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Hemolysins are lytic exotoxins expressed in most strains of S. aureus , but hemolytic activity varies between strains. We have previously reported several novel anti-virulence compounds that disrupt the S. aureus transcriptome, including hemolysin gene expression. This report delves further into our two lead compounds, loratadine and a structurally related brominated carbazole, and their effects on hemolysin production in MRSA. To gain understanding into how these compounds affect hemolysis, we analyzed these exotoxins at the DNA, RNA, and protein level after in vitro treatment. While lysis of red blood cells varied between strains, DNA sequence variation did not account for it. We hypothesized that our compounds would modulate gene expression of multiple hemolysins in a laboratory strain and a clinically relevant hospital-acquired strain of MRSA, both with SCC mec type II. RNA-seq analysis of differential gene expression in untreated and compound-treated cultures revealed hundreds of differentially expressed genes, with a significant enrichment in genes involved in hemolysis. The brominated carbazole and loratadine both displayed the ability to reduce hemolysis in the laboratory strain, but displayed differential activity in a hospital-acquired strain. These results corroborate gene expression studies as well as western blots of alpha hemolysin. Together, this work suggests that small molecules may alter exotoxin production in MRSA, but that the directionality and/or magnitude of the difference is likely strain-dependent.
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Methicillin-resistant Staphylococcus aureus (MRSA) has evolved to become resistant to multiple classes of antibiotics. New antibiotics are costly to develop and deploy, and they have a limited effective lifespan. Antibiotic adjuvants are molecules that potentiate existing antibiotics through nontoxic mechanisms. We previously reported that loratadine, the active ingredient in Claritin, potentiates multiple cell-wall active antibiotics in vitro and disrupts biofilm formation through a hypothesized inhibition of the master regulatory kinase Stk1. Loratadine and oxacillin combined repressed the expression of key antibiotic resistance genes in the bla and mec operons. We hypothesized that additional genes involved in antibiotic resistance, biofilm formation, and other cellular pathways would be modulated when looking transcriptome-wide. To test this, we used RNA-seq to quantify transcript levels and found significant effects in gene expression, including genes controlling virulence, antibiotic resistance, metabolism, transcription (core RNA polymerase subunits and sigma factors), and translation (a plethora of genes encoding ribosomal proteins and elongation factor Tu). We further demonstrated the impacts of these transcriptional effects by investigating loratadine treatment on intracellular ATP levels, persister formation, and biofilm formation and morphology. Loratadine minimized biofilm formation in vitro and enhanced the survival of infected Caenorhabditis elegans. These pleiotropic effects and their demonstrated outcomes on MRSA virulence and survival phenotypes position loratadine as an attractive anti-infective against MRSA.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Loratadina/farmacología , Virulencia , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Farmacorresistencia Microbiana , BiopelículasRESUMEN
Antibiotic tolerance within a biofilm community presents a serious public health challenge. Here, we report the identification of a 2-aminoimidazole derivative that inhibits biofilm formation by two pathogenic Gram-positive bacteria, Streptococcus mutans and Staphylococcus aureus. In S. mutans, the compound binds to VicR, a key response regulator, at the N-terminal receiver domain, and concurrently inhibits expression of vicR and VicR-regulated genes, including the genes that encode the key biofilm matrix producing enzymes, Gtfs. The compound inhibits S. aureus biofilm formation via binding to a Staphylococcal VicR homolog. In addition, the inhibitor effectively attenuates S. mutans virulence in a rat model of dental caries. As the compound targets bacterial biofilms and virulence through a conserved transcriptional factor, it represents a promising new class of anti-infective agents that can be explored to prevent or treat a host of bacterial infections. IMPORTANCE Antibiotic resistance is a major public health issue due to the growing lack of effective anti-infective therapeutics. New alternatives to treat and prevent biofilm-driven microbial infections, which exhibit high tolerance to clinically available antibiotics, are urgently needed. We report the identification of a small molecule that inhibits biofilm formation by two important pathogenic Gram-positive bacteria, Streptococcus mutans and Staphylococcus aureus. The small molecule selectively targets a transcriptional regulator leading to attenuation of a biofilm regulatory cascade and concurrent reduction of bacterial virulence in vivo. As the regulator is highly conserved, the finding has broad implication for the development of antivirulence therapeutics that selectively target biofilms.
