Determining Cellular and Molecular Targets of the Yersinia Tyrosine Phosphatase YopH During Animal Infection.
Durand, Enrique.
2011
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Abstract: The
gram-negative enteric pathogen Yersinia pseudotuberculosis employs a type III secretion
system that is required to translocate proteins essential for virulence, called Yops
(Yersinia outer proteins), into host cells. Yops are efficiently translocated to a wide
variety of cells grown in culture and most interact with many different mammalian
proteins in different cell types; ... read morehowever their cellular and molecular targets in animal
infection were unknown. Using a fluorescence-based assay that employs a membrane
permeable dye, CCF2-AM, that is a substrate for a YopH-β-lactamase fusion protein,
I identified the types of cell targeted by Yersinia by FACS analysis. Professional
phagocytes, i.e. neutrophils, macrophages and dendritic cells, were selectively targeted
by Yersinia for translocation of Yops in the Peyer's patches, mesenteric lymph nodes and
spleen after oral-gastric inoculation. In the absence of these cells, only low levels of
Yop translocation were detected indicating that during infection Yersinia specifically
chooses these cells. Interestingly, when the architecture of the spleen was destroyed,
Yersinia retained its preference for translocation into professional phagocytes compared
to B and T cells from splenocytes at low multiplicity of infection (MOI) but not at high
MOI. In addition, Yersinia bound preferentially to professional phagocytes versus B and
T cells from splenocytes and used two adhesins, YadA and Invasin, to enhance its
association with all splenocytes. Together these results indicate that Yersinia
discriminates among cells it encounters during infection and selectively delivers Yops
to phagocytes while refraining from translocation to other cell types. After identifying
neutrophils as a significant cell target of Yersinia, I determined a molecular target of
YopH in neutrophils during murine infection. YopH is a tyrosine phosphatase that
inhibits diverse cellular functions such as phagocytosis and ROS production in tissue
culture models. To identify the molecular target(s) of YopH during mouse infection,
neutrophils containing translocated with Yops from mice infected with wild-type Yersinia
or ∆yopH Yersinia were collected and analyzed using an anti-phospho tyrosine
antibody by Western blot. SLP-76 was dephosphorylated in the presence of YopH indicating
that YopH interferes with signal-transduction in Slp-76 controlled pathway(s) in
neutrophils. Changes in Syk phosphorylation, an upstream activator of Slp-76 were not
detected suggesting that Slp-76 was a direct target of YopH. We also show that
neutrophils translocated with YopH have reduced ability to flux calcium, which is
required for ROS production, and that YopH translocation leads to a reduction of IL-10
production during infection. In general, phagocytes producing ROS have increased levels
of secreted IL-10. Finally, a yopH mutant was better able to colonize spleens in mice
lacking neutrophils indicating that YopH inactivates neutrophils in infection. Combined
these data strongly suggest that Slp-76 is a molecular target of YopH and its function
in neutrophils is required to advert the immune system during animal infection. To our
knowledge, we have identified for first time cellular targets of Type III secretion
during murine infection.
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Ralph Isberg.
Committee: Carol Kumamoto, and Alexander Poltorak.
Keywords: Microbiology, Immunology, and Molecular biology.read less - ID:
- 7h14b184w
- Component ID:
- tufts:20314
- To Cite:
- TARC Citation Guide EndNote
