Dual infection system identifies a crucial role for PKA-mediated serine phosphorylation of the EPEC-Tir-injected effector protein in regulating Rac1 function.
Many Gram-negative pathogenic bacteria possess type-III or type-IV secretion systems to inject 'effector' proteins directly into host cells to modulate cellular processes in their favour. A common target is the actin-cytoskeleton linked to the delivery of a single (CagA) effector by Helicobacter pylori and multiple effectors by enteropathogenic Escherichia coli (EPEC) respectively. Here we report co-infection as a powerful strategy for defining effector protein function and mechanisms by which they modulate cellular responses. This is exemplified by our finding that EPEC inhibits H. pylori-induced AGS cell elongation, a disease-related event linked to Rac1 activation. While this inhibitory process is dependent on the translocated Intimin receptor, Tir, and the outer-membrane protein, Intimin, it unexpectedly revealed evidence for Tir signalling independent of Intimin interaction and tyrosine phosphorylation of Tir. Furthermore, the work defined a long awaited role for protein kinase A (PKA)-mediated phosphorylation of Tir at serine-434 and serine-463. Our data are consistent with a model whereby EPEC activates PKA for Tir phosphorylation. Activated PKA then phosphorylates Rac1 at serine-71 associated with reduced GTP-load and inhibited cell elongation. Thus, the co-infection approach is a powerful strategy for defining novel effector function with important implications for characterizing mechanisms and regulatory signalling pathways in bacterial pathogenesis.
Department of Medical Microbiology, Otto von Guericke University, Leipziger Str. 44, D-39120 Magdeburg, Germany.
Sabine
S
Brandt
Brendan
B
Kenny
Manfred
M
Rohde
Narcisa
N
Martinez-Quiles
Steffen
S
Backert
D018829:Adhesins, Bacterial / Q000378:metabolism; D000942:Antigens, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D054308:Enteropathogenic Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000150:complications*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D016481:Helicobacter Infections / Q000150:complications*; D016480:Helicobacter pylori / Q000378:metabolism / Q000472:pathogenicity; D054884:Host-Pathogen Interactions; D006801:Humans; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D014774:Virulence; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D018829:Adhesins, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C107953:RAC1 protein, human; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; C080866:cagA protein, Helicobacter pylori; C060296:cytotoxic necrotizing factor type 1; C073819:eaeA protein, E coli; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D020830:rac1 GTP-Binding Protein