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Antiinfecciosos , Caries Dental , Infecciones Estafilocócicas , Ratas , Animales , Virulencia , Staphylococcus aureus/genética , Caries Dental/tratamiento farmacológico , Caries Dental/prevención & control , Biopelículas , Antibacterianos/metabolismo , Antiinfecciosos/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Streptococcus mutans/metabolismoRESUMEN
Bacterial infections due to biofilms account for up to 80% of bacterial infections in humans. With the increased use of antibiotic treatments, indwelling medical devices, disinfectants, and longer hospital stays, antibiotic resistant infections are sharply increasing. Annual deaths are predicted to outpace cancer and diabetes combined by 2050. In the past two decades, both chemical and physical strategies have arisen to combat biofilm formation on surfaces. One such promising chemical strategy is the formation of a self-assembled monolayer (SAM), due to its small layer thickness, strong covalent bonds, typically facile synthesis, and versatility. With the goal of combating biofilm formation, the SAM could be used to tether an antibacterial agent such as a small-molecule antibiotic, nanoparticle, peptide, or polymer to the surface, and limit the agent's release into its environment. This review focuses on the use of SAMs to inhibit biofilm formation, both on their own and by covalent grafting of a biocidal agent, with the potential to be used in indwelling medical devices. We conclude with our perspectives on ongoing challenges and future directions for this field.
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Methicillin-resistant Staphylococcus aureus (MRSA) is a major threat to human health, as the US mortality rate outweighs those from HIV, tuberculosis, and viral hepatitis combined. In the wake of the COVID-19 pandemic, antibiotic-resistant bacterial infections acquired during hospital stays have increased. Antibiotic adjuvants are a key strategy to combat these bacteria. We have evaluated several small molecule antibiotic adjuvants that have strong potentiation with ß-lactam antibiotics and are likely inhibiting a master regulatory kinase, Stk1. Here, we investigated how the lead adjuvant (compound 8) exerts its effects in a more comprehensive manner. We hypothesized that the expression levels of key resistance genes would decrease once cotreated with oxacillin and the adjuvant. Furthermore, bioinformatic analyses would reveal biochemical pathways enriched in differentially expressed genes. RNA-seq analysis showed 176 and 233 genes significantly up- and downregulated, respectively, in response to cotreatment. Gene ontology categories and biochemical pathways that were significantly enriched with downregulated genes involved carbohydrate utilization, such as the citrate cycle and the phosphotransferase system. One of the most populated pathways was S. aureus infection. Results from an interaction network constructed with affected gene products supported the hypothesis that Stk1 is a target of compound 8. This study revealed a dramatic impact of our lead adjuvant on the transcriptome that is consistent with a pleiotropic effect due to Stk1 inhibition. These results point to this antibiotic adjuvant having potential broad therapeutic use in combatting MRSA.
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COVID-19 , Staphylococcus aureus Resistente a Meticilina , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Carbazoles/farmacología , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Pruebas de Sensibilidad Microbiana , Pandemias , Staphylococcus aureus , TranscriptomaRESUMEN
Antibiotic-resistant infections are a pressing global concern, causing millions of deaths each year. Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections in healthcare settings and is increasingly responsible for community-acquired infections that are often more difficult to treat. Antibiotic adjuvants are small molecules that potentiate antibiotics through nontoxic mechanisms and show excellent promise as novel therapeutics. Screening of low-molecular-weight compounds was employed to identify novel antibiotic adjuvant scaffolds for further elaboration. Brominated carbazoles emerged from this screening as lead compounds for further evaluation. Lead carbazoles were able to potentiate several ß-lactam antibiotics in three medically relevant strains of MRSA. Gene expression studies determined that these carbazoles were dampening the transcription of key genes that modulate ß-lactam resistance in MRSA. The lead brominated carbazoles represent novel scaffolds for elaboration as antibiotic adjuvants.
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Antibiotic resistance is a global and pressing concern. Our current therapeutic arsenal is increasingly limited as bacteria are developing resistance at a rate that far outpaces our ability to create new treatments. Novel approaches to treating and curing bacterial infections are urgently needed. Bacterial kinases have been increasingly explored as novel drug targets and are poised for development into novel therapeutic agents to combat bacterial infections. This review describes several general classes of bacterial kinases that play important roles in bacterial growth, antibiotic resistance, and biofilm formation. General features of these kinase classes are discussed and areas of particular interest for the development of inhibitors will be highlighted. Small molecule kinase inhibitors are described and organized by phenotypic effect, spotlighting particularly interesting inhibitors with novel functions and potential therapeutic benefit. Finally, we provide our perspective on the future of bacterial kinase inhibition as a viable strategy to combat bacterial infections and overcome the pressures of increasing antibiotic resistance.
