Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag

Abstract

Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3␤ kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3␤ antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.Many bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate effector proteins into eukaryotic cells (12,23,34). Injected effector proteins function to disrupt host cell signaling pathways that normally function to limit bacterial growth. The T3S apparatus is a complex supramolecular structure that spans the bacterial inner and outer membranes and is topped by a needle-like structure. T4SSs also utilize a multicomponent membrane-bound secretion apparatus to translocate effector proteins or protein-DNA complexes into targeted eukaryotic cells. The mechanism by which T3SSs and T4SSs recognize substrates and transport these substrates across both bacterial and host membranes is not well understood.The injection process can be divided into two distinct steps, (i) secretion of effector proteins across the bacterial membranes and (ii) translocation of effector proteins across a eukaryotic membrane. Recognition of cytosolic T3S substrates requires N-terminal signals, whereas recent analyses suggest that recognition of T4S substrates relies upon C-terminal signals (31,55,60,61). A role for specific chaperone-like proteins has also been confirmed for some, but not all, T3S and T4S substrates (16,17). Secretion chaperones may assist in the targeting of secretion substrates and/or maintain secretion substrates in a secretion-competent state.The identity of the T3S or T4S apparatus components that specifically recognize T3S or T4S signals is not known. Recent studies suggest that a conserved T3S ATPase plays an important role early in the T3S process. The T3S ATPase has been shown to directly interact with both secretion substrates and T3S chaperones (1, 22). ATPase activity is required for unfolding of T3S substrates and for the release of chaperones from their substrates (1). Not s...

The Helicobacter pylori CagF protein is a type IV secretion chaperone-like molecule that binds close to the C-terminal secretion signal of the CagA effector protein

“…To exclude the possibility that CagF is co-translocated with CagA into epithelial cells and is required there for CagA tyrosine phosphorylation, we constructed a fusion of the phosphorylatable GSK-3b reporter tag (Hohlfeld et al, 2006;Torruellas-Garcia et al, 2006) to the N terminus of CagF. Production of GSK-tagged proteins in H. pylori is assessed by immunoblotting using an anti-GSK-3b antiserum, and translocation of these proteins into AGS cells by measuring serine phosphorylation of the GSK tag (Fig.…”

Regulation of the Yersinia type III secretion system: traffic control

“…Differential detergent fractionation was also unable to accurately decipher YopK localization as it is found in the digitonin pellet fraction containing adherent bacteria, along with host cell membranes and organelles (Lee and Schneewind, 1999). With the creation of a GSK tag reporter system, Garcia et al finally demonstrated that YopK from Y. pestis is injected into host cells during infection (Garcia et al, 2006). Y. pseudotuberculosis YopK translocation was also observed recently using a β-lactamase (Bla) reporter (Thorslund et al, 2011).…”

Modulation of innate immune responses byYersiniatype III secretion system translocators and effectors

“…The mechanism by which YopK regulates translocation is not entirely clear. YopK itself is translocated into host cells (Brodsky et al ., 2010, Garcia et al ., 2006), and this translocation is required for YopK to exert its regulatory function (Dewoody et al ., 2013a, Dewoody et al ., 2013b). YopK deficiency is also associated with increased lysis of erythrocytes, which typically correlates with pore size, suggesting that YopK somehow modulates the size or structural properties of the translocon (Holmstrom et al ., 1997).…”