical and biophysical research communications, 285(4), pp.1046–1051.