|Reinhardt, Timothy - Tim|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/23/2013
Publication Date: N/A
Citation: N/A Interpretive Summary:
Technical Abstract: Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. Typically this infection is transient in nature and lasts 2-3 days. However, in a minority of cases, E. coli can cause a persistent intramammary infection. The mechanisms that enable certain strains of E. coli to cause a persistent infection are not known. Three E. coli strains that cause a persistent infection and three E. coli strains that cause transient infections were compared using isobaric-labeled shotgun proteomics to determine protein differences. Bacteria were isolated from milk by centrifugation and purified on a sucrose gradient to limit somatic cell and milk protein contamination. Bacteria were lysed and proteins isolated. Equal amount of proteins were digested with trypsin and iTRAQ labeled (8-plex). Samples were fractionated on an offline high pH RP-HPLC and resulting fractions were run on a nano RP-HPLC directly linked to an LTQ-OrbiTrap. Each sample was run twice, and each experiment was repeated 4 times. We found 1125 E. coli proteins with a confidence score of at least 99%, and 2 peptides were identified. The data show substantial variation in protein expression among the strains. However, 19 proteins had expression changes that differed based on whether the bacterial stain caused a transient or persistent infection. One protein of interest, methyl-accepting chemotaxis protein II (MCPII), is known to be involved in bacteria swimming and swarming; bacterial swimming and swarming are thought to be indicators of bacterial virulence. Motility assays were performed, and the persistent strains demonstrated significantly better swimming and swarming characteristics compared to the transient strains. Our data revealed a correlation between higher MCPII expression in persistent strains of E. coli compared to transient strains, as well as the persistent strains of E. coli exhibiting better swimming and swarming phenotypes. These data highlight specific strain protein expression differences that may contribute to whether a strain causes a transient or a persistent intramammary infection.