Surface anchoring of a bacterial adhesin secreted by the two-partner secretion pathway.
In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. In the present study we sought to determine the mechanism by which HMW1 is anchored to the bacterial surface. In initial experiments we found that HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. Further analysis established that HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Mutagenesis and polyethylene glycol maleimide labelling revealed that anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region. Immunolabelling studies demonstrated that the immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains in the periplasm or is buried in HMW1B. Coexpression of HMW1 lacking the C-terminal 20 amino acids and wild-type HMW1 supported the conclusion that the C-terminus of HMW1 occupies the HMW1B pore. These observations may have broad relevance to proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.
Buscher
Amy Z
AZ
Edward Mallinckrodt Department of Pediatrics , Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
Grass
Susan
S
Heuser
John
J
Roth
Robyn
R
St Geme
Joseph W
JW
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004847:Epithelial Cells / Q000382:microbiology; D006193:Haemophilus influenzae / Q000166:cytology / Q000502:physiology*; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data
D018829:Adhesins, Bacterial; D004220:Disulfides; C117431:HMW1 protein, bacteria; C117432:HMW2 protein, bacteria; D026901:Membrane Transport Proteins; D003545:Cysteine