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66100.9897A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase. The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.201627601569
843410.9897A potent and selective antimicrobial poly(amidoamine) dendrimer conjugate with LED209 targeting QseC receptor to inhibit the virulence genes of gram negative bacteria. The pandemic of multidrug-resistant Gram negative bacteria (GNB) is a worldwide healthcare concern, and very few antibiotics are being explored to match the clinical challenge. Recently, amino-terminated poly(amidoamine) (PAMAM) dendrimers have shown potential to function as broad antimicrobial agents. However, PAMAM displays a generation dependent cytotoxicity to mammalian cells and low selectivity on bacterial cells, which limits PAMAM to be developed as an antibacterial agent for systemic administration. We conjugated G3 PAMAM with LED209, a specific inhibitor of quorum sensor QseC of GNB, to generate a multifunctional agent PAMAM-LED209. Intriguingly, PAMAM-LED209 showed higher selectivity on GNB and lower cytotoxicity to mammalian cells, yet remained strong antibacterial activity. PAMAM-LED209 also inhibited virulence gene expression of GNB, and did not induce antibiotic-resistance. The present work firstly demonstrated that PAMAM-LED209 conjugate had a highly selective anti-GNB activity and low cytotoxicity, which offered a feasible strategy for combating multidrug-resistant GNB infections. FROM THE CLINICAL EDITOR: This research team demonstrated that a novel PAMAM-LED209 conjugate had highly selective activity against Gram-negative bacteria, coupled with low cytotoxicity, offering a potential strategy for combating multidrug-resistant infections.201525461286
55320.9897Single-cell analysis of glycopeptide resistance gene expression in teicoplanin-resistant mutants of a VanB-type Enterococcus faecalis. The vanB gene cluster confers resistance to vancomycin but not to the related antibiotic teicoplanin, as the VanRB SB two-component regulatory system triggers expression of the glycopeptide resistance genes only in response to vancomycin. The VanRB regulator activates promoters PRB and PYB for transcription of the regulatory (vanRB SB) and resistance (vanYB WHB BXB) genes respectively. The gfpmut1 gene encoding a green fluorescent protein was fused to PYB to analyse promoter activation in single cells by fluorescence microscopy and flow cytometry. Characterization of 17 teicoplanin-resistant mutants indicated that amino acid substitutions on either side of the VanSB autophosphorylation site led to a constitutive phenotype. Substitutions in the membrane-associated domain resulted in a gain of function, as they allowed induction by teicoplanin. A vanSB null mutant expressed gfpmut1 at various levels under non-inducing conditions, and the majority of the bacteria were not fluorescent. Bacteria grown in the presence of vancomycin or teicoplanin were homogeneously fluorescent. The increase in the number of fluorescent bacteria resulted from induction in negative cells rather than from selection of a resistant subpopulation, indicating that VanRB was activated by cross-talk. Transglycosylase inhibition was probably the stimulus for the heterologous kinase, as moenomycin was also an inducer.199910216856
374130.9897The fib locus in Streptococcus pneumoniae is required for peptidoglycan crosslinking and PBP-mediated beta-lactam resistance. Penicillin resistance in pneumococci is mediated by modified penicillin-binding proteins (PBPs) that have decreased affinity to beta-lactams. In high-level penicillin-resistant transformants of the laboratory strain Streptococcus pneumoniae R6 containing various combinations of low-affinity PBPs, disruption of the fib locus results in a collapse of PBP-mediated resistance. In addition, crosslinked muropeptides are highly reduced. The fib operon consists of two genes, fibA and fibB, homologous to Staphylococcus aureus femA/B which are also required for expression of methicillin resistance in this organism. FibA and FibB belong to a family of proteins of Gram-positive bacteria involved in the formation of interpeptide bridges, thus representing interesting new targets for antimicrobial compounds for this group of pathogens.200010867238
