MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS
Location: Molecular Characterization of Foodborne Pathogens
Title: The CsgA and Lpp proteins affect HEp-2 cell invasion, motility, and biofilm formation in a strain of Escherichia coli O157:H7
Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 1, 2009
Publication Date: April 1, 2009
Citation: Uhlich, G.A., Gunther, N.W., Bayles, D.O., Mosier, D.A. 2009. The CsgA and Lpp proteins affect HEp-2 cell invasion, motility, and biofilm formation in a strain of Escherichia coli O157:H7. Infection and Immunity. 77(4):1543-1552.
Interpretive Summary: In a previous study, we identified a strain of Escherichia coli O157:H7 that can exist in two biological states, one of which has an unusual ability to form biofilm, persist on solid surfaces, and invade cultured human epithelial cells. The two states differ genetically only in the chromosomal sequence of a regulatory gene known to control aspects of the biofilm formation process. In the present study, we used proteomic techniques to compare the total proteins produced in the two different states in an attempt to identify additional proteins which contribute to biofilm formation, human cell invasion, and other properties. We identified two proteins, CsgA and Lpp, which were produced in different quantities in one of the states as compared to the other. The genes encoding CsgA and Lpp were deleted from the chromosome of bacteria existing in the state that could form biofilm and invade cells. The deletion mutant strains for both CsgA and Lpp became defective in biofilm formation and cell invasion, proving that both proteins are necessary for those properties. We also showed that while the CsgA protein may be physically involved in biofilm formation and invasion, the Lpp protein most likely acts by controlling the production of CsgA. In addition, we identified an intermediate regulatory protein, CpxR, which was being used by Lpp in the regulation of CsgA. The results of this study provide new information on mechanisms that E. coli serotype O157:H7 may use to persist on food products and processing equipment, and/or to cause disease in susceptible people. These studies may be important in the development of future intervention technologies.
In Escherichia coli O157:H7 strain ATCC 43895, a guanine to thymine transversion in the csgD promoter created strain 43895OR. Strain 43895OR produces an abundant extracellular matrix rich in curli fibers, forms biofilm on solid surfaces, invades cultured epithelial cells, and is more virulent in mice than strain 43895. In this study we compared the formic acid-soluble proteins expressed by strains 43895OR and 43895 using one-dimensional SDS-PAGE analysis and identified two differentially expressed proteins. A 15-kDa protein unique to strain 43895OR was identified from MALDI-TOF combined MS+MS/MS spectra as the curli subunit encoded by csgA. A <10-kDa protein, more highly expressed in strain 43895, was identified as the Lpp lipoprotein. Mutants of strain 43895OR with disruption of lpp, csgA, and both lpp/csgA were created and tested for changes in phenotype and function. The results of this study show that both Lpp and CsgA contribute to the observed colony morphology, Congo red binding, motility, and biofilm formation. We also show that both CsgA and Lpp were required by strain 43895OR for the invasion of cultured HEp-2 cells. These studies suggest that in strain 43895OR, the murein lipoprotein, Lpp, indirectly regulates CsgA expression through the CpxAR system by a post-transcriptional mechanism.