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Antiinfecciosos/química , Antiinfecciosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Biopelículas/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Histidina Quinasa/antagonistas & inhibidores , Histidina Quinasa/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Staphylococcus epidermidis and Staphylococcus aureus are important human pathogens responsible for two-thirds of all postsurgical infections of indwelling medical devices. Staphylococci form robust biofilms that provide a reservoir for chronic infection, and antibiotic-resistant isolates are increasingly common in both healthcare and community settings. Novel treatments that can simultaneously inhibit biofilm formation and antibiotic-resistance pathways are urgently needed to combat the increasing rates of antibiotic-resistant infections. Herein we report that loratadine, an FDA-approved antihistamine, significantly inhibits biofilm formation in both S. aureus and S. epidermidis. Furthermore, loratadine potentiates ß-lactam antibiotics in methicillin-resistant strains of S. aureus and potentiates both ß-lactam antibiotics and vancomycin in vancomycin-resistant strains of S. aureus. Additionally, we elucidate loratadine's mechanism of action as a novel inhibitor of the regulatory PASTA kinases Stk and Stk1 in S. epidermidis and S. aureus, respectively. Finally, we describe how Stk1 inhibition affects the expression of genes involved in both biofilm formation and antibiotic resistance in S. epidermidis and S. aureus.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Loratadina/farmacología , Fosfotransferasas/antagonistas & inhibidores , Staphylococcus aureus/efectos de los fármacos , Staphylococcus epidermidis/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Staphylococcus aureus/enzimología , Staphylococcus epidermidis/enzimología , Vancomicina/farmacología , Factores de Virulencia/antagonistas & inhibidores , beta-Lactamas/farmacologíaRESUMEN
A simple electrochemical assay to monitor the dispersion of Pseudomonas aeruginosa PA01 biofilm is described. Pyrolytic graphite (PG) electrodes were modified with P. aeruginosa PA01 using layer-by-layer (LbL) methods. The presence of the bacteria on the electrodes was directly monitored using square wave voltammetry (SWV) via the electrochemical reduction of electroactive phenazine compounds expressed by the bacteria, which indicate the presence of biofilm. Upon treatment of bacteria-modified electrodes with a 2-aminoimidazole (2-AI) derivative with known Pseudomonas anti-biofilm properties, the bacteria-related electrochemical reduction peaks decreased in a concentration dependent manner, indicating dispersal of the biofilm on the electrode surface. A similar 2-AI compound with negligible anti-biofilm activity was used as a comparative control and produced muted electrochemical results. Electrochemical responses mirrored previously established bioassay-derived half maximal inhibition concentration (IC50) and half maximal effective concentration (EC50) values.. Biofilm dispersal detection via the electrochemical response was validated by monitoring crystal violet absorbance after its release from electrode confined P. aeruginosa biofilm. Mass spectrometry data showing multiple redox active phenazine compounds are presented to provide insight into the surface reaction complexity. Overall, we present a very simple assay to monitor the anti-biofilm activity of compounds of interest.
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Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of recurrent infections in humans including endocarditis, pneumonia, and toxic shock syndrome. Novel therapeutics to treat MRSA and other resistant bacteria are urgently needed. Adjuvant therapy, which uses a non-toxic compound to repotentiate the toxic effects of an existing antibiotic, is an attractive response to the growing resistance crisis. Herein, we describe the evaluation of structurally related, FDA-approved tricyclic amine antidepressants that selectively repotentiate MRSA to ß-lactam antibiotics. Our results identify important structural features of the tricyclic amine class for ß-lactam adjuvant activity. Furthermore, we describe the mechanism of action for our lead compound, amoxapine, and illustrate that it represses the mRNA levels of key ß-lactam resistance genes in response to ß-lactam treatment. This work is novel in that it highlights an important class of small molecules with the ability to simultaneously inhibit production of both ß-lactamase and penicillin binding protein 2a.
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Antidepresivos Tricíclicos/farmacología , Farmacorresistencia Bacteriana/genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , ARN Mensajero/metabolismo , Antibacterianos/farmacología , Antidepresivos Tricíclicos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Pruebas de Sensibilidad Microbiana , Oxacilina/farmacología , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
The rapid increase in bacterial resistance to antibiotics is a global healthcare crisis. Non-antibiotic pharmaceuticals that have attained approval by the United States Food and Drug Administration have the potential to be repurposed as bacterial resistance-modifying agents and therefore could become valuable resources in our battle against antibiotic-resistant microbes. Amoxapine is a tetracyclic antidepressant used in the treatment of major depressive disorder. Here we demonstrate the ability of amoxapine to resensitize methicillin-resistant Staphylococcus aureus strain ATCC 43300 to oxacillin in both agar diffusion and broth microdilution assays. Amoxapine also reduced the bacterial cleavage of nitrocefin in a dose-dependent manner, suggesting that it may exert its adjuvant effects through reduction of beta-lactamase activity.
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There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment.