60740.9896A novel copper-sensing two-component system for inducing Dsb gene expression in bacteria. In nature, bacteria must sense copper and tightly regulate gene expression to evade copper toxicity. Here, we identify a new copper-responsive two-component system named DsbRS in the important human pathogen Pseudomonas aeruginosa; in this system, DsbS is a sensor histidine kinase, and DsbR, its cognate response regulator, directly induces the transcription of genes involved in protein disulfide bond formation (Dsb) (i.e., the dsbDEG operon and dsbB). In the absence of copper, DsbS acts as a phosphatase toward DsbR, thus blocking the transcription of Dsb genes. In the presence of copper, the metal ion directly binds to the sensor domain of DsbS, and the Cys82 residue plays a critical role in this process. The copper-binding behavior appears to inhibit the phosphatase activity of DsbS, leading to the activation of DsbR. The copper resistance of the dsbRS knock-out mutant is restored by the ectopic expression of the dsbDEG operon, which is a DsbRS major target. Strikingly, cognates of the dsbRS-dsbDEG pair are widely distributed across eubacteria. In addition, a DsbR-binding site, which contains the consensus sequence 5'-TTA-N(8)-TTAA-3', is detected in the promoter region of dsbDEG homologs in these species. These findings suggest that the regulation of Dsb genes by DsbRS represents a novel mechanism by which bacterial cells cope with copper stress.202236546013
21150.9895Preclinical pharmacology of GAR-936, a novel glycylcycline antibacterial agent. GAR-936 is an analog of minocycline, a semisynthetic derivative of tetracycline. It has broad-spectrum antibacterial activity in vitro and in vivo. The new class of tetracyclines to which GAR-936 belongs is named the glycylcyclines. Tetracyclines act by inhibiting protein translation in bacteria, presumably by binding to the 30S ribosomal subunit and blocking entry of amino-acyl transfer RNA molecules into the A site of the ribosome. This prevents incorporation of amino acid residues into elongating peptide chains. In general, tetracyclines are considered bacteriostatic and the critical therapeutic parameter is the area under the concentration-time curve. GAR-936 has bactericidal activity; at 4 times the minimum inhibitory concentration, a 2- to 3-log reduction in colony counts was seen against Streptococcus pneumoniae, Neisseria gonorrhoeae, Haemophilus influenzae, Escherichia coli, and Staphylococcus aureus. GAR-936 is active against the antibiotic-resistant gram-positive bacteria methicillin-resistant Staphylococcus aureus, penicillin-resistant S. pneumoniae, and vancomycin-resistant enterococci. It is most significant that GAR-936 and other glycylcyclines are active against bacterial strains carrying either or both of the two major forms of tetracycline resistance: efflux and ribosomal protection. Using isogenic panels of bacteria carrying various tetracycline-resistance determinants, a series of more than 300 analogs was tested for antibacterial activity, which allowed for structure-activity relationships to be determined. Results indicated that certain substituents at the 9 position of the tetracycline molecule restored activity against bacteria harboring genes encoding either or both efflux and ribosomal protection. A single chemical modification overcame the two molecularly distinct forms of resistance while maintaining activity against susceptible gram-positive, gram-negative, aerobic, and anaerobic bacteria. Although mutants can be generated that are less susceptible to previously studied glycylcyclines, only marginal differences in susceptibility to GAR-936 were noted. Therefore, whereas emergence of resistance to any widely administered antibiotic is a foregone conclusion, resistance to GAR-936 will not readily arise by trivial mutations in existing resistance genes.200011001329
65560.9894Identification of a cell envelope protein (MtrF) involved in hydrophobic antimicrobial resistance in Neisseria gonorrhoeae. The mtrCDE-encoded efflux pump of Neisseria gonorrhoeae provides gonococci with a mechanism to resist structurally diverse antimicrobial hydrophobic agents (HAs). Strains of N. gonorrhoeae that display hypersusceptibility to HAs often contain mutations in the efflux pump genes, mtrCDE. Such strains frequently contain a phenotypically suppressed mutation in mtrR, a gene that encodes a repressor (MtrR) of mtrCDE gene expression, and one that would normally result in HA resistance. We have recently examined HA-hypersusceptible clinical isolates of gonococci that contain such phenotypically suppressed mtrR mutations, in order to determine whether genes other than mtrCDE are involved in HA resistance. These studies led to the discovery of a gene that we have designated mtrF, located downstream of the mtrR gene, that is predicted to encode