Emerging literature suggests that innate immunity serves as a
sophisticated system for sensing signals of “danger”, such as pathogens.
The success of a pathogen depends on its ability to mount an effective
anti-immune response. Vertebrates have evolved systems of immune defense
to eliminate pathogens and, in turn pathogens have evolved complex and
efficient methods to overcome innate and adaptive immune responses.
Although pathogens use different virulence strategies, there are several
general mechanisms that are shared between diverse microrganisms.
Shigella flexneri
is a human intestinal pathogen which uses a type III secretion (TTS)
system to deliver effectors to enter into colonic epithelial cells as
well as to exploit and subvert host immune responses. Large amounts of
pro-inflammatory molecules are released upon S. flexneri infection
leading to a strong inflammatory response. Our study is focused on the
characterization of the role of the ospB-phoN2 operon in the mechanism of pathogenicity of S. flexneri. ospB codes for a TTS effector of undefined activity secreted into host cell, while phoN2
codes for a periplasmic ATP-diphosphohyrolase (apyrase) involved in the
polar IcsA localization on the surface of S. flexneri. We recently
discovered that the periplasmic PhoN2 localized, as IcsA, at the old
pole of S. flexneri. Recent results have demonstrated that OspB
plays an important role in manipulating host cell signaling in order to
subvert the innate immune response. It has been reported that OspB might
be involved in attenuating inflammatory cytokine production. However,
the mechanism through which OspB exerts its role in the pathogenicity of
S. flexneri has not been completely elucidated yet. We plan to further
characterize the role of these two genes and:
1-To identify the
domains responsible of the polar localization of PhoN2. To this end we
will construct HA-tagged PhoN2 mutants presenting deletions of the PPPP
and of the catalytic domains, as well as to introduce selected amin
oacid substitutions (P to S) within the PPPP domain of PhoN2-HA. Mutants
will be assayed for the polar localization of PhoN2, for their ability
to complement a phoN2 mutant for the production of actin tails, plaque
assay, etc.
2– To identify and characterize putative protein(s) which
interact with PhoN2 and OspB. This point will be accomplished by
co-purification experiments and by two-hybrid technique in yeast.
Confirmation of putative interactors will be achieved by cross-linking
experiments.
3- To assess the role of ospB in the inflammatory response of epithelial cells infected by S. flexneri.
Since anti-OspB specific antibodies are not available, we will generate
HA-tagged OspB in order to better define the activity and the
localization of OspB. The production of inflammatory mediators (TNF-a,
IL-6 and IL-8), positively regulated by MAPKs, will be investigated by
infecting HeLa and/or CaCo-2 cell lines with parental S. flexneri strain M90T and with an ospB
deletion derivative. The activation state of endogenous MAPKs, will
also be determined. Difference of phospho-active levels of ERK1/2, JNK
and p38 will be evaluated. Specific activators and/or inhibitors which
are known to interfere with single MAPK pathways will be used to
evaluate MAPKs modulation. Moreover, the expression of different
cytokines involved in inflammation will be analyzed by Real Time RT-PCR
assays or by ELISA assays.
4- To study the effect of SurA, Skp and
DegP on the polar localization of PhoN2. Recent evidence indicates that
IcsA need to be assisted by the specific periplasmic chaperones Skp,
DegP, and SurA. Mutants of the three genes will be contructed in S. flexneri to analyze their involvement on the polar localization of PhoN2.
5-
To assess whether OspB or PhoN2 affect the inflammatory response. The
Sereny test model of infection will be used. Eyes of guinea pigs will be
infected with wild-type and mutants strains carrying deletions
encompassing ospB, phoN2 or the entire operon. Increased or decreased
inflammatory responses will be evaluated.
Principal Investigator:
Mauro Nicoletti, Associate Professor of Microbiology, Department of
Experimental and Clinical Sciences, University G. D'Annunzio. Chieti.
Italy