Transcriptional Regulation of a major virulence operon in Staphylococcus aureus: Insights from biochemcial genomic and proteomic studies

Staphylococcus aureus is a bacterium that is often referred to as a 'superbug' because of its ability to cause a diverse range of human and animal diseases and to resist antibiotic treatment (e.g. the MRSA-strain). S. aureus is a major human and animal pathogen because it can produce a wide range of virulence factors, which allow the bacterium to attach itself to the host, evade the host immune system and to rapidly degrade host tissue through the production of potent toxins, e.g. haemolysins which break down blood cells. The expression of genes in S. aureus responsible for the production of virulence factors are tightly controlled by several gene regulatory systems. It is generally accepted that the so called 'agr system' is the 'master' regulatory system involved in the control and expression of virulence genes in S. aureus. Here, we propose to study how the expression of the 'agr system' itself is controlled in the different stages of growth of S. aureus cells and in response to the environmental conditions encountered by S. aureus cells during growth and infection. In particular, we will describe and indentify the component interactions between proteins and DNA that contribute to 'switching on' the agr system. This study will provide one of the fewest examples, where a major virulence-linked gene regulatory system is described at the molecular level in a clinically significant bacterial pathogen. Thus, the outcome of this research will generate valuable resources and a knowledge base for the academic and clinical research communities and those in industrial settings seeking to exploit the agr system to create novel drugs for the treatment of staphylococcal infections.

The Gram-positive bacterium Staphylococcus aureus is an opportunistic human and animal pathogen responsible for a wide array of diseases of various severities depending on the site of infection and immune status of the host. The ability of S. aureus to cause disease is due to its ability to express a variety of virulence factors in a highly co-ordinated and growth phase and environment dependent manner, which enables the bacterium to successfully invade the host and evade host immune response. The expression of virulence genes in S. aureus is subject to tight control by several global gene regulatory systems. However, the accessory gene regulatory (agr) operon is considered to be the 'master' system responsible for the growth phase and environment dependent co-ordination and regulation of virulence gene expression in S. aureus. The agr operon consists of two divergently transcribed promoters, P2 and P3, which encode a quorum-sensing system and a pleiotropic regulatory RNA molecule, respectively. Previous in vivo studies with agr operon-deleted S. aureus strains and recent mathematical modelling of the agr operon mediated impact on virulence gene expression at the whole-genome level clearly underscore the central role of the agr operon in S. aureus biology and pathogenesis. However, how the expression of the agr operon itself is regulated at the transcriptional level - the first and mostly controlled step during bacterial gene expression - is not well understood. Here, we will take advantage of an in-house developed fully native S. aureus in vitro transcription system to unravel the mechanisms that govern expression of the agr operon at the transcriptional level using bespoke biochemical, genomic and proteomic approaches. Results will provide the first comprehensive molecular description of the regulatory events that lead to the expression of a major virulence-linked operon in this clinically significant Gram-positive bacterial pathogen.

The research proposed focuses on how a major virulence-associated gene regulatory system- the agr operon in Staphylococcus aureus- is regulated at the transcriptional level in a clinically relevant Gram-positive bacterial pathogen. Further, it seeks to indentify novel virulence-associated or essential genes in S. aureus at a genome-wide and, to a certain extent, at the proteome-wide scale. In addition to contributing to advancing our understanding of bacterial gene regulatory processes and systems in general, we expect that results will also be beneficial to those seeking to exploit such knowledge for the identification and/or development of novel targets for antibacterial drugs to combat or prevent diseases caused by S. aureus. Thus, the proposed research will impact basic and clinical microbiology research in the academic and industrial settings. To this end, we will ensure that results are rapidly published in peer-reviewed journals and by presenting our work at national and international research meetings. Where possible, we will make our findings available to the wider public (by publication in popular science magazine etc.). Any possibilities for commercial exploitation (i.e. evaluation of targets for drug discovery etc.) will be realised via locally existing structures (e.g. Imperial Drug Discovery Facility, IC Innovations etc.) and established contacts. In the proposed research we will employ state-of-the-art biochemical and post-genomic methods such as ChIP-chip and SELDI-MS to evaluate biological processes, not only at the molecular level but also at the system-wide level. This provides an excellent platform to enhance training of the associated RA and undergraduate/postgraduate students, who will contribute to the proposed research by conducting e.g. mini-projects etc. Such trained RAs (and associated PhD, masters and undergraduate students) will represent the 'next generation' of experimental bio-scientists and are likely to benefit the biotechnology and pharmaceutical industries, as well as the academic base in the UK and its representation abroad. We therefore anticipate medium term economic benefits arising from a well-trained UK research base, reflected in maintaining internationally competitive research intensive universities and associated industries. Since our research concerns a clinically significant bacterial pathogen, outcome of the proposed research has the potential to impact the government and healthcare policies in the long term.