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Antiinfecciosos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Imidazoles/farmacología , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/enzimología , beta-Lactamasas/metabolismo , beta-Lactamas/farmacología , Carbenicilina/farmacología , Colorantes/química , Lípidos/análisis , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Ácidos Nucleicos/metabolismo , Penicilina V/farmacología , Dodecil Sulfato de Sodio/farmacología , Coloración y Etiquetado , Transcripción Genética/efectos de los fármacos , Vancomicina/farmacologíaRESUMEN
A library of 2-aminobenzimidazole derivatives was screened for the ability to suppress ß-lactam resistance in Mycobacterium smegmatis. Several non-bactericidal compounds were identified that reversed intrinsic resistance to ß-lactam antibiotics in a manner distinct from ß-lactamase inhibitors. Activity also translates to M.â tuberculosis, with a lead compound from this study potently suppressing carbenicillin resistance in multiple M.â tuberculosis strains (including multidrug-resistant strains). Preliminary mechanistic studies revealed that the lead compounds act through a mechanism distinct from that of traditional ß-lactamase inhibitors.
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Antibacterianos/farmacología , Bencimidazoles/farmacología , Lactamas/farmacología , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Bencimidazoles/química , Descubrimiento de Drogas , Lactamas/síntesis química , Lactamas/química , Estructura Molecular , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
Acinetobacter baumannii has quickly become one of the most insidious and prevalent nosocomial infections. Recently, the reverse-amide class of 2-aminoimidazole compounds (RA-2AI) was found both to prevent A. baumannii biofilm formation and also to disperse preexisting formations, putatively through interactions with cytosolic response regulators. Here we focus on how this class of antibiofilm agent traverses cellular membranes. Following the discovery of dosage-dependent growth rate changes, the cellular effects of RA-2AI were investigated using a combination of molecular assays and microscopic techniques. It was found that RA-2AI exposure has measureable effects on the bacterial membranes, resulting in a period of increased permeability and visible structural aberrations. Based on these results, we propose a model that describes how the structure of RA-2AI allows it to insert itself into and disrupt the fluidity of the membrane, creating an opportunity for increased molecular permeability.
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Acinetobacter baumannii/efectos de los fármacos , Amidas/química , Biopelículas/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Imidazoles/química , Antibacterianos/química , Antibacterianos/farmacología , Células Cultivadas , Infección Hospitalaria , Imidazoles/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Infections caused by multidrug-resistant bacteria are a considerable and increasing global problem. The development of new antibiotics is not keeping pace with the rapid evolution of resistance to almost all clinically available drugs, and novel strategies are required to fight bacterial infections. One such strategy is the control of pathogenic behaviors, as opposed to simply killing bacteria. Bacterial two-component system (TCS) signal transduction pathways control many pathogenic bacterial behaviors, such as virulence, biofilm formation and antibiotic resistance and are, therefore, an attractive target for the development of new drugs. This review presents an overview of TCS that are potential targets for such a strategy, describes small-molecules inhibitors of TCS identified to date and discusses assays for the identification of novel inhibitors. The future perspective for the identification and use of inhibitors of TCS to potentially provide new therapeutic options for the treatment of drug-resistant bacterial infections is discussed.
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Bacterias/patogenicidad , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Virulencia/efectos de los fármacos , Bacterias/metabolismo , Infecciones Bacterianas/tratamiento farmacológico , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Histidina Quinasa , Humanos , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
Infections caused by bacterial biofilms are a significant global health problem, causing considerable patient morbidity and mortality and contributing to the economic burden of infectious disease. This review describes diverse strategies to combat bacterial biofilms, focusing firstly on small molecule interference with bacterial communication and signaling pathways, including quorum sensing and two-component signal transduction systems. Secondly we discuss enzymatic approaches to the degradation of extracellular matrix components to effect biofilm dispersal. Both of these approaches are based upon non-microbicidal mechanisms of action, and thereby do not place a direct evolutionary pressure on the bacteria to develop resistance. Such approaches have the potential to, in combination with conventional antibiotics, play an important role in the eradication of biofilm based bacterial infections.
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Antibacterianos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Fenómenos Fisiológicos Bacterianos/efectos de los fármacos , Biopelículas/efectos de los fármacos , Animales , Antibacterianos/uso terapéutico , Infecciones Bacterianas/metabolismo , Humanos , Percepción de Quorum/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Aberrant gene expression is responsible for a myriad of human diseases from infectious diseases to cancer. Precise regulation of these genes via specific interactions with the DNA double helix could pave the way for novel therapeutics. Pyrrole-imidazole polyamides are small molecules capable of binding to pre-determined DNA sequences up to 16 base pairs with affinity and specificity comparable to natural transcription factors. In the three decades since their development, great strides have been made relating to synthetic accessibility and improved sequence specificity and binding affinity. This perspective presents a brief history of early seminal developments in the field and highlights recent reports of the utility of polyamides as both genetic modulators and molecular probes.