Inhibition of Bacterial Type III Secretion by Salicylanilides

The emergence and spread of antibiotic resistance among microbes is a serious threat to the control of disease in humans, animals and plants. One alternative to the use of conventional antibiotics is the development of agents that disable specific virulence factors of the microbial pathogen without affecting survival or growth. A selection of salicylanilide compounds has been made available to us which are known to inhibit expression of the Type III secretion system (TTSS) of Y. pseudotuberculosis. TTSS have been identified in a variety of pathogenic bacteria. This complex apparatus is composed of approximately 20 different proteins and functions to 'inject' bacterial virulence proteins directly into the host cell. Our preliminary data indicates that salicylanilides also affect the SPI-1 encoded Type-III secretion system (TTSS-1) of the zoonotic pathogen Salmonella enterica. TTSS-1 is required for invasion of non- phagocytic cells and for induction of enteropathogenic responses in vivo. <P>Our primary aim is to assess the potential of these compounds to inhibit TTSS activity, to investigate the molecular mechanism underlying the inhibition, and to use these compounds to study fundamental aspects of bacterial virulence. Our studies will primarily focus on the S. enterica serovars Dublin and Typhimurium, however we will also assess if salicylanilides could inhibit Type III secretion in enteropathogenic E. coli and Burkholderia pseudomallei. <P>Our objectives are: <OL> <LI> To investigate the molecular mechanisms underlying inhibition of Salmonella TTSS-1 by salicylanilides. Evaluating the level at which salicylanilides act by assessing transcription of genes encoding structural components of TTSS-1 and TTSS-1 secreted proteins (Sips and Sops), assessing assembly of the needle complex, assessing secretion of Sips and Sops, and assessing translocation of Sops into eukaryotic cells. <LI>. To employ salicylanilides as molecular probes in a chemical genetic approach to study the importance of TTSS-mediated secretion in bacterial pathogenesis. Determine whether inhibition of the function of TTSS-1 translates directly to reductions in Salmonella invasion of non-phagocytic cell lines, in intracellular growth of bacteria, and also in Salmonella-induced enteropathogenic responses. Use compounds to "switch off" virulence-associated TTSS at different stages of the infection to aid studies of fundamental mechanisms underlying bacterial pathogenicity. <LI>To evaluate if the inhibitory effect of salicylanilides could be alleviated by mutational inactivation of Salmonella gene(s). Screen an existing mutant library of Salmonella in the invasion assay in the presence of inhibitory concentrations of salicylanilides to identify mutants that are more invasive than the wild type strain and assess the potential of generating resistance to salicylanilides treatment through the inactivation of genes. <LI> To assess the potential for use of salicylanilides as novel broad spectrum anti- bacterial infection agents. Salicylanilides may also inhibit Type-III secretion in other pathogenic bacteria such as (EPEC) and B. pseudomallei. If so, it may be possible to identify compounds with broad range anti-pathogen activity. This will be achieved by using in vitro assays developed in our lab.