Determining bacterial RNA polymerase functionalities required for sigma factor specific escape from antibiotic action.

Basic research best contributes to social and economic goals when targeted to areas that can benefit from additional fundamental knowledge, as reported in: 1) Nelson, R.R., 2004. The market economy and the scientific commons. Res. Policy 33: 455-471; 2) Rosenberg, Nathan, and Richard R. Nelson (1994), "American Universities and Technical Advance in Industry," Res. Policy 23:323-348; 3) Rosenberg, Nathan (1982), "How Exogenous is Science?" in Inside the Black Box (NY: Cambridge University Press), p. 141-159. In this proposal we seek to establish how bacteria respond to the agents that are used to control their growth in infection settings.

Knowing the basis of relative sensitivities of bacteria to antimicrobials is important for managing infection in live stock and in humans, and informs strategies to manage infection and design new antimcrobials. One validated target for antimicrobials is the central machinery of gene expression, the enzyme RNA polymerase (RNAP) that decodes the DNA. Knowing the basis of relative sensitivities of various RNAP holoenzymes to antimicrobials is important, and speaks to how the intrinsic levels of sensitivity may be accounted for - something of practical significance.

The application was motivated by the gap in basic knowledge which currently exists simply because the range of RNAP states - reflected by the use of differing accessory factors called sigma factors - are not often taken into account when assessing the efficacies of antimicrobials that target bacterial RNAPs. The response of various RNAPs to antimicrobials is important, and speaks to how the intrinsic levels of sensitivity of an organism or gene set might be accounted for. Emerging evidence shows that transcription factors, such as RNAP sigma factors, can be members of a group of factors that form the resistome. Showing that is, that the basic make up of the cell that can confer significant insensitivity to an antimicrobial without invoking the action of specific resistance mechanisms.

Since transcription factors such as RNAP sigma factors can be members of a resistome, we wish to work out how these factors confer upon RNAP tolerance to antimicrobials. Outcomes arising are expected to enlarge our knowledge of the action of antibiotics that target bacterial RNAP, as well as how the RNA functions to access the genetic information in DNA. We will use methods established by us to examine which steps in the DNA decoding process are sensitive or resistant to the actions of antibiotics, in particular those agents which are emerging as new candidates for antimicrobial therapies.

For more than a decade, genomic approaches have been used with only limited success to identify new potential antibiotic targets essential for bacterial viability. The bacterial RNA polymerase (bRNAP); is a validated target and is an attractive target for antimicrobial therapy given (i) RNAP is an essential enzyme in transcription ; (ii) bRNAP-subunit sequences are conserved (permitting broad-spectrum activities); and (iii) bacterial and eukaryotic RNAP-subunit sequences are less well conserved (permitting therapeutic selectivity). Currently there is only one class of clinical antibiotics that target bRNAP, rifamycins (Rif). However, emergence of bacterial strains resistant to rifamycins has greatly compromised treatment of many bacterial pathogens including Mycobacterium (tuberculosis), Staphylococcus (MRSA) and Listeria. There is therefore an urgent need to develop new antibiotics to treat infections caused by Rif-resistant and multidrug-resistant bacteria. A detailed mechanistic understanding of transcription, in particular knowledge of the various mechanisms by which RNAP-associated antibiotic resistance arise is of immense importance. These findings will guide essential steps in the development and use of future therapeutic agents. Knowledge of intrinsic resistance mechanisms is of growing importance in developing new antimicrobial strategies. Here we will study one intrinsic resistance mechanism associated with bRNAP function. The intrinsic resistance mechanism relies on the association of the bRNAP with one of its sigma factors, and how this form of bRNAP holoenzyme confers tolerance to antibiotics will be dissected biochemically and genetically using purified components and readouts of the various stages of the transcription process. Outcomes are expected to enlarge our understanding of the mechanisms operating for gene specific expression by bRNAP as well as provide a basis to rationalise intrinsic mechanisms of antimicrobial resistance in bacteria.

For more than a decade, genomic approaches have been used with only limited success to identify new potential antibiotic targets essential for bacterial viability. The bacterial RNA polymerase (bRNAP) is a validated and attractive target for antimicrobial therapy given (i) RNAP is an essential enzyme in transcription; (ii) bRNAP-subunit sequences are conserved (permitting broad-spectrum activities); and (iii) bacterial and eukaryotic RNAP-subunit sequences are less well conserved (permitting therapeutic selectivity). Currently there is only one class of clinical antibiotics that target bRNAP, rifamycins (Rif). However, emergence of bacterial strains resistant to rifamycins has greatly compromised treatment of many bacterial pathogens including Mycobacterium (tuberculosis), Staphylococcus (MRSA) and Listeria. There is therefore an urgent need to develop new antibiotics to treat infections caused by Rif-resistant and multidrug-resistant bacteria. A detailed mechanistic understanding of transcription, in particular knowledge of the various mechanisms by which RNAP-associated antibiotic resistance arise is of immense importance. These findings will guide essential steps in the development and use of future therapeutic agents. An emerging alternative to essential functions as drug targets is to target a set of gene products whose inactivation enhances the activity of existing antibiotics - potentially acting as combination therapy targets. Knowledge of intrinsic resistance mechanisms is of growing importance in developing new antimicrobial strategies.

The overall aim of the research outlined in this proposal is to determine the basis by which one particular sigma factor, called sigma54, confers greater tolerance to new and emerging antimicrobials that directly target bRNAPs. Dissecting the bRNAP functionalities that underpin the mechanisms by which antibiotic resistance is acquired at the protein level, in combination with current and developing structural models will allow the improved rational design of antibiotics - as well as suggest new features of the transcription complex that can be targeted for future antimicrobial drug development. The work will add to the emerging awareness of needing to understand how intrinsic resistance mechanisms operate.

The proposed research through the training of the named RA and connectivity to pharma will generate a valuable knowledge-base, a variety of non-invasive experimental approaches (e.g. use of low-MW inhibitors to interrogate structure-function relationships) and useful resources for investigating RNAP function from single molecule through to genomic levels. The research should uncover novel vulnerabilities in bRNAP that can be exploited for rational design of antimicrobial compounds. This is important since bRNAP is a proven target for antimicrobials, but Rif is currently the only commercially available anti-bRNAP antibiotic - to which many bacteria have become resistant. The application of low-MW inhibitors as chemical genetic probes to interrogate structure-function relationships in E. coli bRNAP is particularly well-aligned to the latter point and is well suited to clinical and commercial exploitation. The study of RNAP in the context of the sigma 54 system is significant, since genes under the control of sigma54-bRNAP are employed to express a wide-range of virulence determinants in several important animal and plant pathogens such as Chlamydia trachomatis, Borrelia burgdorferi, Vibrio cholerae and Pseudomonas spp.