Abstract
ABSTRACTBorrelia burgdorferi HtrA (HtrABb) is a serine protease that targets damaged or improperly folded proteins. In our previous studies, HtrABb specifically degraded basic membrane protein BmpD, chemotaxis phosphatase CheX, and outer membrane protein P66. In addition, HtrABb degrades virulence factor BB0323 and components of the extracellular matrix fibronectin and aggrecan. A proteomics-based analysis (two-dimensional difference gel electrophoresis [2-D DIGE], liquid chromatography-mass spectrometry [LC-MS]) of an HtrABb-overexpressing strain of B. burgdorferi (A3HtrAOE) revealed that protein levels of P66 were reduced in comparison to wild-type B. burgdorferi, confirming its status as an HtrABb substrate. Hbb, a P66-DNA-binding transcription factor, was specifically degraded by HtrABb, providing supportive evidence for a role for both in the regulation of P66. A3HtrAOE exhibited reduced motility in swarm assays, a possible link between overabundance of HtrABb and its enzymatic specificity for P66. However, the ΔP66 strain did not have reduced motility in the swarm assays, negating a role for this protein. The proteomics analyses also identified three enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerol-3-phosphate dehydrogenase (GPDH), and glycerol kinase (GK), and one enzyme involved in carbohydrate metabolism, diphosphate-fructose-6-phosphate 1-phosphotransferase, which were reduced in A3HtrAOE. Consistent with its reduced protein levels of these glycolytic enzymes, A3HtrAOE was also deficient in production of pyruvate. We propose a model for a role for HtrABb in contributing to a decrease in metabolic activity of B. burgdorferi.
Highlights
Borrelia burgdorferi HtrA (HtrABb) is a serine protease that targets damaged or improperly folded proteins
We previously reported that standard 2-D electrophoresis analysis of B. burgdorferi B31A3 wild type and HtrA-overexpressing strain A3HtrAOE identified a likely HtrA substrate candidate with a pI, molecular mass, and MALDI-TOF mass spectrometry (MS) spectrum consistent with those of outer membrane protein P66 [20]
Our results confirm unequivocally that outer membrane protein P66 is an in vivo substrate for HtrABb as shown by 2-D DIGE
Summary
Borrelia burgdorferi HtrA (HtrABb) is a serine protease that targets damaged or improperly folded proteins. HtrABb promotes invasiveness through degradation of the extracellular matrix components aggrecan [7] and fibronectin [8], an unexpected role for a periplasmic protease. HtrABb is released in vesicles [9] This exogenous role is not unique to the HtrA of B. burgdorferi. Overexpression of HtrA in Helicobacter pylori leads to increased HtrA secretion and to cleavage of E-cadherin This function increases bacterial transmigration and delivery of the type IV secretion system (T4SS) effector protein CagA into polarized epithelial cells [10]. H. pylori secretes HtrA through a T4SS to deliver CagA to cells In this pathway, HtrA degrades epithelial junctional proteins necessary for this bacterium to reach the specific cell basolateral locations where CagA is injected [11]. It is evident that the HtrAs of these two pathogens have important exogenous proteolytic roles that result in enhanced invasiveness
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