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Modifying the Amino Acid Sequence in the Surface-Exposed Loops of the Omptin Family of Proteins to Determine Their Effect on Function Natiera Magnuson, Eun-Hae Kim*, Christian Ross and Helen J Wing Introduction The omptin family of proteins consists of proteases which lie in the outer membrane of some gram-negative, pathogenic bacteria such as Escherichia coli (OmpT), Shigella flexneri (IcsP), Salmonella typhimurium (PgtE), and Yersinia pestis (Pla). These proteases are highly conserved, sharing approximately 50% sequence identity and a β-barrel shape (fig. 1D). The differences in the structure of these four proteins are in the surface-exposed loop region surrounding the active site, but not in the active site itself . These proteases are important for the virulence of many bacteria. For example, OmpT of E. coli cleaves an antimicrobial peptide secreted by epithelial cells of the urinary tract ; IcsP of S. flexneri regulates IcsA, which uses the host’s actin to allow motility of the bacterium ; PgtE of S. typhimurium helps the bacterium evade the immune system by cleaving the α-helical cationic antimicrobial peptides ; and, Pla of Y. pestis enhances bacterial migration through tissue barriers by cleaving plasminogen . Previous work  has shown that the omptin proteins of E. coli and S. typhimurium do not cleave IcsA in the same manner as IcsP in S. flexneri. Differences in the cleavage of IcsA may be due to the differences in surface-exposed loops of the protease or in its LPS binding motif . Determining whether the surface-exposed loops of a protein affects its function could lead to a better understanding of this protein’s function and how it has evolved to serve different functions in different bacterial pathogens. University of Nevada Las Vegas *University of Arizona Hypothesis The objective of this work is to determine whether or not the differences in the amino acid sequence of the surfaceexposed loops of OmpT, PgtE, Pla, and IcsP allow for differential cleavage of IcsA. We hypothesize that the modified IcsP protein will function similar to the protein it was mutated to resemble. A B Figure 2: IcsP cleavage of IcsA in Shigella. Whole cell and supernatant proteins were separated by SDS-PAGE, and transferred to a PVDF membrane. IcsA was detected by immunoblotting with an anti-IcsA antibody. A: IcsA was detected in the whole cell as well as the supernatant of 2457T (wild-type). However, it was only detected in the whole cell of MBG341 (2457T-ΔicsP). This shows that IcsP cleaves IcsA, removing a fragment of approximately 95kDa from the bacterial surface. B: pMBG270 (icsA) was introduced into the Shigella strain MBG283 (2457T-ΔicsA). IcsA was detected in the whole cell as well as in the supernatant. The IcsA expressed by pMBG270 was cleaved similarly to the wild type.  Figure 5: Comparison of the amino acid sequence for IcsP, OmpT, and PgtE. Outlined in green is the specific amino acid in IcsP changed to resemble the corresponding amino acid in OmpT and PgtE (fig. 1, L2). B A D Methods • Site Directed Mutagenesis of icsP gene: ~ Key nucleotides are changed in the gene to produce a change in the amino acid sequence ~AAC changed to ATG ⇒ asparagine into methionine ~ IcsP now appears the same as OmpT and PgtE in that region (fig. 5) • pBAD33 (ΔicsP), pAJH02 (icsP), and pNKM02 (mutated icsP) were introduced into Shigella strain MBG341 •SDS-PAGE (PolyAcrylamide Gel Electrophoresis) and Western Blot ~SDS-PAGE to separate whole cell and supernatant proteins according to their size ~ proteins then transferred to a PVDF membrane ~ IcsA detected by immunoblotting with and anti-IcsA antibody •Imaged with UVP Imaging System Figure 3: OmpT cleavage of IcsA in E. coli. pMBG270 (icsA) was introduced into the E.coli strains MC1061 (wild-type) and MBG263 (MC1061-ΔompT). Whole cell and supernatant proteins were separated by SDS-PAGE, and transferred to a PVDF membrane. IcsA was detected by immunoblotting with an anti-IcsA antibody. IcsA was detected only in the whole cell of MBG263 (lacking OmpT). IcsA was detected in the supernatant of MC1061 (wild type producing OmpT). This shows that OmpT cleaves IcsA, removing a fragment of approximately 85kDa from the bacterial surface.  C Results and Future Direction • It is expected for IcsP to produce the same cleavage fragments as OmpT • Mutation of additional surface-exposed loops to compare function to other members of the omptin family Acknowledgements I would like to thank everyone in the Wing Lab for their help and support. Funding was provided by NIH grant: R15 AI090573-01. Figure 1: The omptin family. A: Neighbor-joining tree for IcsP and related proteins. Constructed using ClustaIW with Gonnett 250 matrix and no distance correction, gaps ignored. B: Transmembrane spanning segments and external loops of omptins, based on structure of E. coli OmpT. Segment correlate to the protein tree on the left. C: Key to figure 1B. Shows conserved sequences along with the differing amino acid sequences. Domain structure corresponds to branches in figure1A, as determined by MEME/MAST analysis. D: Predicted 3D structure of IcsP.  Figure 4: PgtE cleavage of IcsA in Salmonella. pMBG270 (icsA) was introduces into the Salmonella strains CS022 (wild-type) and TG61 (CS022-ΔpgtE). Whole cell and supernatant proteins were separated by SDS-PAGE, and transferred to a PVDF membrane. IcsA was detected by immunoblotting with an anti-IcsA antibody. IcsA was detected only in the whole cell of TG61 (lacking PgtE). IcsA was detected in the supernatant of CS022 (wild type producing PgtE). This shows that PgtE cleaves IcsA, removing two fragments of approximately 95kDa and 85kDa from the bacterial surface.  Sources 1. Guina T., Yi E.C., Wang H., Hackett M., and Miller S.I. (2000) A Pho-P regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides. Journal of Bacteriology 182: 4077-4086. 2. Kim, EH. The conserved mechanism of IcsA polar targeting among proteobacteria, characterization of the omptin family, and the roles and regulation of IcsP in Shigella flexneri [Master’s thesis]. University of Nevada, Las Vegas; 2004 3. Kim EH., Ross C., Struve T., and Wing H.J. (2007) Roles and regulation of the Shigella outer membrane protease, IcsP. Poster presentation at the American Society for Microbiology General Meeting. 4. Kukkonen M., and Korhonen T.K. (2004) The omptin family of enterobacterial surface proteases/adhesins: From housekeeping in Escherichia coli to systemic spread of Yersinia pestis. International Journal of Medical Microbiology 294: 7-14. 5. Marrs C.F., Zhang L., Tallman P., Manninf S.D, Somsel P., Raz P., et al. (2002) Variations in 10 putative uropathogen virulence genes among urinary, faecal and peri-urethral Escherichia coli. Journal of Medical Microbiology 51: 138-142. 6. SteinhauerJ., Agha R., Pham T., Varga A.W., and Goldberg M.B. (1999) The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface. Molecular Microbiology 32: 367-377.