a 56.1 kDa cytoplasmic membrane protein containing 12 transmembrane domains. Expression of mtrF was enhanced in a strain deficient in MtrR production, indicating that this gene, together with the closely linked mtrCDE operon, is subject to MtrR-dependent transcriptional control. Orthologues of mtrF were identified in a number of diverse bacteria. Except for the AbgT protein of Escherichia coli, their products have been identified as hypothetical proteins with unknown function(s). Genetic evidence is presented that MtrF is important in the expression of high-level detergent resistance by gonococci. We propose that MtrF acts in conjunction with the MtrC-MtrD-MtrE efflux pump, to confer on gonococci high-level resistance to certain HAs.200312493784
11270.9893Glycopeptide resistance determinants from the teicoplanin producer Actinoplanes teichomyceticus. In enterococci and other pathogenic bacteria, high-level resistance to vancomycin and other glycopeptide antibiotics requires the action of the van genes, which direct the synthesis of peptidoglycan terminating in the depsipeptide D-alanyl-D-lactate, in place of the usual D-Ala-D-Ala. The Actinoplanes teichomyceticus tcp cluster, devoted to the biosynthesis of the glycopeptide antibiotic teicoplanin, contains van genes associated to a murF-like sequence (murF2). We show that A. teichomyceticus contains also a house-keeping murF1 gene, capable of complementing a temperature sensitive Escherichia coli murF mutant. MurF1, expressed in Streptomyces lividans, can catalyze the addition of either D-Ala-D-Ala or D-Ala-D-Lac to the UDP-N-acetyl-muramyl-L-Ala-D-Glu-d-Lys. However, similarly expressed MurF2 shows a small enzymatic activity only with D-Ala-D-lactate. Introduction of a single copy of the entire set of van genes confers resistance to teicoplanin-type glycopeptides to S. coelicolor.200415500981
55480.9893VanZ Reduces the Binding of Lipoglycopeptide Antibiotics to Staphylococcus aureus and Streptococcus pneumoniae Cells. vanZ, a member of the VanA glycopeptide resistance gene cluster, confers resistance to lipoglycopeptide antibiotics independent of cell wall precursor modification by the vanHAX genes. Orthologs of vanZ are present in the genomes of many clinically relevant bacteria, including Enterococcus faecium and Streptococcus pneumoniae; however, vanZ genes are absent in Staphylococcus aureus. Here, we show that the expression of enterococcal vanZ paralogs in S. aureus increases the minimal inhibitory concentrations of lipoglycopeptide antibiotics teicoplanin, dalbavancin, oritavancin and new teicoplanin pseudoaglycone derivatives. The reduction in the binding of fluorescently labeled teicoplanin to the cells suggests the mechanism of VanZ-mediated resistance. In addition, using a genomic vanZ gene knockout mutant of S. pneumoniae, we have shown that the ability of VanZ proteins to compromise the activity of lipoglycopeptide antibiotics by reducing their binding is a more general feature of VanZ-superfamily proteins.202032318043
374090.9892Stp1 Loss of Function Promotes β-Lactam Resistance in Staphylococcus aureus That Is Independent of Classical Genes. β-Lactam resistance in Staphylococcus aureus limits treatment options. Stp1 and Stk1, a serine-threonine phosphatase and kinase, respectively, mediate serine-threonine kinase (STK) signaling. Loss-of-function point mutations in stp1 were detected among laboratory-passaged β-lactam-resistant S. aureus strains lacking mecA and blaZ, the major determinants of β-lactam resistance in the bacteria. Loss of Stp1 function facilitates β-lactam resistance of the bacteria.202032179529
9035100.9892Involvement of a novel efflux system in biofilm-specific resistance to antibiotics. Bacteria growing in biofilms are more resistant to antibiotics than their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874 to PA1877 (PA1874-1877) genes, in an P. aeruginosa PA14 background results in an increase in sensitivity to tobramycin, gentamicin, and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than in planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (T. F. Mah, B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 gene deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics.200818469108
