Developing a Cell Based Screen for Inhibitors of Two Component Signal Transduction in Mycobacteria

The growing number of drug resistant strains of Mycobacterium tuberculosis appearing worldwide has had an enormous impact on the ability to control and treat Tuberculosis (TB). Discovering new anti-TB drugs is of paramount importance to the global effort for TB eradication. The success of the pathogen is largely due to its inherent ability to remain in a non-replicating or latent state for extended periods of time. In order to achieve this shift it requires tightly controlled signal transduction mechanisms to respond to its host environment. Two component systems (TCS) are one example of signalling mechanisms employed by prokaryotes and are ideal candidates for antibacterial drug targets. It is understood that many TCS are conserved in a large number of organisms, they are often essential to the virulence and persistence of pathogens and they are virtually exclusive to prokaryotes. In this study three Mycobacterium smegmatis TCS were selected; DevS/DevR, MtrB/MtrA and SenX3/RegX3. Promoters under the control of these systems were cloned into an optimised mycobacterial high copy number GFP reporter plasmid and subject to a number of in vitro stress conditions to ascertain induction conditions for these systems. As expected the DevS/DevR controlled hspX promoter was responsive to oxygen starvation and the SenX3/RegX3 controlled phoA was induced by phosphate starvation. Interestingly, phoA and mtrA were also induced by magnesium chelator EDTA in minimal media. The phoA and mtrA promoter constructs were then used for in vitro high throughput bioassays with a number of compound libraries in order to screen for any inhibitory activity on each of the target systems. A phosphorylation inhibitor included in one of the screens, oleic acid, indicated that this assay could potentially be used to screen for TCS inhibitors, but no novel compounds were found in this study. As a proof of principle, known TCS inhibitors palmitoleic and oleic acid were employed to show a dose dependent inhibition mtrA expression. This method could potentially be expanded to other TCS of Mycobacterium smegmatis and Mycobacterium bovis BCG, or other signal transduction systems such as one component regulators and serine threonine kinases

Extracellular matrix derived peptides and mesenchymal stem cell motility

Biomaterials derived from decellularised extracellular matrices have shown promise as tools in tissue regeneration and wound healing. Such materials display biocompatibility as well as inherent bioactivity, promoting constructive remodelling in healing tissues. In this study, the bioactivity of ovine forestomach matrix (a decellularised extracellular matrix biomaterial) is assessed based on its ability to affect the proliferation and migration of wound healing cells. This material supported cell attachment and proliferation, but did not allow cell infiltration in vitro. Enzymatic digestion of the material rendered soluble components that were able to induce proliferation and migration of some cell types. Cell-mediated processing of the material generated a protein or proteins with chemotactic activity for mesenchymal stem cells in vitro. Mass spectrometry analysis indicated the bioactive component consisted of the proteoglycan decorin, or fragments thereof. Decorin has not previously been shown to induce mesenchymal stem cell motility, and these findings may add to what is known about decorin and its role in constructive remodelling. Furthermore, this cell-mediated approach for ECM breakdown could lead to the discovery of other bioactive peptides involved in ECM remodelling and wound healing

Developing a Cell Based Screen for Inhibitors of Two Component Signal Transduction in Mycobacteria

The growing number of drug resistant strains of Mycobacterium tuberculosis appearing worldwide has had an enormous impact on the ability to control and treat Tuberculosis (TB). Discovering new anti-TB drugs is of paramount importance to the global effort for TB eradication. The success of the pathogen is largely due to its inherent ability to remain in a non-replicating or latent state for extended periods of time. In order to achieve this shift it requires tightly controlled signal transduction mechanisms to respond to its host environment. Two component systems (TCS) are one example of signalling mechanisms employed by prokaryotes and are ideal candidates for antibacterial drug targets. It is understood that many TCS are conserved in a large number of organisms, they are often essential to the virulence and persistence of pathogens and they are virtually exclusive to prokaryotes. In this study three Mycobacterium smegmatis TCS were selected; DevS/DevR, MtrB/MtrA and SenX3/RegX3. Promoters under the control of these systems were cloned into an optimised mycobacterial high copy number GFP reporter plasmid and subject to a number of in vitro stress conditions to ascertain induction conditions for these systems. As expected the DevS/DevR controlled hspX promoter was responsive to oxygen starvation and the SenX3/RegX3 controlled phoA was induced by phosphate starvation. Interestingly, phoA and mtrA were also induced by magnesium chelator EDTA in minimal media. The phoA and mtrA promoter constructs were then used for in vitro high throughput bioassays with a number of compound libraries in order to screen for any inhibitory activity on each of the target systems. A phosphorylation inhibitor included in one of the screens, oleic acid, indicated that this assay could potentially be used to screen for TCS inhibitors, but no novel compounds were found in this study. As a proof of principle, known TCS inhibitors palmitoleic and oleic acid were employed to show a dose dependent inhibition mtrA expression. This method could potentially be expanded to other TCS of Mycobacterium smegmatis and Mycobacterium bovis BCG, or other signal transduction systems such as one component regulators and serine threonine kinases

Extracellular matrix derived peptides and mesenchymal stem cell motility

Biomaterials derived from decellularised extracellular matrices have shown promise as tools in tissue regeneration and wound healing. Such materials display biocompatibility as well as inherent bioactivity, promoting constructive remodelling in healing tissues. In this study, the bioactivity of ovine forestomach matrix (a decellularised extracellular matrix biomaterial) is assessed based on its ability to affect the proliferation and migration of wound healing cells.  This material supported cell attachment and proliferation, but did not allow cell infiltration in vitro. Enzymatic digestion of the material rendered soluble components that were able to induce proliferation and migration of some cell types. Cell-mediated processing of the material generated a protein or proteins with chemotactic activity for mesenchymal stem cells in vitro. Mass spectrometry analysis indicated the bioactive component consisted of the proteoglycan decorin, or fragments thereof. Decorin has not previously been shown to induce mesenchymal stem cell motility, and these findings may add to what is known about decorin and its role in constructive remodelling. Furthermore, this cell-mediated approach for ECM breakdown could lead to the discovery of other bioactive peptides involved in ECM remodelling and wound healing.</p

A novel chemotactic factor derived from the extracellular matrix protein decorin recruits mesenchymal stromal cells in vitro and in vivo.

Soft tissue is composed of cells surrounded by an extracellular matrix that is made up of a diverse array of intricately organized proteins. These distinct components work in concert to maintain homeostasis and respond to tissue damage. During tissue repair, extracellular matrix proteins and their degradation products are known to influence physiological processes such as angiogenesis and inflammation. In this study we developed a discovery platform using a decellularized extracellular matrix biomaterial to identify new chemotrophic factors derived from the extracellular matrix. An in vitro culture of RAW.264 macrophage cells with the biomaterial ovine forestomach matrix led to the identification of a novel ~12 kDa chemotactic factor, termed 'MayDay', derived from the N-terminal 31-188 sequence of decorin. The recombinant MayDay protein was shown to be a chemotactic agent for mesenchymal stromal cells in vitro and in vivo. We hypothesize that the macrophage-induced cleavage of decorin, via MMP-12, leads to the release of the chemotactic molecule MayDay, that in turn recruits cells to the site of damaged tissue