Experimental validation of the predicted binding site of Escherichia coli K1 outer membrane protein A to human brain microvascular endothelial cells: identification of critical mutations that prevent E. coli meningitis.
Escherichia coli K1, the most common cause of meningitis in neonates, has been shown to interact with GlcNAc1-4GlcNAc epitopes of Ecgp96 on human brain microvascular endothelial cells (HBMECs) via OmpA (outer membrane protein A). However, the precise domains of extracellular loops of OmpA interacting with the chitobiose epitopes have not been elucidated. We report the loop-barrel model of these OmpA interactions with the carbohydrate moieties of Ecgp96 predicted from molecular modeling. To test this model experimentally, we generated E. coli K1 strains expressing OmpA with mutations of residues predicted to be critical for interaction with the HBMEC and tested E. coli invasion efficiency. For these same mutations, we predicted the interaction free energies (including explicit calculation of the entropy) from molecular dynamics (MD), finding excellent correlation (R(2) = 90%) with experimental invasion efficiency. Particularly important is that mutating specific residues in loops 1, 2, and 4 to alanines resulted in significant inhibition of E. coli K1 invasion in HBMECs, which is consistent with the complete lack of binding found in the MD simulations for these two cases. These studies suggest that inhibition of the interactions of these residues of Loop 1, 2, and 4 with Ecgp96 could provide a therapeutic strategy to prevent neonatal meningitis due to E. coli K1.
Pascal
Tod A
TA
From the Materials and Process Simulation Center, California Institute of Technology, Pasadena, California 91125.
Abrol
Ravinder
R
Mittal
Rahul
R
Wang
Ying
Y
Prasadarao
Nemani V
NV
Goddard
William A
WA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000098:blood supply / Q000378:metabolism / Q000382:microbiology*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D042783:Endothelial Cells / Q000378:metabolism / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D005260:Female; D006801:Humans; D008297:Male; D008581:Meningitis / Q000378:metabolism / Q000382:microbiology*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D014774:Virulence
D001425:Bacterial Outer Membrane Proteins; C464645:GPATCH1 protein, human; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; C078167:OMPA outer membrane proteins