209110.9891Targeting quinolone- and aminocoumarin-resistant bacteria with new gyramide analogs that inhibit DNA gyrase. Bacterial DNA gyrase is an essential type II topoisomerase that enables cells to overcome topological barriers encountered during replication, transcription, recombination, and repair. This enzyme is ubiquitous in bacteria and represents an important clinical target for antibacterial therapy. In this paper we report the characterization of three exciting new gyramide analogs-from a library of 183 derivatives-that are potent inhibitors of DNA gyrase and are active against clinical strains of gram-negative bacteria (Escherichia coli, Shigella flexneri, and Salmonella enterica; 3 of 10 wild-type strains tested) and gram-positive bacteria (Bacillus spp., Enterococcus spp., Staphylococcus spp., and Streptococcus spp.; all 9 of the wild-type strains tested). E. coli strains resistant to the DNA gyrase inhibitors ciprofloxacin and novobiocin display very little cross-resistance to these new gyramides. In vitro studies demonstrate that the new analogs are potent inhibitors of the DNA supercoiling activity of DNA gyrase (IC(50)s of 47-170 nM) but do not alter the enzyme's ATPase activity. Although mutations that confer bacterial cells resistant to these new gyramides map to the genes encoding the subunits of the DNA gyrase (gyrA and gyrB genes), overexpression of GyrA, GyrB, or GyrA and GyrB together does not suppress the inhibitory effect of the gyramides. These observations support the hypothesis that the gyramides inhibit DNA gyrase using a mechanism that is unique from other known inhibitors.201730034678
6176120.9891Involvement of GcvB small RNA in intrinsic resistance to multiple aminoglycoside antibiotics in Escherichia coli. Deleting the gene for small RNA GcvB in Escherichia coli was found to increase the sensitivity to several aminoglycoside antibiotics, such as neomycin, streptomycin, kanamycin, kasugamycin and spectinomycin, at low concentrations. GcvB, conserved in gram-negative enteric bacteria, is known to negatively control the expression of many genes for amino acid incorporation systems, especially the periplasmic ABC-transporter proteins. Deletions of several amino acid transporter genes in ΔgcvB cells decreased the antibiotic sensitivity to the wild-type level, suggesting that those genes are involved in uptake of aminoglycosides into the cell. Since GcvB is constitutively synthesized in growing cells, repressing synthesis of amino acid transporters, it contributes to the intrinsic resistance to several aminoglycoside antibiotics.202133169170
6175130.9891Phenotype microarray analysis of the drug efflux systems in Salmonella enterica serovar Typhimurium. A large number of drug efflux transporters have been identified in Salmonella enterica serovar Typhimurium, and increased expression of these transporters confers drug resistance in this organism. Here we compared the respiration activities of the wild-type strain and a mutant with nine deleted transporters by phenotype microarray analysis. The mutant was susceptible to 66 structurally unrelated compounds including many antibiotics, dyes, detergents, antihistamine agents, plant alkaloids, antidepressants, antipsychotic drugs, and antiprotozoal drugs. To investigate the effect of each transporter on the susceptibilities to these drugs, we used the single transporter mutants, several multiple deletion mutants, and the transporter overexpressor strains to determine minimum inhibitory concentrations of ampicillin, erythromycin, minocycline, ciprofloxacin, orphenadrine, amitriptyline, thioridazine, and chlorpromazine. The data indicate that the increased susceptibilities of the mutant lacking nine transporter genes are mainly dependent on the absence of the acrAB efflux genes as well as the tolC gene. In addition to the AcrAB-TolC efflux system, the results from the overexpressor strains show that AcrEF confers resistance to these compounds as well as AcrAB of Escherichia coli, MexAB-OprM and MexXY-OprM of Pseudomonas aeruginosa. The results highlight the importance of the efflux systems not only for resistance to antibiotics but also for resistance to antihistamine agents, plant alkaloids, antidepressants, antipsychotic drugs, and antiprotozoal drugs.201627210311
3744140.9891Vancomycin resistance VanS/VanR two-component systems. Vancomycin is a member of the glycopeptide class of antibiotics. Vancomycin resistance (van) gene clusters are found in human pathogens such as Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus, glycopeptide-producing actinomycetes such as Amycolotopsis orientalis, Actinoplanes teichomyceticus and Streptomyces toyocaensis and the nonglycopeptide producing actinomycete Streptomyces coelicolor. Expression of the van genes is activated by the VanS/VanR two-component system in response to extracellular glycopeptide antibiotic. Two major types of inducible vancomycin resistance are found in pathogenic bacteria; VanA strains are resistant to vancomycin itself and also to the lipidated glycopeptide teicoplanin, while VanB strains are resistant to vancomycin but sensitive to teicoplanin. Here we discuss the enzymes the van genes encode, the range of different VanS/VanR two-component systems, the biochemistry of VanS/VanR, the nature of the effector ligand(s) recognised by VanS and the evolution of the van cluster.200818792691
