Direct class switching to IgE generates IgE+ GC cells that are highly apoptotic and do not contribute to the memory compartment, while sequential switching through an IgG+ intermediate results in the generation of long-lived IgE+ plasma cells.

Numerous Gram-negative bacterial pathogens employ type III secretion systems (T3SSs) to inject effector proteins into eukaryotic cells. The activation of the type III secretion (T3S) process is tightly controlled in all T3SSs. In Yersinia pestis, the secretion of effector proteins, termed Yersinia outer proteins (Yops), is regulated by the activity of the YopN/SycN/YscB/TyeA complex. YopN is a secreted protein that interacts with the SycN/YscB chaperone via an N-terminal chaperone-binding domain (CBD) and with TyeA via a C-terminal TyeA-binding domain (TBD). Efficient YopN secretion is dependent upon its N-terminal secretion signal (SS), CBD, and the SycN/YscB chaperone. In this study, we investigate the role of the YopN CBD in the regulation of Yop secretion. Analysis of YopE/YopN hybrid proteins in which the YopN SS or SS and CBD were replaced with the analogous regions of YopE indicated that the YopN CBD or SycN/YscB chaperone play a role in the regulation of Yop secretion that is independent of their established roles in YopN secretion. To further analyze the role of the YopN CBD in the regulation of Yop secretion a series of tetra-alanine substitution mutants were generated throughout the YopN CBD. A number of these mutants exhibited a defect in the regulation of Yop secretion but showed no defect in YopN secretion or in the interaction of YopN with the SycN/YscB chaperone. Finally, conditions were established that enabled YopN and TyeA to regulate Yop secretion in the absence of the SycN/YscB chaperone. Importantly, a number of the YopN CBD mutants maintained their defect in the regulation of Yop secretion even under the established SycN/YscB chaperone-independent conditions. These studies establish a role for the CBD region of YopN in the regulation of Yop secretion that is independent from its role in YopN secretion or in the binding of the SycN/YscB chaperone.

The Chlamydia pneumoniae CopN protein is a member of the YopN/TyeA/InvE/MxiC family of secreted proteins that function to regulate the secretion of type III secretion system (T3SS) translocator and effector proteins. In this study, the Scc1 (CP0432) and Scc4 (CP0033) proteins of C. pneumoniae AR-39 were demonstrated to function together as a type III secretion chaperone that binds to an N-terminal region of CopN. The Scc1/Scc4 chaperone promoted the efficient secretion of CopN via a heterologous T3SS, whereas, the Scc3 chaperone, which binds to a C-terminal region of CopN, reduced CopN secretion.

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...