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Structure/function studies on the autotrasporter BrkA and investigation of the role of periplasmic chaperones in BrkA secretion Yue, Jody
Abstract
BrkA is a virulence factor in Bordetella pertussis that belongs to the ATI subfamily of autotransporter proteins in Gram negative bacteria. ATI members have: (i) an N-terminal signal peptide for secretion across the inner membrane, (ii) a surface expressed passenger domain, and (iii) a C-terminal translocation unit for passenger secretion across the outer membrane. There are many questions about ATI secretion across the periplasm and the outer membrane. Studies on the ATI member IcsA suggests rapid periplasmic transit, and that DegP's chaperone activity is involved. Comparison of passenger size with estimated and known pore sizes of transporter domains suggest that the passenger is too large for translocation across the outer membrane in a folded state. The mechanism by which an ATI passenger might maintain an unfolded state in the periplasm while resisting proteolysis is unknown. BrkA secretion in Escherichia coli was used to investigate periplasmic transit and outer membrane translocation of an ATI passenger. Previous studies revealed the junction domain in BrkA, which functioned in passenger folding. A highly conserved subdomain (region 3) was identified by ClustalW alignment of the junction, and was dispensable for in vitro folding. This subdomain might function in secretion by keeping the passenger unfolded for translocation by binding to a periplasmic chaperone. The role of region 3 in BrkA surface expression and in vivo folding was tested by trypsin accessibility and immunofluorescence microscopy, and limited proteolysis assays, respectively. In parallel studies, the involvement of periplasmic chaperones in BrkA secretion was investigated by trypsin accessibility assays on Skp, DegP, and SurA knockout strains expressing BrkA. Residues A⁶⁸¹ -E⁶⁹³ of region 3 were important for secretion of a folding-competent BrkA passenger, but were dispensable for in vivo folding. We termed A⁶⁸¹-E⁶⁹³ the "hydrophobic secretion facilitation" (HSF) domain, after the term coined by Velarde and Nataro for the corresponding region in the ATI member EspP, which was important for passenger secretion. SurA, a peptidyl prolyl isomerase (PPIase) that functions in porin secretion, was implicated in BrkA secretion. The PPIase activity of SurA was dispensable, suggesting that SurA's chaperone activity is the necessary activity for BrkA secretion.
Item Metadata
Title |
Structure/function studies on the autotrasporter BrkA and investigation of the role of periplasmic chaperones in BrkA secretion
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2005
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Description |
BrkA is a virulence factor in Bordetella pertussis that belongs to the ATI subfamily of
autotransporter proteins in Gram negative bacteria. ATI members have: (i) an N-terminal
signal peptide for secretion across the inner membrane, (ii) a surface expressed passenger
domain, and (iii) a C-terminal translocation unit for passenger secretion across the outer
membrane.
There are many questions about ATI secretion across the periplasm and the outer
membrane. Studies on the ATI member IcsA suggests rapid periplasmic transit,
and that DegP's chaperone activity is involved. Comparison of passenger size with estimated
and known pore sizes of transporter domains suggest that the passenger is too large for
translocation across the outer membrane in a folded state. The mechanism by which an ATI
passenger might maintain an unfolded state in the periplasm while resisting proteolysis is
unknown.
BrkA secretion in Escherichia coli was used to investigate periplasmic transit and outer
membrane translocation of an ATI passenger. Previous studies revealed the junction domain
in BrkA, which functioned in passenger folding. A highly conserved subdomain (region 3)
was identified by ClustalW alignment of the junction, and was dispensable for in vitro folding.
This subdomain might function in secretion by keeping the passenger unfolded for
translocation by binding to a periplasmic chaperone. The role of region 3 in BrkA surface
expression and in vivo folding was tested by trypsin accessibility and immunofluorescence
microscopy, and limited proteolysis assays, respectively. In parallel studies, the involvement
of periplasmic chaperones in BrkA secretion was investigated by trypsin accessibility assays
on Skp, DegP, and SurA knockout strains expressing BrkA.
Residues A⁶⁸¹ -E⁶⁹³ of region 3 were important for secretion of a folding-competent
BrkA passenger, but were dispensable for in vivo folding. We termed A⁶⁸¹-E⁶⁹³ the
"hydrophobic secretion facilitation" (HSF) domain, after the term coined by Velarde and
Nataro for the corresponding region in the ATI member EspP, which was important for
passenger secretion.
SurA, a peptidyl prolyl isomerase (PPIase) that functions in porin secretion, was
implicated in BrkA secretion. The PPIase activity of SurA was dispensable, suggesting that
SurA's chaperone activity is the necessary activity for BrkA secretion.
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Genre | |
Type | |
Language |
eng
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Date Available |
2009-12-11
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0092037
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2005-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.