Inhibition of bacterial Type III secretion by salicylanilides

Background Most bacteria exist harmlessly in the environment; many millions actually on or within our own bodies, but a few species are capable of causing severe disease. It is only with the advent of antibiotics in the last half century that humanity has gained the advantage. Antibiotics are compounds which destroy bacteria or inhibit their growth. Sometimes bacteria can become naturally resistant to the antibiotic used due to a genetic change such as a mutation in a gene or acquisition of a new gene. Regrettably, we came to rely too heavily on antibiotics and their misuse has lead to a major problem: some bacteria have become resistant to almost all known antibiotics. There is a high possibility of encountering more untreatable bacterial infections in the future. The food-poisoning pathogen, Salmonella enterica, can infect both humans and animals causing stomach pain and diarrhoea. Infected animals can pass the disease to humans by direct contact or through contaminated meat, milk or eggs. Ingested Salmonella reach the intestines and must then compete with the millions of 'good' bacteria that inhabit the gut. Salmonella invades the intestine wall by switching on several 'virulence factors'. These are factors which are not necessary for day-to-day existence in the environment but are absolutely required to cause disease in the host. One of the most striking is the Type-III secretion system. This is a syringe-like structure used to 'inject' bacterial molecules directly into the host cell. These molecules then hijack the machinery of the host cell and enable Salmonella to enter the cell. From here Salmonella can move through the tissues deeper into the body. This invasion triggers pain and diarrhoea as the body detects the attack and tries to flush out the Salmonella. It may be possible to identify new types of antibacterial compound that will specifically disable bacterial virulence factors, thus preventing disease. Ideally, these compounds will not affect survival or growth of the bacteria so resistance of bacteria to the compounds should be less likely to emerge. Summary of proposed research. We have developed laboratory assays to measure Salmonella Type III secretion and functions. We will use these assays to screen a number of compounds for inhibition of Type III secretion and/or expression. The best candidates will be used in assays to investigate how the compounds work and to see whether they can reduce Salmonella invasion of cultured cells and gut tissues. We will also measure if these compounds are effective in reducing the amount of fluid secretion and gut inflammation that occurs in response to the invasion. As Salmonella actually has two TTSS, the second being mostly necessary for later stages of the disease, targeting the Type-III secretion system could therefore be very beneficial because a successful antibacterial compound may act on two systems to inhibit disease. Furthermore, other pathogenic microbes are also known to use a Type III secretion systems that are very similar to that found in Salmonella, so it is possible that some compounds may provide broad-range anti-pathogen treatments with minimal effects on commensal 'good' bacteria. Finally, compounds inhibiting Type III secretion systems could be used to study the process of how bacteria interact with host cells and cause diseases as they allow the type III secretion system to be 'switched off' at any stage of infectious process. All these studies will be of interest to scientists and non-scientists because antimicrobial agents that target virulence factors have the potential to revolutionise human and veterinary medicine. Such compounds may be used to prevent disease in individuals at risk of infection or alternatively used to treat established disease.

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. 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. Our objectives are: 1. 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. 2. 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. 3. 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. 4. 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.