1. Towards the Purifications of MxiK, an Essential Component of the Shigella Type III
Secretion System
Ryan R. Skaar, Michael L. Barta, Prashant Kumar, William D. Picking
1 Department of Pharmaceutical Chemistry, University of Kansas
2Simpson College, Indianola IA
Abstract
Bacterial infections caused by Shigella, called shigellosis, result in diarrhea and in
some cases dysentery. There are approximately 164.7 million cases of shigellosis each
year worldwide annually and 163.2 million of these cases occur in developing
countries. Children under the age of 5 account for 69% of all cases and 61% of the 2.6
million deaths that occur annually (2). Shigella are Gram-negative bacteria that
invade the epithelial cell lining of the colon via a Type 3 Secretion System (T3SS).
The T3SS is essential for invasion by Shigella and it delivers virulence proteins
through an apparatus that consists of external needle and tip complex, a basal body
and a cytoplasmic sorting platform that energizes and controls secretion (1). The
objective of this project is to examine what role MxiK plays in the sorting platform
and how it might interact with MxiN, Spa33, and/or MxiG within the sorting
platform. MxiK and its homologs in other T3SS bacteria are essential for T3SS
activity. The sorting platform in MxiK null mutants is lost from the basal body which
results in complete loss of virulence. This effect on the apparatus is also apparent in a
Spa33 null mutant strain which suggests that Spa33 and MxiK may be interacting
within the apparatus. To date, there have been no reports describing the purification
of MxiK (or its homologs), an outcome likely hindered by insoluble expression in E.
coli cloning strains. The goal of this project is to overcome this limitation and obtain
refolded MxiK for biophysical analyses and protein-protein interaction studies.
Results & Discussion
Contact Info
Email: ryan.skaar31@gmail.com
Phone: 515-520-0438
Funding
University of Kansas Pharmaceutical Chemistry Department
References
Conclusions
Background
Recent studies on the T3SS in Shigella have been focused on the interactions of
the proteins that make up the structure of the injectisome and more specifically
the cytoplasmic sorting platform. Previous research has shown that there are
many similarities between the injectisome and bacterial flagellar “C ring” (1).
Each of these platforms contains an ATPase, Spa47 (orange) in the injectisome,
that aligns with the central complex (Fig. 1, left and center). It is thought that
substrates for the formation of the external needle complex and/or for virulence
are delivered by the sorting platform to Spa47, which then transfers them through
the rest of injectisome. This function is dependent on the presence of Spa33,
MxiN, and MxiK (Fig. 1, center). This conclusion was made by examining
mutants that lacked each of these proteins. In Figure (top right) it is clear by cryo-
electron tomography that the removal of MxiN from the sorting platform
prevents the Spa47 and the external needle complex from forming resulting in a
loss of virulence. However, Spa33 is still conserved within the platform. When
Spa33 is mutated and removed from the injectisome, virulence is again lost (Fig.
1, bottom right). Virulence is also lost for ΔmxiK. There is very little structural
and biological data available for MxiK and since the presence of MxiK is
essential for T3SS it is important that research be done to better understand what
role MxiK plays in the cytoplasmic sorting platform of the T3SS injectisome.
1. Hu, Bo, et al. "Visualization of the type III secretion sorting platform of Shigella flexneri." Proceedings of the National Academy of Sciences 112.4 (2015): 1047-
1052.
2. Kotloff, Karen L., et al. "Global burden of Shigella infections: implications for vaccine development and implementation of control strategies." Bulletin of the World
Health Organization 77.8 (1999): 651-666.
3. Hemschemeier, Dr. Susanne Katharina, and Dr. Alfred Maelicke. "Zum Directory-modus." Chromatographie Von Proteinen. N.p., n.d. Web. 25 July 2016.
A
MxiK and its homologs in other T3SS
bacteria are essential for T3SS activity.
Therefore, in order to obtain structural,
protein-protein interaction data for
MxiK, a purified, soluble and
biologically active MxiK inclusion body
(IB) is needed. This is done by cloning
the mxiK[1-175] C-His pT7HmT gene
into E. coli Tuner Expression Cells.
After cultivation, and induce expression
of the MxiK IB, the expression cells
were lysed by sonication releasing the
IB. The presence of MxiK within the
IBs in sonicated lysis sample (SL) is
confirmed by SDS-PAGE (Fig. 2). Any
cellular material or IBs that were
insoluble after this point were then
pelleted by centrifugation. If the MxiK
IB was soluble one would expect to see
expression in the supernatant (SS),
however, Fig. 2 shows that the presence
of MxiK in the IB is lost when
compared to the SL and the SS samples.
The SL pellet was resuspended
and analyzed by SDS-page for
IB expression (Fig. 3 a). A series
of sonication, centrifugation, and
resuspension wash steps were
performed in order to dissolve
and remove cellular
contaminants. Samples b-e (Fig.
3) were taken from supernatants
after each centrifuge step to
confirm the removal of
contaminants and not the IBs.
These wash steps are also
extremely important for reducing
the viscosity of the IB solution.
However, the wash steps were
not efficient for reducing cellular
contaminants. In order to purify
and solubilize MxiK, the IB
must be denatured. This was
done using 8M urea followed by
incubating and washing the IB
pellet in 6M GdnHCl.
Figure 2: SDS-PAGE analysis of the
solubility of MxiK IB upon sonicated lysis
from expression cells. The loss of our
expression band between SL and SS samples
and shows that the IB is contained in the pellet
of centrifuged SL sample and not the
supernatant
Figure 3: SDS-page analysis of the supernatants
taken after each resuspension, sonication, and
centrifugation wash steps. Sample a: Resuspension of
SL pellet in lysis buffer, b&c: 1% Triton X-100
detergent washes to help dissolve cellular membranes,
d: lysis buffer to remove residual detergent from pellet,
e: 1.5M NaCl wash to break up nucleic acids. 2a:
Resuspention after denaturing in 8M Urea.
The next step once the IB is denatured
was to use a denaturing Nickel
Immobilized Metal Affinity
Chromatography (Ni-IMAC), which
takes advantage of the C-His tag that was
coded into the MxiK IB sequence. The
His tag has a high affinity for the charge
resign of the Ni-IMAC column. Fig. 4 on
the right is a diagram that shows the
chemical interactions that are occurring
between the protein from the IBs and the
charged resign.
Figure 4: Shows the chemical interactions occurring between the C-His tag of MxiK and the NiIMAC resign (3).
The cellular contaminants also may have an
affinity for the Ni-IMAC, however, there
affinity is much lower. In order to remove
the contaminants from the column before the
IB an increasing imidazole (IMID)
concentration gradient (20 mM to 500 mM)
was used. Using lower concentrations first
allowed for the displacement of
contaminants from the column without
affecting the binding of the MxiK. When the
[IMID] rose above 80 mM the bound protein
began to elute from the column, purified
from the other contaminants (Fig. 5).
Figure 5: SDS-PAGE analysis of fractions taken from each step of
the IMID elution of the IB and contaminants from the Ni-IMAC.
Figure 1: Cryo-electrotomography images of the T3SS and sorting platform found in Shigella.
• Recombinant expression of MxiK in E. coli
results in insoluble protein
• Highly purified, denatured MxiK obtained
through a series of IB washing steps
• Denatured MxiK failed to properly refold
under an extensive array of refolding methods
• Closely related homolog PscK from
Pseudomonas aeruginosa can be
recombinantly expressed in the soluble form Figure 6: SDS-PAGE analysis of the SL and
SS samples, along with NiIMAC purifaction
fraction. In comparison with Figure 2 we can
see that the PscK is soluble and present after
centrifugation.