You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Novel Riboswitch Ligand Analogs as Selective Inhibitors of Guanine-Related Metabolic Pathways}
Article:
Abstract:
Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5′-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound( PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.
Introduction:
Multiple drug resistance( MDR) has been a growing problem during the last decade, partly due to excessive use of antibiotics in human medicine and food animal production. MDR also stems from the fact that drug design has been largely based on limited chemical scaffolds leaving an opportunity for pathogens to circumvent antibiotic action mechanisms[1]. Staphylococcus aureus and Clostridium difficile are nosocomial pathogens responsible for a significant mortality rate in hospitals and increased health care costs[2]. Recently, community-acquired methicillin-resistant S. aureus( MRSA) infections have emerged and are commonly responsible for skin and soft-tissue infections that may rapidly evolve in severe and life-threatening infections[3],[4]. Moreover, some emerging clones were shown to be resistant to vancomycin, which is considered as the last chance antibiotic[5]. The pathogen C. difficile has also dramatically increased the hospital-associated deaths in recent years due to the MDR emergence and spreading of the hypervirulent and high toxin-producing strain BI/NAP1/027[4],[5],[6]. This particular strain is spreading in North America and Europe with currently little therapeutic solutions besides the use of metronidazole and vancomycin, which are increasingly associated with relapses and poor treatment outcome[7]. Previous attempts to discover alternative antibacterial drugs targeting RNA were mainly based on a fortuitous interaction between an exogenous ligand and its RNA target[1],[8],[9],[10]. Metabolite-responsive riboswitches represent a novel solution to MDR since they could be considered as antimicrobial targets when agonistic ligands are employed as demonstrated for lysine, thiamine pyrophosphate( TPP), flavin mononucleotide( FMN) and guanine responsive riboswitches[1],[11],[12],[13],[14],[15]. In the case of lysine and TPP riboswitches, previously described ligand analogs were reported to have a multitude of cellular effects in addition to inhibition of gene expression via riboswitch binding[16],[17],[18],[19]. Pleiotropic effects were also observed for compounds targeting the guanine riboswitch and at least one analog was reported to be possibly incorporated in DNA during replication[15],[20]. Thus, while it is of interest to select antibiotics that are chemically distinct from natural ligands to avoid cellular efflux or chemical modification, it is important to consider that these chemical differences will potentially help avoid patient toxicity due to off-target binding. It is also important that the antibiotic provokes a bacteriostatic or bactericidal effect either by targeting a single gene, or a collection of genes, that is necessary for growth, or essential for bacterial survival or virulence. Thus, because modified pyrimidines can specifically bind the purine riboswitch with affinities in the low nanomolar range[21], they make excellent candidates to target purine riboswitches which are likely potent drug targets given their role in regulating purine metabolic pathways( Figure S1). For instance, the inactivation of the E. coli GMP synthetase guaA leads to guanine auxotrophy[22] whereas the inactivation of the B. subtilis IMP dehydrogenase guaB is lethal[23]. Here we show that the guanine riboswitch in S. aureus and C. difficile controls the expression of guaA and that this gene appears essential for virulence in a murine model.