621150.9890Activation of ChvG-ChvI regulon by cell wall stress confers resistance to β-lactam antibiotics and initiates surface spreading in Agrobacterium tumefaciens. A core component of nearly all bacteria, the cell wall is an ideal target for broad spectrum antibiotics. Many bacteria have evolved strategies to sense and respond to antibiotics targeting cell wall synthesis, especially in the soil where antibiotic-producing bacteria compete with one another. Here we show that cell wall stress caused by both chemical and genetic inhibition of the essential, bifunctional penicillin-binding protein PBP1a prevents microcolony formation and activates the canonical host-invasion two-component system ChvG-ChvI in Agrobacterium tumefaciens. Using RNA-seq, we show that depletion of PBP1a for 6 hours results in a downregulation in transcription of flagellum-dependent motility genes and an upregulation in transcription of type VI secretion and succinoglycan biosynthesis genes, a hallmark of the ChvG-ChvI regulon. Depletion of PBP1a for 16 hours, results in differential expression of many additional genes and may promote a stress response, resembling those of sigma factors in other bacteria. Remarkably, the overproduction of succinoglycan causes cell spreading and deletion of the succinoglycan biosynthesis gene exoA restores microcolony formation. Treatment with cefsulodin phenocopies depletion of PBP1a and we correspondingly find that chvG and chvI mutants are hypersensitive to cefsulodin. This hypersensitivity only occurs in response to treatment with β-lactam antibiotics, suggesting that the ChvG-ChvI pathway may play a key role in resistance to antibiotics targeting cell wall synthesis. Finally, we provide evidence that ChvG-ChvI likely has a conserved role in conferring resistance to cell wall stress within the Alphaproteobacteria that is independent of the ChvG-ChvI repressor ExoR.202236480495
770160.9890Mutations in the efflux pump regulator MexZ shift tissue colonization by Pseudomonas aeruginosa to a state of antibiotic tolerance. Mutations in mexZ, encoding a negative regulator of the expression of the mexXY efflux pump genes, are frequently acquired by Pseudomonas aeruginosa at early stages of lung infection. Although traditionally related to resistance to the first-line drug tobramycin, mexZ mutations are associated with low-level aminoglycoside resistance when determined in the laboratory, suggesting that their selection during infection may not be necessarily, or only, related to tobramycin therapy. Here, we show that mexZ-mutated bacteria tend to accumulate inside the epithelial barrier of a human airway infection model, thus colonising the epithelium while being protected against diverse antibiotics. This phenotype is mediated by overexpression of lecA, a quorum sensing-controlled gene, encoding a lectin involved in P. aeruginosa tissue invasiveness. We find that lecA overexpression is caused by a disrupted equilibrium between the overproduced MexXY and another efflux pump, MexAB, which extrudes quorum sensing signals. Our results indicate that mexZ mutations affect the expression of quorum sensing-regulated pathways, thus promoting tissue invasiveness and protecting bacteria from the action of antibiotics within patients, something unnoticeable using standard laboratory tests.202438519499
571170.9890Alternative periplasmic copper-resistance mechanisms in Gram negative bacteria. Bacteria have evolved different systems to tightly control both cytosolic and envelope copper concentration to fulfil their requirements and at the same time, avoid copper toxicity. We have previously demonstrated that, as in Escherichia coli, the Salmonella cue system protects the cytosol from copper excess. On the other hand, and even though Salmonella lacks the CusCFBA periplasmic copper efflux system, it can support higher copper concentrations than E. coli under anaerobic conditions. Here we show that the Salmonella cue regulon is also responsible for the control of copper toxicity in anaerobiosis. We establish that resistance in this