MP02-11 CHARACTERIZATION OF COMPLEMENT RESISTANCE FACTOR TRAT ENCODED IN DRUG RESISTANCE PLASMIDS OF UROPATHOGENIC ESCHERICHIA COLI

Abstract

You have accessJournal of UrologyCME1 May 2022MP02-11 CHARACTERIZATION OF COMPLEMENT RESISTANCE FACTOR TRAT ENCODED IN DRUG RESISTANCE PLASMIDS OF UROPATHOGENIC ESCHERICHIA COLI Chae Hee Lim, Aryana Bhati, Katelyn Lott, Jazmyn Whitfield, John Land, and Tatyana Sysoeva Chae Hee LimChae Hee Lim More articles by this author , Aryana BhatiAryana Bhati More articles by this author , Katelyn LottKatelyn Lott More articles by this author , Jazmyn WhitfieldJazmyn Whitfield More articles by this author , John LandJohn Land More articles by this author , and Tatyana SysoevaTatyana Sysoeva More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002514.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The TraT protein is a multifunctional virulence factor that is encoded by conjugative F-like plasmids. A majority of multidrug-resistant uropathogenic Escherichia coli (UPEC) carry F-like plasmids and thus encode for this TraT factor. In non-pathogenic E. coli, TraT is a highly expressed outer membrane lipoprotein that protects the bacterial cell from excessive rounds of plasmid transfer, from a few bacteriophages, and from the host innate immune response via serum complement killing. Our objectives in this study were (1) to measure the protective effect of TraT in UPEC background and (2) to establish whether extracellular TraT domain is sufficient to provide serum resistance. METHODS: We used conjugation to transfer the model F’ plasmid in different E. coli backgrounds. Recombinant extracellular soluble domain TraT33-244 was purified from BL21(DE3) E. coli strain using Ni-affinity chromatography. Exponentially growing E. coli cultures with and without traT-encoding plasmids or the purified TraT domain were subjected to different doses of blood serum for 2 hours at 37°C. Viable cells were quantified as colony forming units by plating serial dilutions at 0 and 2 hours after addition of serum. RESULTS: The traT-encoding plasmid provides variable level of protection that depends on serum type, bacterial cells, time, and ratios of bacteria and serum, and can reach several orders of magnitude. We have developed a method of expression and purification of soluble TraT33-244 protein and showed that this extracellular domain forms stable oligomers in solution with five-to-six subunits. Testing this purified extracellular domain in serum resistance assays revealed that it is insufficient to exhibit the full protective action of TraT. CONCLUSIONS: In this study, we measured the protective effect of traT-encoding plasmid F’ in model E. coli strains, which displayed strong but variable serum resistance. The extracellular soluble domain of TraT protein oligomerizes, but does not strongly protect against serum killing when added to bacterial cells in trans. These results suggest that membrane localization, N-terminus, or lipidation itself are important for function of the TraT factor. Overall, this study sets the stage for further investigation into the mechanism of how TraT assists UPEC bacteria in evading innate immune responses within the host organism. Source of Funding: The University of Alabama in Huntsville startup funds to Tatyana Sysoeva © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e14 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chae Hee Lim More articles by this author Aryana Bhati More articles by this author Katelyn Lott More articles by this author Jazmyn Whitfield More articles by this author John Land More articles by this author Tatyana Sysoeva More articles by this author Expand All Advertisement PDF DownloadLoading ...