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Inhibition of the Initiation of Bacterial Chromosome Replication As an Antimicrobial Strategy

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Inhibition of the Initiation of Bacterial Chromosome Replication As an Antimicrobial Strategy antibiotics Review Blocking the Trigger: Inhibition of the Initiation of Bacterial Chromosome Replication as an Antimicrobial Strategy Julia E. Grimwade * and Alan C. Leonard Program in Biological Sciences, Department of Biomedical and Chemical Engineering and Sciences, Florida Institute of Technology, 150 West. University Blvd., Melbourne, FL 32901, USA * Correspondence: grimwade@fit.edu; Tel.: +1-321-674-7152 Received: 30 June 2019; Accepted: 4 August 2019; Published: 6 August 2019 Abstract: All bacterial cells must duplicate their genomes prior to dividing into two identical daughter cells. Chromosome replication is triggered when a nucleoprotein complex, termed the orisome, assembles, unwinds the duplex DNA, and recruits the proteins required to establish new replication forks. Obviously, the initiation of chromosome replication is essential to bacterial reproduction, but this process is not inhibited by any of the currently-used antimicrobial agents. Given the urgent need for new antibiotics to combat drug-resistant bacteria, it is logical to evaluate whether or not unexploited bacterial processes, such as orisome assembly, should be more closely examined for sources of novel drug targets. This review will summarize current knowledge about the proteins required for bacterial chromosome initiation, as well as how orisomes assemble and are regulated. Based upon this information, we discuss current efforts and potential strategies and challenges for inhibiting this initiation pharmacologically. Keywords: antibiotic targets; orisome; oriC; DnaA; DnaB; DnaC; initiation of bacterial DNA replication 1. Introduction Since the 1940s, antibiotics have become an integral part of global health care, and combined with advances in food handling, sewage treatment and other improvements in general hygiene, antibiotics have led to a dramatic decrease in mortality and morbidity caused by bacterial infections [1]. The ability to control bacterial infections has also stimulated advances in surgical techniques and chronic disease management [2]. However, the use of antibiotics is accompanied by a rise in the number of drug-resistant bacteria in the environment. The emergence of antibiotic resistance is attributed largely to an overuse of antibiotics in human medicine and in agriculture, which puts enormous selective pressure upon bacterial populations, such that only those bacteria that have resistance mechanisms can survive [2]. Resistance comes from mutations in the bacterial molecular target of the drug, and by acquisition, via the horizontal gene transfer (HGT), of genes that can inactivate or remove the drugs from the bacterial cell [1]. HGT, in particular, has allowed the emergence of the so-called “Superbugs”: Bacteria that are resistance to multiple antibiotics. The origin of all transferred resistance elements is still speculative; however, antibiotic resistance determinants have been discovered in ancient DNA samples [3,4], and there is evidence that at least some of the resistance genes originated in antibiotic producers as part of their survival strategy [5,6]. Resistance determinants may have also evolved in non-producers to allow them to survive in environments shared with antimicrobial producers [7]. The prevalence of antibiotic-resistant bacteria has resulted in a global health crisis, and there is an urgent need for new antibiotics to treat resistant infections. Logically, when developing these new drugs, effort should be made to delay any development of resistance to them. However, many recent Antibiotics 2019, 8, 111; doi:10.3390/antibiotics8030111 www.mdpi.com/journal/antibiotics Antibiotics 2019, 8, x 2 of 17 The prevalence of antibiotic-resistant bacteria has resulted in a global health crisis, and there is an urgent need for new antibiotics to treat resistant infections. Logically, when developing these Antibioticsnew drugs,2019, 8effort, 111 should be made to delay any development of resistance to them. However,2 many of 17 recent drug discovery efforts have focused upon modifying existing scaffolds [8], and any new agents that are developed in this way risk being inactivated by the same mechanisms that affected drugthe parental discovery compounds. efforts have Further focusedmore upon, although modifying microbial existing extracts scaffolds have [8], andbeen any a valuable new agents source that of areantibiotics developed in inthe this past, way riskall antibiotics being inactivated from natural by the samesources mechanisms risk inactivation that affected by the the parentalresistance compounds.mechanisms Furthermore, originally used although by the microbial antibiotic extracts