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The beast and its Achilles heel:
A novel target to fight multi-resistant pathogenic bacteria
The lack of novel antibiotics: the problem that we address
The discovery of penicillin by Alexander Fleming in 1928 and the broad application of antibiotics marked a major victory of mankind in the battle against infectious diseases.
However, shortly after the beginning of industrial penicillin production in 1942 some human pathogens already acquired resistance against penicillin and related antibiotics.
Moreover, the frequent use of antibiotics in the past and the rapid adaptation of bacteria to these compounds led to the development of many multi-resistant pathogens.
Some of the pathogenic bacteria are even resistant against most commercially available antibiotics. Therefore, both the discovery and the development of new antibacterial substances are extremely important to fight these threats of human health. Unfortunately, the pipeline of novel compounds with antibiotic activity and appropriate pharmaceutical properties is empty. This makes the challenge of finding new compounds even more urgent!
The first step in designing efficient novel antibacterial compounds is the identification of a suitable target. Very recently, a potentially interesting target has been identified. The emerging signaling molecule cyclic dinucleotide c-di-AMP is essential in the Gram-positive model organism B. subtilis and in closely related pathogenic bacteria such as Listeria monocytogenes, Streptococcus pneumoniae, and Staphylococcus aureus.
These bacteria cause serious diseases and are often multi-resistant. c-di-AMP is needed for the control of vital cellular processes in these pathogens because both: lack and accumulation of c-di-AMP, strongly inhibit growth of the bacteria.
Therefore, any substance that disturbs the homeostasis of the important signaling molecule c-di-AMP and the interaction with its target is of substantial interest to fight human pathogens. The next important step in identifying efficient antibacterial compounds, which either inhibit c-di-AMP biosynthesis or interfere with the essential function of c-di-AMP in the cell, is the development of a powerful screening system.
Our project:
Our project is aimed at the development of a simple screening system, which allows the rapid identification and characterization of substances that disturb c-di-AMP homeostasis in pathogenic bacteria.
The principle of how such a screening system could look like is illustrated in Figure 1. The screening system will be established in the non-pathogenic bacterium E. coli.
First, we want to construct a promoter-reporter gene fusion which allows us to monitor the activity of a transcription factor that only binds to a specific DNA sequence (operator) in the presence of c-di-AMP. The operator sequence will be placed between a constitutively active promoter and a reporter gene, such as lacZ and gfp encoding the -galactosidase and the green-fluorescent protein (GFP), respectively.
The activity of either of the two proteins is very easy to detect. Then, we will evaluate whether binding of the transcription factor and thus inhibition of the promoter-reporter gene fusion can be controlled by exogenous c-di-AMP.
Finally, we want to express a diadenylate cyclase from B. subtilis to inhibit the promoter reporter gene fusion by endogenously synthesized c-di-AMP. There are two big advantages of using E. coli as a host for the development of a screening system to identify antibacterial compounds that interfere with c-di-AMP homoeostasis in Gram-positive pathogenic bacteria.
First, c-di-AMP is not synthesized in E. coli. Thus, compounds that inhibit c-di-AMP synthesis will specifically inhibit growth of Gram-positive bacteria. Second, the use of a nonpathogenic E. coli strain, which is easy to cultivate will keep the costs very low.
We are confident that our screening system will facilitate the identification of novel antibacterial substances because any change in the activity of the c-di-AMP-dependent promoter-reporter gene fusion, either by inhibition of c-di-AMP synthesis or by activation of DNA-binding activity of the transcription factor will indicate perturbation of c-di-AMP homeostasis.
