by Infect Immun. 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 Mibampator compounds with a broad spectrum of activity against Gram-negative bacterial secretion systems could be developed to prevent and treat bacterial diseases. INTRODUCTION In the twentieth century the treatment of infectious diseases was revolutionized by the development of antibiotics (Morens et al., 2004). However, due to their widespread use resistance to antibiotics is increasing on a global scale, such that adequate therapies are lacking for both previously controlled and emerging bacterial diseases (Levy and Marshall, 2004; Marra, 2006; Morens et al., 2004). Moreover, the molecular targets and mechanisms of action of most newly developed antibiotics are similar to current ones (Levy and Marshall, 2004; Nathan, 2004), Mibampator reducing their efficacy in the face of resistance. Mibampator The effective treatment of infectious diseases in the face of increasing antibiotic resistance will likely require the development of pharmaceuticals that act upon previously unutilized conserved targets (Levy and Marshall, 2004). Recently, bacterial virulence properties have been proposed and explored as attractive targets for the development of new therapeutic agents (Marra, 2006). This strategy could decrease the likelihood for selection of resistance, because in contrast to currently available antibiotics these agents would presumably not require inhibition of general bacterial growth. Such compounds would likely have the advantage of sparing commensals, reducing the likelihood of side effects. A potential disadvantage of pathogenic mechanisms as therapeutic targets is that many are microbial specific, necessitating more rapid pathogen identification than currently is in clinical practice. Gram-negative bacterial virulence Mibampator secretion systems represent particularly appealing virulence factor targets; because they are essential for a wide array of animal and plant infectious diseases and have some functionally conserved components. Two prominent examples of Gram-negative bacterial virulence associated secretion systems, termed type II secretion (T2S) and type III secretion (T3S), are responsible for the pathogenesis of many infectious diseases including plague, gastroenteritis, Gram-negative pneumonia, dysentery, enteric fever, tularemia, trachoma, endometritis and a variety of plant diseases. T2S is also known as the terminal component of the (EPEC), and (Mattick, 2002). T3S systems are complex multi-protein organelles that assemble in the bacterial membrane of more than 25 Gram-negative animal and plant pathogens to deliver multiple virulence proteins, or effector proteins, directly from the bacterial cytosol into host cells. These secreted Rabbit polyclonal to PTEN proteins influence host cell physiology by altering a variety of antibacterial functions with resultant disease (Cornelis and Van Gijsegem, 2000). In recent years whole-cell based high-throughput screens have been performed to identify inhibitors of T3S systems (Gauthier et al., 2005; Kauppi et al., 2003; Pan et al., 2007). These screens have identified several classes of synthetic compounds and the natural product glycolipid caminosides, as active for inhibition of T3S in a broad range of Gram-negative bacterial pathogens, including and (Gauthier et al., 2005; Kauppi et al., 2003; Linington et al., 2006; Linington et al., 2002; Negrea et al., 2007; Nordfelth et al., 2005; Wolf et al., 2006). The salicylanilides likely inhibit T3 gene transcription while the salicylideneacylhydrazides, sulfonylaminobenzanilides and caminosides have unknown targets. In this study, we designed and implemented a tractable high-throughput screen (HTS) for the identification of compounds that could function to inhibit T3S secretion systems and Mibampator found a small molecule that broadly inhibits T3 and T2 bacterial secretion systems. RESULTS A high throughput screen using engineered identifies a 2-imino-5-arylidene thiazolidinone as an inhibitor of T3S To screen biological and chemical small molecule libraries for inhibitors of bacterial secretion; we designed and employed a whole-cell HTS for inhibitors of T3S. T3S systems, which are evolutionarily related to flagella, are complex multi-protein organelles that assemble in the bacterial membrane to deliver virulence proteins directly from the bacterial cytosol into host cells. A strain of was constructed that secretes a phospholipase A2 reporter construct in a T3S-dependent manner. Specifically, this strain contains a protein fusion between the T3S substrate SipA and the T3 secreted substrate YplA. The first 59 amino acids were removed from YplA, because they contain the signal sequence for its secretion by strain, fluorescence is proportional to the amount of phospholipase reporter secreted by the T3S system. Using this HTS, 92,000.
