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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 [4]. 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 [5];
IcsP of S. flexneri regulates IcsA, which uses the host’s
actin to allow motility of the bacterium [6]; PgtE of S.
typhimurium helps the bacterium evade the immune system
by cleaving the α-helical cationic antimicrobial peptides [1];
and, Pla of Y. pestis enhances bacterial migration through
tissue barriers by cleaving plasminogen [4]. Previous work
[2] 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 [2]. 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.
[2]
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.
[2]
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.
[3]
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.
[2]
Sources
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(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.
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omptin family, and the roles and regulation of IcsP in
Shigella flexneri [Master’s thesis]. University of Nevada, Las
Vegas; 2004
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and regulation of the Shigella outer membrane protease,
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Microbiology General Meeting.
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uropathogen virulence genes among urinary, faecal and
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Molecular Microbiology 32: 367-377.