During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II beta-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a critical role in negatively regulating T cell responses and has also been implicated in the development and function of natural FOXP3+ regulatory T cells. CTLA-4–deficient mice develop fatal, early onset lymphoproliferative disease. However, chimeric mice containing both CTLA-4–deficient and –sufficient bone marrow (BM)–derived cells do not develop disease, indicating that CTLA-4 can act in trans to maintain T cell self-tolerance. Using genetically mixed blastocyst and BM chimaeras as well as in vivo T cell transfer systems, we demonstrate that in vivo regulation of Ctla4−/− T cells in trans by CTLA-4–sufficient T cells is a reversible process that requires the persistent presence of FOXP3+ regulatory T cells with a diverse TCR repertoire. Based on gene expression studies, the regulatory T cells do not appear to act directly on T cells, suggesting they may instead modulate the stimulatory activities of antigen-presenting cells. These results demonstrate that CTLA-4 is absolutely required for FOXP3+ regulatory T cell function in vivo.

SUMMARY Bacterial virulence mechanisms are attractive targets for antibiotic development, because they are required for the pathogenesis of numerous global infectious disease agents. The bacterial secretion systems used to assemble the surface structures that promote adherence and deliver protein virulence effectors to host cells could comprise one such therapeutic target. In this study, we developed and performed a high-throughput screen (HTS) of small molecule libraries and identified a small molecule, a 2-imino-5-arylidene thiazolidinone that blocked secretion and virulence functions of a wide array of animal and plant Gram-negative bacterial pathogens. This compound inhibited type III secretion-dependent functions, with the exception of flagellar motility, and Type II secretion-dependent functions, suggesting that the target of the compound could be an outer membrane component conserved between these two secretion systems. This work provides a proof of concept that compounds with a broad spectrum of activity against Gram-negative bacterial secretion systems could be developed to prevent and treat bacterial diseases.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory receptor on T cells essential for maintaining T cell homeostasis and tolerance to self. Mice lacking CTLA-4 develop an early onset, fatal breakdown in T cell tolerance. Whether this autoimmune disease occurs because of the loss of CTLA-4 function in regulatory T cells, conventional T cells, or both is unclear. We show here that lack of CTLA-4 in regulatory T cells leads to aberrant activation and expansion of conventional T cells. However, CTLA-4 expression in conventional T cells prevents aberrantly activated T cells from infiltrating and fatally damaging nonlymphoid tissues. These results demonstrate that CTLA-4 has a dual function in maintaining T cell tolerance: CTLA-4 in regulatory T cells inhibits inappropriate naïve T cell activation and CTLA-4 in conventional T cells prevents the harmful accumulation of self-reactive pathogenic T cells in vital organs.

Regulatory T cells (T reg cells) constitute a population of CD4+ T cells that limits immune responses. The transcription factor Foxp3 is important for determining the development and function of T reg cells; however, the molecular mechanisms that trigger and maintain its expression remain incompletely understood. In this study, we show that mice deficient for the Ets-1 transcription factor (Ets-1−/−) developed T cell–mediated splenomegaly and systemic autoimmunity that can be blocked by functional wild-type T reg cells. Spleens of Ets-1−/− mice contained mostly activated T cells, including Th2-polarized CD4+ cells and had reduced percentages of T reg cells. Splenic and thymic Ets-1−/− T reg cells expressed low levels of Foxp3 and displayed the CD103 marker that characterizes antigen-experienced T reg cells. Thymic development of Ets-1−/− T reg cells appeared intrinsically altered as Foxp3-expressing cells differentiate poorly in mixed fetal liver reconstituted chimera and fetal thymic organ culture. Ets-1−/− T reg cells showed decreased in vitro suppression activity and did not protect Rag2−/− hosts from naive T cell–induced inflammatory bowel disease. Furthermore, in T reg cells, Ets-1 interacted with the Foxp3 intronic enhancer and was required for demethylation of this regulatory sequence. These data demonstrate that Ets-1 is required for the development of natural T reg cells and suggest a role for this transcription factor in the regulation of Foxp3 expression.