producers, have beenwhich a valuablecan be transferred source of antibioticsto pathogens in the via past,HGT. all Therefore, antibiotics fromalthough natural it is sources tempting risk to inactivation continue with by the past resistance antibiotic mechanisms discovery originallystrategies, used if we bywish the antibioticto avoid producers,a rapid development which can beof transferredresistance, toit pathogensmay be advantageous via HGT. Therefore, to avoid although pre-existing it is temptingresistanc toe continueelements withby broadening past antibiotic the discovery search to strategies, new areas. if weFor wish example, to avoid since a rapid current development antibiotics oftarget resistance, only a it handful may be of advantageous the approximately to avoid 300 pre-existing–400 essential resistance bacterial elements genes [9– by11] broadening, one solution the to searchdeveloping to new new, areas. effective For example, antibiotics since is current to target antibiotics novel pathways. target only It a might handful also of be the advantageous approximately to 300–400examine essential pathways bacterial that are genes normally [9–11], avoided one solution by microbial to developing antibiotic new, producers. effective antibiotics is to target novel pathways.One unexploited It might and also ecologically be advantageous-avoided to examinepathway pathwaysis the one that that are triggers normally an avoidedinitiation by of microbialbacterial antibioticchromosome producers. replication. This initiation is mediated by the assembly of the orisome complexOne unexploited that unwinds and DNA ecologically-avoided in the unique chromosomal pathway is the replication one that triggers origin an (oriC initiation) and prepares of bacterial for chromosomethe establishment replication. of new This repl initiationication forks. is mediated In this review, by the we assembly summarize of the current orisome knowledge complex about that unwindsorisome DNA assembly in the and unique the chromosomal initiator proteins replication used originin this (oriC process.) and preparesWe also for discuss the establishment progress on ofidentifying new replication initiation forks. inhibitors, In this review, and discuss we summarize new strategies current for knowledge targeting about replication orisome initia assemblytion. and the initiator proteins used in this process. We also discuss progress on identifying initiation inhibitors, and2. Proteins discuss newInvolved strategies in Initiation for targeting of Bacterial replication Chromosome initiation. Replication 2.2.1. Proteins Overview Involved in Initiation of Bacterial Chromosome Replication 2.1. OverviewOrigin DNA strand separation, followed by the loading of two opposing molecules of replicative DNA helicase, are prerequisites for starting new replication forks in all bacteria [12]. Origin DNA strand separation, followed by the loading of two opposing molecules of replicative Based on studies using Escherichia coli, these activities require three proteins: DnaA, the primary DNA helicase, are prerequisites for starting new replication forks in all bacteria [12]. Based on studies initiator protein; DnaB (using E. coli nomenclature), the replicative helicase; and DnaC, the helicase using Escherichia coli, these activities require three proteins: DnaA, the primary initiator protein; DnaB loader. These are described in more detail below. (using E. coli nomenclature), the replicative helicase; and DnaC, the helicase loader. These are described in2.1.1. more DnaA detail below. 2.1.1. DnaADnaA is a highly conserved protein that has been identified genetically and biochemically as the primary initiator protein in eubacteria [13–15]. DnaA is the primary chromosomal initiator DnaA is a highly conserved protein that has been identified genetically and biochemically as the protein in almost all bacteria studied, although some bacteria, such as Vibrio cholerae, have two primary initiator protein in eubacteria [13–15]. DnaA is the primary chromosomal initiator protein in chromosomes, and contain distinct and unrelated initiators for the second chromosome [16]. DnaA almost all bacteria studied, although some bacteria, such as Vibrio cholerae, have two chromosomes, is responsible for binding to specific sequences in oriC and unwinding an A-T rich region termed the and contain distinct and unrelated initiators for the second chromosome [16]. DnaA is responsible for DNA Unwinding Element (DUE) [17]. In many bacterial types, DnaA also recruits DnaB and DnaC, binding to specific sequences in oriC and unwinding an A-T rich region termed the DNA Unwinding and helps position the helicase onto the newly-formed single strands of the unwound DUE [12]. Element (DUE) [17]. In many bacterial types, DnaA also recruits DnaB and DnaC, and helps position DnaA accomplishes its tasks as an initiator using four domains, each with
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