condition requires a novel CueR-controlled gene named cueP. A DeltacueP mutant is highly susceptible to copper in the absence of oxygen, but shows a faint phenotype in aerobic conditions unless other copper-resistance genes are also deleted, resembling the E. coli CusCFBA behaviour. Species that contain a cueP homologue under CueR regulation have no functional CusR/CusS-dependent Cus-coding operon. Conversely, species that carry a CusR/CusS-regulated cus operon have no cueP homologues. Even more, we show that the CueR-controlled cueP expression increases copper resistance of a Deltacus E. coli. We posit that CueP can functionally replace the Cus complex for periplasmic copper resistance, in particular under anaerobic conditions.200919538445
622180.9890Small-Molecule Antibiotics Inhibiting tRNA-Regulated Gene Expression Is a Viable Strategy for Targeting Gram-Positive Bacteria. Bacterial infections and the rise of antibiotic resistance, especially multidrug resistance, have generated a clear need for discovery of novel therapeutics. We demonstrated that a small-molecule drug, PKZ18, targets the T-box mechanism and inhibits bacterial growth. The T-box is a structurally conserved riboswitch-like gene regulator in the 5' untranslated region (UTR) of numerous essential genes of Gram-positive bacteria. T-boxes are stabilized by cognate, unacylated tRNA ligands, allowing the formation of an antiterminator hairpin in the mRNA that enables transcription of the gene. In the absence of an unacylated cognate tRNA, transcription is halted due to the formation of a thermodynamically more stable terminator hairpin. PKZ18 targets the site of the codon-anticodon interaction of the conserved stem I and reduces T-box-controlled gene expression. Here, we show that novel analogs of PKZ18 have improved MICs, bactericidal effects against methicillin-resistant Staphylococcus aureus (MRSA), and increased efficacy in nutrient-limiting conditions. The analogs have reduced cytotoxicity against eukaryotic cells compared to PKZ18. The PKZ18 analogs acted synergistically with aminoglycosides to significantly enhance the efficacy of the analogs and aminoglycosides, further increasing their therapeutic windows. RNA sequencing showed that the analog PKZ18-22 affects expression of 8 of 12 T-box controlled genes in a statistically significant manner, but not other 5'-UTR regulated genes in MRSA. Very low levels of resistance further support the existence of multiple T-box targets for PKZ18 analogs in the cell. Together, the multiple targets, low resistance, and synergy make PKZ18 analogs promising drugs for development and future clinical applications.202033077662
6188190.9889Quinolone mode of action. Physical studies have further defined interactions of quinolones with their principal target, DNA gyrase. The binding of quinolones to the DNA gyrase-DNA complex suggests 2 possible binding sites of differing affinities. Mutations in either the gyrase A gene (gyrA) or the gyrase B gene (gyrB) that affect quinolone susceptibility also affect drug binding, with resistance mutations causing decreased binding and hypersusceptibility mutations causing increased binding. Combinations of mutations in both GyrA and GyrB have further demonstrated the contribution of both subunits to the quinolone sensitivity of intact bacteria and purified DNA gyrase. A working model postulates initial binding of quinolones to proximate sites on GyrA and GyrB. This initial binding then produces conformational changes that expose additional binding sites, possibly involving DNA. Quinolones also inhibit the activities of Escherichia coli topoisomerase IV (encoded by the parC and parE genes), but at concentrations higher than those inhibiting DNA gyrase. The patterns of resistance mutations in gryA and parC suggest that topoisomerase IV may be a secondary drug target in E. coli and Neisseria gonorrhoeae. In contrast, in Staphylococcus aureus these patterns suggest that topoisomerase IV may be a primary target of quinolone action. Regulation of expression of membrane efflux transporters may contribute to quinolone susceptibility in both Gram-positive and Gram-negative bacteria. The substrate profile of the NorA efflux transporter of S. aureus correlates with the extent to which the activity of quinolone substrates is affected by overexpression of NorA. In addition, the Emr transporter of E. coli affects susceptibility to nalidixic acid, and the MexAB OprK transport system of Pseudomonas aeruginosa affects susceptibility to ciprofloxacin.(ABSTRACT TRUNCATED AT 250 WORDS)19958549276