Abstract
Biofilm infections have no approved effective medical treatments and can only be disrupted via physical means. This means that any biofilm infection that is not addressable surgically can never be eliminated and can only be managed as a chronic disease. Therefore, there is an urgent need for the development of new classes of drugs that can target the metabolic mechanisms within biofilms which render them recalcitrant to traditional antibiotics. Persister cells within the biofilm structure may play a large role in the enhanced antibiotic recalcitrance of bacteria biofilms. Biofilm persister cells can be resistant to up to 1000 times the minimal inhibitory concentrations of many antibiotics, as compared to their planktonic envirovars; they are thought to be the prokaryotic equivalent of metazoan stem cells. Their metabolic resistance has been demonstrated to be an active process induced by the stringent response that is triggered by the ribosomally-associated enzyme RelA in response to amino acid starvation. This 84-kD pyrophosphokinase produces the “magic spot” alarmones, collectively called (p)ppGpp. These alarmones act by directly regulating transcription by binding to RNA polymerase. These transcriptional changes lead to a major shift in cellular function to both upregulate oxidative stress-combating enzymes and down regulate major cellular functions associated with growth and replication. These changes in gene expression produce the quiescent persister cells. In this work, we describe a hybrid in silico laboratory pipeline for identifying and validating small-molecule inhibitors of RelA for use in the combinatorial treatment of bacterial biofilms as re-potentiators of classical antibiotics.
Highlights
Biofilms can be defined as a multicellular stage in the bacterial life cycle wherein bacteria, through multiple intercellular communication systems, create densely populated communities embedded within a self-extruded extracellular polymeric matrix [1,2,3,4,5]
Using the structural information gained from the in silico and laboratory studies, we developed a computationally-based pipeline to identify RelA inhibitors from large databases of known compounds that provided for the screening of compounds in a relatively timely and cost-effective manner
We have previously reported that E. coli strain C [50] is the only one of the five major “laboratory strains” of E. coli that is a superior biofilm former; this strain was used in our biofilm assays
Summary
Biofilms can be defined as a multicellular stage in the bacterial life cycle wherein bacteria, through multiple intercellular communication systems, create densely populated communities embedded within a self-extruded extracellular polymeric matrix [1,2,3,4,5]. The activation of the ribosomally-associated RelA enzyme via amino acid starvation triggers the bacterial stringent response that leads to the phenotypic changes that underlie the extreme recalcitrance that biofilms exhibit towards antibiotics [12] This ancient bacterial stress response produces an active metabolic state that results in the inability to treat chronic infections resulting from biofilms. There are only a very limited number of inhibitors known for RelA and (p)ppGpp that have been identified principally through traditional drug discovery methods, such as substrate analog design and high-throughput compound screening, none of which are candidates for clinical trials for the control of biofilm infections. It has become possible through alignment and homology studies to determine the active residues within the catalytic center of these enzymes and to target this region to predict and understand the ligand binding events for the rational identification of inhibitors
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