Display of beta-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions.
Bacterial cell-surface exposure of foreign peptides and soluble proteins has been achieved recently by employing a fusion protein methodology. An Lpp'-OmpA(46-159)-Bla fusion protein has been shown previously to display the normally periplasmic enzyme beta-lactamase (Bla) on the cell surface of the Gram-negative bacterium Escherichia coli. Here, we have investigated the role of the OmpA domain of the tripartite fusion protein in the surface display of the passenger domain (Bla) and have characterized the effects of the fusion proteins on the integrity and permeability of the outer membrane. We show that in addition to OmpA(46-159), a second OmpA segment, consisting of amino acids 46-66, can also mediate the display of Bla on the cell surface. Other OmpA domains of various lengths (amino acids 46-84, 46-109, 46-128, 46-141 and 46-145) either anchored the Bla domain on the periplasmic face of the outer membrane or caused a major disruption of the outer membrane, allowing the penetration of antibodies into the cell. Detergent and antibiotic sensitivity and periplasmic leakage assays showed that changes in the permeability of the outer membrane are an unavoidable consequence of displaying a large periplasmic protein on the surface of E. coli. This is the first systematic report on the effects that cell surface engineering may have on the integrity and permeability properties of bacterial outer membranes.
Georgiou
G
G
Department of Chemical Engineering, University of Texas, Austin 78712, USA.
Stephens
D L
DL
Stathopoulos
C
C
Poetschke
H L
HL
Mendenhall
J
J
Earhart
C F
CF
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins; D002473:Cell Wall / Q000201:enzymology; D004492:Edetic Acid / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000235:genetics; D007150:Immunohistochemistry; D008074:Lipoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010539:Permeability; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism / Q000494:pharmacology; D001618:beta-Lactamases / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C099932:Lpp protein, E coli; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate; D004492:Edetic Acid; D001618:beta-Lactamases