Discussion:
Despite previous large scale screen data suggesting that guaA is not essential for S. aureus growth[23],[35] in relatively rich media, we show here that blocking guaA expression can lead to bactericidal activity in various bacterial species. In support of guaA for cell viability, it has been recently reported that mutations occurring in guaA prevent Streptococcus suis[36] and Salmonella thyphimurium[37] from properly infecting porcine and murine models, respectively, suggesting that GMP bioavailability may be reduced during host infection and that guaA is likely to be crucial for bacterial infection in mammals. Thus, together with studies showing the importance of guaA for bacterial growth in urine or blood[38],[39], our data suggest that mammalian infection sites may significantly differ in their nutrient compositions from those used in large scale screens[23],[35], and that care should be taken when assessing the “essentiality” of a gene. Furthermore, when assessing whether S. aureus could develop resistance to PC1, no resistant bacteria were obtained after more than 30 passages suggesting that maintaining a functional guaA-regulated riboswitch is a vital process( Figure S6). Taken together, the demonstration that guaA expression is normally maintained in S. aureus grown in vivo and the strong therapeutic effect resulting from PC1 treatment indicate that guaA is an important contributor to the survival of S. aureus during infection and that it can be used as an antibiotic target. The major limitation to validate an antibiotic that targets riboswitches is to evaluate the antibiotic specificity of action. In this particular case, PC1 is not a broad-spectrum antibacterial drug given that it does not target all bacteria containing guanine riboswitches, but only those in which guaA is under the control of a riboswitch. It is not excluded that other riboswitch-controlled genes may participate in the PC1-dependent bacterial growth inhibition( e. g., guaB), or that PC1 may bind other cellular targets, which alone or in combination with the riboswitch-controlled gene repression, would repress bacterial growth. Nevertheless, the restricted nature of growth inhibition likely indicates that PC1 inhibits bacterial growth through riboswitch binding and not via an alternative mechanism such as DNA incorporation( Figure 6). For instance, when performing antibiograms using the guanine analog 6-thioguanine, a general growth inhibition was observed in E. coli and S. aureus( Figure S7), consistent with its incorporation into DNA that perturbs the epigenetic pathway of gene regulation[40]. The selective antibacterial activity of PC1 toward S. aureus was also supported by the lack of apparent toxicity for mice treated with the experimental compound at concentrations as high as 100 µg/gland with no sign of discomfort including vocalizations, curved back, piloerection and hypothermia. There was also no apparent cytotoxicity upon histological observations of mammary tissues in PC1-treated mice compared to PBS-treated glands( Figure S8). Recently, the Breaker group used purine derivatives modified in position 2 or 4 to target guanine riboswitches and found three molecules that inhibited bacterial growth[15]. Among these molecules, only one was able to repress the expression of a guanine riboswitch-controlled reporter gene suggesting that at least two molecules inhibited cell growth through a different mechanism of action. It is possible that the mode of action of these two molecules involves nucleic acids incorporation following ribosylation at position 9 of the purine analog, as demonstrated for 6-thioguanine. It is also interesting to mention that the antibiotic activity was only observed using B. subtilis strains cultivated in minimal medium whereas no growth inhibition was detected in rich media. In our study, we selected guanine riboswitch ligands that cannot be ribosylated to prevent alternative modes of action, and favored a pyrimidine compound( PC1) that retained most of the key functional groups. By testing various bacterial species, we observed that the bactericidal activity of PC1 was only seen against bacteria using a guanine riboswitch to control guaA expression, and this even when bacteria were grown in a rich medium. This demonstrates that analog binding to riboswitch aptamers is not the only determinant to achieve selective and efficient bactericidal effects. This study shows for the first time that antibiotics targeting riboswitches may be efficient to kill bacterial pathogens in vitro as well as in mammalian infection models. We found here that the selective bacterial killing of PC1 is only achieved when guaA, a GMP synthetase, is under the control of the riboswitch and when the antimicrobial agent cannot be ribosylated. The narrow spectrum of activity we demonstrated here for PC1 is very interesting since two of the target bacteria, S. aureus and C. difficile, are among the most problematic nosocomial pathogens. The spread of MDR in those bacterial species also stresses the importance to develop new antibiotics that avoid current mechanisms of resistance. The use of narrow spectrum drugs should be encouraged whenever possible to reduce any selective pressure for resistance in non-targeted bacteria. Here, we also showed that the development of resistance toward PC1 is likely to be infrequent.
During the last 30 years , bacterial resistance to antibiotics has become a major problem . This situation is partly because today's antibiotics are mainly based on a limited selection of chemical scaffolds , which makes it easier for bacterial pathogens to quickly develop resistance against new drug derivatives . This recurrent problem of multiple drug resistance implies a constant need to search for novel microbial targets and to modulate their activity using artificial molecules . Riboswitches are newly discovered gene regulatory elements that represent attractive targets for antimicrobial drugs . Riboswitches are RNA structures located in untranslated regions of messenger RNAs that regulate the expression of genes involved in the transport and metabolism of small metabolites . We have identified a new antibiotic specifically targeting riboswitches found in a subgroup of bacteria including Staphylococcus aureus and Clostridium difficile , which are nosocomial pathogens responsible for a significant mortality rate in hospitals , and increased health care costs . The riboswitch controls the expression of guaA that appears essential for virulence in the mammalian host . A murine model was used as a proof of principle to show that such an antibiotic could inhibit the growth of S . aureus in a mammal . Our work provides new insights into the discovery and design of novel antimicrobial agents against bacterial pathogens .