title: Borrelia burgdorferi HtrA: evidence for twofold proteolysis of outer membrane protein p66.
abstract: In prokaryotes, members of the High Temperature Requirement A (HtrA) family of serine proteases function in the periplasm to degrade damaged or improperly folded membrane proteins. Borrelia burgdorferi, the agent of Lyme disease, codes for a single HtrA homolog. Two-dimensional electrophoresis analysis of B. burgdorferi B31A3 and a strain that overexpresses HtrA (A3HtrAOE) identified a downregulated protein in A3HtrAOE with a mass, pI and MALDI-TOF spectrum consistent with outer membrane protein p66. P66 and HtrA from cellular lysates partitioned into detergent-resistant membranes, which contain cholesterol-glycolipid-rich membrane regions known as lipid rafts, suggesting that HtrA and p66 may reside together in lipid rafts also. This agrees with previous work from our laboratory, which showed that HtrA and p66 are constituents of B. burgdorferi outer membrane vesicles. HtrA degraded p66 in vitro and A3HtrAOE expressed reduced levels of p66 in vivo. Fluorescence confocal microscopy revealed that HtrA and p66 colocalize in the membrane. The association of HtrA and p66 establishes that they could interact efficiently and their protease/substrate relationship provides functional relevance to this interaction. A3HtrAOE also showed reduced levels of p66 transcript in comparison with wild-type B31A3, indicating that HtrA-mediated regulation of p66 may occur at multiple levels.
authors.affiliation: New York State Department of Health, Stony Brook University, Stony Brook, NY, USA.|Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA.
authors.forename: James L
authors.initials: JL
authors.lastname: Coleman
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Toledo
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Jorge L
authors.initials: JL
authors.lastname: Benach
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D025065:Borrelia burgdorferi / Q000201:enzymology* / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018272:Porins / Q000378:metabolism*; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C107893:Oms66 protein, Borrelia; D018272:Porins; D012697:Serine Endopeptidases