Transcript The Rho GTPase activators CNF1 and DNT bacterial toxins have

The Rho GTPase activators CNF1 and DNT bacterial toxins have
mucosal adjuvant properties
Patrick Munro, et al.
Presented by Jared Nelson
Introduction
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There is, even today, a demand for more effective vaccines containing
adjuvants that help stimulate an IgA(and IgG) response. Cholera toxin (CT)
and other enterotoxins have been identified as potent IgA-stimulating
adjuvants that could be used in vaccines.
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Cytotoxic Necrotizing Factor 1(CNF1), found in uropathogenic E. coli, is
structurally and functionally related to E. coli CNF2, Yersinia
pseudotuberculosis CNFy, and Bordetella Dermonecrotic toxin(DNT). CNF1 is
an A-B toxin. CNF1 and DNT both use similar mechanisms to invade and
intoxicate host cells and to allow bacterial cells to invade and persist in host
cells.
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CNF1 and DNT are both taken up by host cells via their aminoterminal domains into vesicles. Their translocation domains are used
in acidic pHs to eject the toxins out of their vesicles into the
cytoplasm. After this, these toxins convert Rho proteins into their
active forms by stopping their GTPase functioning via deamidation or
transglutamination. Then ubiquitin regulates the digestion of these
Rho proteins by proteases/proteasomes.
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The activation of Rho proteins, such as Rac and Cdc42 (particularly
via CNF1), is all that is needed to send a signal cascade that leads to
generating IL-8, MCP-1, and MIP-3-alpha (cytokines that are involved
in inflammation and directly or indirectly in the regulation of the
adaptive immune response).
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This experiment points to CNF1 and DNT as being agents used to
elicit a strong T-cell mediated and B-cell mediated immune reaction
with a hydrophilic protein antigen named ovalbumin (OVA). They
claim that Rho proteins(GTPases), when activated by CNF1 and DNT,
are directly responsible for the regulation and activation of the above
adaptive immunity.
Materials and Methods
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I. CNF1 and CNF1-C866S(mutant inactivated form of CNF1) toxins
were made and purified. CNF1 samples were subjected to a Hep2
multinucleation assay to determine their activity. CNF1-CD and DNTCD (CD stands for ‘catalytic domain’) were also made and purified.
Rac (a Rho GTPase) activating and digestion by DNT-CD was
observed with the “GST protein pull-down” experiment.
II. Each lab mouse was given (orally) one of the following toxins:
CNF1, CNF1C866S, CNF1-CD, DNT-CD, or CT along with OVA, except
for the OVA-only controls. Mice were given this cocktail 2 or 3 times,
with 10-12 days between times.
III. Anti-OVA antibodies were detected by “solid phase ELISA” using
HRP-tagged goat anti-mouse antibodies.
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IV. Anti-OVA IgA and IgG were extracted from tissue samples from
mice. Supernatants from these tissue samples and/or sera were
analyzed via ELISA.
V. Mice were given CNF1 or CNF1-C866S, and small intestines were
processed and sectioned using paraffin( The toxins were supposedly
given without OVA, perhaps to confirm that only anti-OVA antibodies
were produced).
Results
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Mice given OVA and 10 ug CNF1 produced a
very high titer of anti-OVA IgG. When the mice
were given cholera toxin, the anti-OVA IgG titer
was almost as high as the above result.
IgGs specific for CNF1 were not found.
NOTE: Only anti-OVA antibodies were detected.
The anti-OVA IgG titer yielded by 1 ug CNF1
was only slightly greater than that produced by
OVA alone (the + control). These titers were
several times less than the CT and CNF1 results.
When mice were given 10 ug of CNF1-C866S
mutant ( - control), the IgG titer was next to
zero.
Therefore, only a high dose of active CNF1 will
give a high IgG titer.
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In figure 2A(GST protein pull-down), DNT
temporarily activated Rac, since Rac is digested
afterwards. The RacGTP band is getting larger
overall as time goes on simply because the GTP
is not being hydrolyzed = no GTPase activity in
Rac. Then notice the last RacGTP band is
smaller compared to the next to last one,
probably because Rac is being digested. The
last lane, showing actin, apparently represents
actin rearrangement, although it doesn’t appear
to prove this.
Contrary to what the diagram tells us verbatim,
fig. 2B shows how effectively CNF1-CD and DNTCD intoxicate (activate Rac within ) host
cells(intestinal endothelial). DNT is clearly much
more effective than CNF1 in terms of its
intoxicating effect and in terms of the IgG titer
DNT stimulates (fig 2C).
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Fig 3 shows no measureable levels of CNF1C866S-stimulated IgA nor IgG, except for a
slight amount of IgG1.
Compare this to varying but similar levels of
IgG subclasses, such as IgG2a, and IgA,
produced from CNF1 and CT stimulation. The
amounts of IgA were low in both of these
conditions; the amounts of all the varieties of
IgGs were more or less significantly higher
than those of IgA.
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When mice were given OVA and CNF1 or
CNF1C866S(10 ug for either one), the CNF1
produced IgA titer was several times greater than
that produced by CNF1C866S or in an OVA-only
control.
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In fig. 5, Intestinal tissue preparations
(with paraffin cross-sections) of CNF1
or CNF-C866S-treated mice were not
very different from those of a
bicarbonate treated control. I’m not
sure why this is, since I would expect
to see signs of inflammation, at least.
Discussion and Conclusions
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CNF1 boosts IgA and IgG-based adaptive immunity with respect to a
protein antigen, OVA. CNF1 is an adjuvant because of its (at least)
deamidase activity. CT has adjuvant effects that are close, in terms
of what degree of titer of which antibodies, whether IgG or IgA, are
produced, to those of CNF1. All antibodies produced were specific
to OVA.
CNF1-C866S, the mutant, doesn’t influence the adaptive immune
reactions very much at all.
Both CNF1 and DNT had a temporary activating effect on Rac in
relating ways; therefore, DNT has the same kinds of adjuvant
effects, in which the active Rho proteins like Rac are directly
responsible for the adaptive immune effects.
Future questions
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They haven’t drawn any conclusions about whether 1. the cytokine
MIP3-alpha activation/regulation of IgA adaptive immunity and 2. an
active Cdc42 influencing the process of dendritic cell maturing are
important parts of CNF1’s adaptive immunity boosting influence.
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Could CNF1’s toxic effect as explained in this study impart any
benefit to E. coli? The only benefit I can think of is one that these
researchers mentioned about active CNF1 rearranging the host cell
cytoskeleton, allowing the E. coli to invade the cells and persist there
through resistance to signals that lead to host cell apoptosis.
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How else could we target or influence Rho proteins to regulate/boost
the adaptive immune activity?